EP1037963A1 - CRYSTAL COMPRISING HUMAN IMMUNODEFICIENCY VIRUS ENVELOPE GLYCOPROTEIN gp120, COMPOUNDS INHIBITING CD4-gp120 INTERACTION, COMPOUNDS INHIBITING CHEMOKINE RECEPTOR-gp120 INTERACTION, MIMICS OF CD4 AND gp120 VARIANTS - Google Patents
CRYSTAL COMPRISING HUMAN IMMUNODEFICIENCY VIRUS ENVELOPE GLYCOPROTEIN gp120, COMPOUNDS INHIBITING CD4-gp120 INTERACTION, COMPOUNDS INHIBITING CHEMOKINE RECEPTOR-gp120 INTERACTION, MIMICS OF CD4 AND gp120 VARIANTSInfo
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- EP1037963A1 EP1037963A1 EP98959406A EP98959406A EP1037963A1 EP 1037963 A1 EP1037963 A1 EP 1037963A1 EP 98959406 A EP98959406 A EP 98959406A EP 98959406 A EP98959406 A EP 98959406A EP 1037963 A1 EP1037963 A1 EP 1037963A1
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- polypeptide
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- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
- G01N23/207—Diffractometry using detectors, e.g. using a probe in a central position and one or more displaceable detectors in circumferential positions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- HIV Human Immunodeficiency Virus
- gpl20 gpl20
- AIDS acquired immunodeficiency syndrome
- CD4 two cellular receptors of the human host
- chemokine receptor primarily CXCR-4 or CCR-5, depending on viral strain
- these high affinity interactions are attractive targets for mimetic drug design.
- the structure of the gpl20-binding domain of CD4 and the identity of residues critical to its interaction with gpl20 have been known for several years (13,14), this has not been sufficient for design of potent antagonists (15-17).
- the major virus-specific antigen accessible to neutralizing antibodies knowledge of the gpl20 structure could also impact considerably on vaccine design.
- the gpl20 protein has been an obvious target for structural investigation, and quantities of pure soluble protein have been available for several years, a byproduct in part from vaccine trials. Nevertheless, despite considerable effort, it has resisted crystallographic analysis for more than a decade.
- the mature gpl20 glycoproteins of different HIV-1 strains have approximately 470-490 amino acids (18). Extensive N-linked glycosylation at approximately 20-25 sites accounts for roughly half its mass (18,19). Sequences from many different viral isolates show that it contains five conserved regions (C1-C5) and five variable regions (V1-V5)' (18, 20) and nine conserved disulfide bridges (19) . Except for limited N- and C- terminal cleavage, proteolytic digestion does not reveal a sub-domain structure. Indeed, even after extensive proteolytic cleavage, the unreduced protein runs near its native molecular weight on SDS-PAGE (Peter D. Kwong: unpublished data) .
- variable regions the V3 loop in particular, appear to be conformationally variable. Conformational change is also evidenced by shedding, the CD4-induced dissociation of gpl20 from the surface of the virus, and by ligand-induced variations in monoclonal antibody binding (21,22). These changes may be related to the functional role of gpl20 in virus entry.
- the subject invention provides a crystal suitable for X- ray diffraction comprising a polypeptide having an amino acid sequence of a portion of a Human Immunodeficiency Virus envelope glycoprotein gpl20, wherein the amino acid sequence is at least 100 amino acids in length.
- the subject invention also provides the above-described crystals, which effectively diffract X-rays for determination of the atomic coordinates of the polypeptide to a resolution of 2.5 angstroms or better than 2.5 angstroms.
- the subject invention additionally provides a method for producing a crystal suitable for X-ray diffraction comprising: (a) deglycosylating a polypeptide having amino acid sequence of a portion of a gpl20 wherein said portion is produced by deleting or replacing part of the gpl20 to reduce the surface loop flexibility; (b) contacting the polypeptide with a ligand so as to form a complex which exhibits restricted conformational mobility; and (c) obtaining crystal from the complex so formed to produce a crystal suitable for X-ray diffraction.
- the subject invention also provides the above-described methods, wherein the VI, V2, or V3 loop of the gpl20 contained in the polypeptide are partially truncated, deleted or replaced.
- the subject invention also provides a method for identifying a compound capable of binding to a portion of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining a binding site on the portion of gpl20 based on the atomic coordinates computed from X-ray diffraction data of a crystal comprising the portion of gpl20; and (b)- determining whether a compound would fit into the binding site, a positive fitting indicating that the compound is capable of binding to the gpl20.
- This invention also provides a method of inhibiting the interaction of HIV-gpl20 with CD4 which comprises administering to a mammal in need thereof a compound capable of disrupting two or more of the contacts between gpl20 and CD4 as set forth in Figure 54.
- This invention also provides a method for identifying a compound capable of binding to the CD4 binding site of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining the CD4 binding site on the gpl20 based on the atomic coordinates computed from X- ray diffraction data of a crystal comprising a polypeptide having amino acid sequence of a portion of gpl20 capable of binding to CD ; and (b) determining whether a compound would fit into the binding site, a positive fitting indicating that the compound is capable of binding to the CD4 binding site of the gpl20.
- This invention also provides a method for designing a compound capable of binding to the CD4 binding site of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining the CD4 binding site on the gpl20 based on the atomic coordinates computed from X- ray diffraction data of a crystal comprising a polypeptide having amino acid sequence of a portion of gpl20 capable of binding to CD4 ; and (b) designing a compound to fit the CD4 binding site.
- This invention also provides a method of inhibiting Human Immunodeficiency Virus infection in a subject comprising adminstering effective of amount of the above-described composition to the subject.
- This invention provides a method for identifying a compound capable of binding to the chemokine receptor binding site of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining the chemokine receptor binding site on the gpl20 based on the atomic coordinates computed from X-ray diffraction data of a crystal comprising a polypeptide having the amino acid sequence of a portion of gpl20 capable of binding to the chemokine receptor; and (b) determining whether a compound would fit into the binding site, a positive fit indicating that the compound is capable of binding to the chemokine receptor binding site of the gpl20.
- This invention also provides a method for designing a compound capable of binding to the chemokine receptor binding site of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining the chemokine receptor binding site on the gpl20 based on the atomic coordinates computed from X-ray diffraction data of a crystal comprising a polypeptide having the amino acid sequence of a portion of gpl20 capable of binding to the chemokine receptor; and (b) designing a compound to fit the chemokine receptor binding site.
- the crystal further comprises a chemokine receptor, a second polypeptide having amino acid sequence of a portion of chemokine receptor, an antibody or a Fab capable of binding to the chemokine receptor binding site or a compound known to be capable of binding to the chemokine receptor binding site, bound to the polypeptide.
- This invention further provides a method of inhibiting the interaction of HIV-gpl20 with chemokine receptor which comprises administering to a mammal in need thereof a compound capable of disrupting two or more of the contacts between gpl20 and chemokine receptor as set forth in Figure 55, thereby inhibiting the interaction of HIV-gpl20 with chemokine receptor.
- This invention provides a substance mimicking the gpl20- binding domain of CD4 wherein the size of the residue or analog thereof at position 43 is bigger than the size of a phenylalanine so as to increase the affinity for human immunodeficiency virus envelope glycoprotein gpl20.
- This invention also provides the above-described substances, wherein the modification results in a o residue or analog thereof larger than 10 A across its longest dimension.
- This invention also provides the above-described substances, wherein the modification results in a residue or analog thereof, wherein the residue's longest dimension is longer than phenylalanine ' s longest dimension.
- This invention also provides the above-described substances, wherein the modification results in a residue or analog thereof larger than 15 A across its longest dimension.
- This invention also provides the above-described substances, wherein the modification involves replacemnet of the residue at position 43 with a cysteine .
- This invention also provides the above-described substances, wherein the modification involves replacement of the residue at position 43 with a tyrosine .
- This invention further provides a pharmaceutical composition capable of inhibiting cell entry by HIV, comprising (a) an effective amount of the substance of claim 1; and (b) a pharmaceutically acceptable carrier.
- This invention further provides a method of inhibiting cell entry by HIV, comprising contacting the cells with an effective amount of the above-described substances, thereby inhibiting cell entry by HIV.
- This invention further provides a method of treating or preventing HIV infection in a subject, comprising administering to the subject an effective amount of the above-described substances, thereby treating or preventing HIV infection.
- the subject invention provides a variant of gpl20 which presents a hidden, conserved, neutralization epitope.
- the subject invention also provides a composition comprising a variant of gpl20 which presents a hidden, conserved, neutralization epitope and a suitable carrier.
- the subject invention further provides a vaccine comprising a variant of gpl20 which presents a hidde conserved, neutralization epitope and a suitable carrier .
- the subject invention also provides an antibody induced by a vaccine comprising a variant of gpl20.
- CD4 is in the top left, gpl20 is toward the right, and Fab 17b interacting.
- CD4 is in the top left, gpl20 is toward the right, and Fab
- 17b is in the bottom left of the figure.
- Crystal types A-F are shown and correspond to the crystal types described in the text and Tables 3 and 4.
- the photomicrograph in A is at twice the magnification.
- the bar in A corresponds to 25 ⁇ m (50 ⁇ m for B-F) .
- Lane 1 (Pharmacia Phast system). Lane 1, 2.5 ug of ternary complex purified by gel filtration. The top band is the deglycosylated ⁇ 82 ⁇ V1/2* ⁇ V3 ⁇ C5 gpl20, the next two bands are the alkylated and reduced heavy and light chains respectively of the Fab 17b, and the bottom band is the two-domain sCD4 (D1D2). Lane 2, standards: 94, 67, 43
- Lane 5 dissolved crystals. The gel is silver stained.
- the HIV-1 entry process The trimeric HIV-1 envelope glycoproteins, anchored in the viral membrane, are depicted, with gpl20 in the lower right and gp41 in the upper right. For simplicity, the gpl20 variable loops are not shown, but would extend over the outer surface of the envelope glycoprotein complex.
- the receptors on the target cell, CD4 and chemokine receptor are also shown.
- the structures of gpl20, gp41, and CD4 are adapted from available X-ray crystallographic studies (5,20,21), whereas the chemokine receptor model is hypothetical .
- Figure 27 The HIV-1 gpl20 surface.
- Figure 27A The HIV-1 gpl20 surface.
- the molecular surface of the HIV-1 gpl20 core (20) is shown, with the arrow pointing towards the viral membrane.
- the inner domain, believed to interact with gp41, and the outer domain, which is probably exposed on the assembled trimer, are on the left and right, respectively.
- the gpl20 surface occluded by CD4 is shown and the gpl20 region thought to be involved in chemokine receptor binding (27) is also shown.
- the location of the base of the V3 loop is shown.
- the relationship of different surfaces of the gpl20 core to the antibody response generated by the gpl20 glycoprotein is depicted.
- the surface of gpl20 that interacts with neutralizing antibodies (32) is shown, spans the inner and outer domains, and includes the V2 and V3 variable loops (not shown) .
- the surface of gpl20 that interacts with non-neutralizing antibodies is located on the inner domain, and includes gp41- interactive N- and C-terminal gpl20 regions (not shown) .
- the heavily glycosylated surface of the gpl20 outer domain, which appears to be minimally immunogenic, is also shown.
- core gpl20 Structure of core gpl20.
- the orientation of gpl20 in each of the panels shown in this figure is related to Figure 17 by a 90° rotation about a vertical axis.
- the viral membrane would be oriented above, the target membrane below, and the C-terminal tail of CD4 coming out of the page.
- the left portion of core gpl20 as the “inner” domain
- the right portion as the “outer” domain
- the 4-stranded sheet at the bottom left of gpl20 as the "bridging sheet.”
- the bridging sheet ( ⁇ 3, ⁇ 2, ⁇ 21, ⁇ 20) can be seen packing primarily over the inner domain, although some surface residues of the outer domain, e.g.
- FIG 29A Ribbon diagram. Helices and ⁇ -strands are depicted, strand ⁇ l5 makes an antiparallel ⁇ -sheet alignment with strand C' 1 of CD4. The dashed line to the right of the diagram represents the disordered V4 loop. Selected parts of the structure are labeled.
- Figure 29B
- Solvent accessibility is indicated for each residue by an open circle if the fractional solvent accessibility is greater than 0.4, a half-closed circle if 0.1 to 0.4, and a closed circle if less than 0.1. Sequence variability observed among primate immunodeficiency viruses is indicated below the solvent accessibility by the number of horizontal hash marks: 1 mark, residues conserved among all primate immunodeficiency viruses; 2 marks, conserved among all HIV-1 isolates; 3 marks, exhibits moderate variation among HIV-1 isolates; and 4 marks, exhibits significant variability among HIV-1 isolates. In accessing conservation, all single atom changes were permitted as well as larger substitutions if the character of the sidechain was conserved (e.g. K to R or F to L) .
- N-linked glycosylation is indicated by "m” for the high mannose additions and "c” for the complex additions observed in mammalian cells (6).
- Residues of gpl20 in direct contact with CD4 are indicated by "*". Direct contact is a more restrictive criterion of interaction than the often used loss of solvent accessible surface; residues of gpl20 which show loss of solvent accessible surface but are not in direct contact are 123, 124, 126, 257, 278, 282, 364, 471, 475, 476 and 477. Parts (a) and (b) were drawn with MOLSCRIPT (P. J. Kraulis) .
- Figure 30 CD4-gpl20 interactions.
- Figure 30A Ribbon diagram of gpl20 binding to CD4. Residue Phe 43 of CD4 is also depicted reaching into the heart of gpl20. From this orientation the recessed nature of the gpl20 binding pocket is evident.
- Electron density in the Phe 43 cavity The 2Fo-Fc electron density map at 2.5 ⁇ , l.l ⁇ contour, is shown. The orientation is the same as in (a). The foreground has been clipped for clarity removing the overlying ⁇ 24- ⁇ 5 connection. In the upper middle of the picture is the central unidentified density. At the bottom of the picture, Phe 43 of CD4 can be seen reaching up to contact the cavity.
- the gpl20 residues are Trp 427 (with its indole ring partially clipped by foreground slabbing) , Trp 112, Val 255, Thr 257, Glu 370 (packing under the Phe 43 ring), lie 371, and Glu 368 (partially clipped in the bottom right corner) . Hydrophobic residues lining the back of the cavity can be partially glimpsed around the central unidentified density.
- Electrostatic surface of CD4 and gpl20 The electrostatic potential is displayed at the solvent accessible surface, which is shaded according to the local electrostatic potential. The slight "puffiness" of the surface arises from the enlarged nature of the solvent accessible surface relative to the standard molecular surface.
- the gpl20 surface On the right, the gpl20 surface is shown in an orientation similar to that of Figures 29A and 29C, but rotated -20° around a vertical axis to depict the recessed binding pocket more clearly.
- a thin yellow C ⁇ worm of CD4 is shown to aid in orientation.
- the CD4 surface is shown, rotated relative to the gpl20 panel by an exact 180° rotation about the vertical axis shown.
- a thin red C ⁇ worm of gpl20 is shown.
- CD4-gpl20 contact surface On the right, the gpl20 surface is shown with the surface within 3.5 A of CD4 (surface-to-atom center distance) . This effectively creates an "imprint" of CD4 on the displayed gpl20 surface. On the left (180° rotation) , the corresponding CD4 surface and gpl20 "imprint” is also shown.
- Figure 30E CD4-gpl20 mutational "hot-spots.”
- the surface of gpl20 On the right, the surface of gpl20 is shown with the surface of gpl20 residues shown by substitution to affect CD4 binding highlighted: substantial effect -- residues 257, 368, 370 and 427; moderate effect -- residue 457.
- Sequence variability mapped to the gpl20 surface The sequence variability observed among primate immunodeficiency viruses (Figure 29D) is depicted mapped onto the gpl20 surface. Also shown is the carbohydrate: N-acetylglucosamine and fucose residues present in the structure; Asn-proximal N-acetylglucosamines modeled at residues 88, 230, 241, 356, 397, 406, 462. Much of the carbohydrate (22 residues) is hidden on the back side of the outer domain. Figure 30H
- Phe 43 cavity The surface of the Phe 43 cavity is shown, buried in the heart of gpl20.
- a worm representation of gpl20 shows the three stretches that are incorrectly predicted by secondary structure prediction: the ⁇ B loop, bending around the top of the cavity, strands ⁇ 20- ⁇ 21 just below the cavity, and strand ⁇ l5, slightly more distal to the cavity right.
- the orientation shown here is the same as for the gpl20 surfaces in Figure 30C-G.
- FIG 31A Worm diagram of Fab 17b and gpl20.
- the Fab 17b is shown binding to gpl20.
- the orientation shown is the same as in Figures 29A and 29C.
- Electrostatic surface The electrostatic potential is displayed at the solvent accessible surface, which is shaded according to the local electrostatic potential.
- the electrostatic shading is the same scale as that shown in Figure 30C.
- the surface that corresponds to the 17b epitope is the most electropositive region of the molecule.
- the V3 loop is truncated here, but sequence analysis shows that it is generally quite positively charged.
- FIG. 29A and 29C Schematic representation of the gpl20 initiation of fusion.
- a single monomer of core gpl20 is depicted in an orientation similar to Figures 29A and 29C.
- the "3" symbolizes the 3-fold axis, from which gp41 interacts with the gpl20 N- and C- termini to generate the functional oligomer.
- the V1/V2 loops are shown partially occluding the CD4 binding site.
- a conformational change is depicted as an inner/outer domain shift, with the dark circle denoting the formation of the Phe 43 cavity.
- HIV-1 gpl20 Structure of HIV-1 gpl20 with neutralizing antibody and human receptor CD .
- Figure 34D View of the molecular surface of the gpl20 core inner domain.
- variability is indicated by the shading scheme used in Figure 34B.
- the CD4-binding site is to the right of the figure, and the protruding V1/V2 stem is indicated.
- the conserved molecular surface, which is associated with the inner domain of the gpl20 core, is devoid of know N-linked glycosylation. These are modeled in the figure on the right, which is shaded as described in Figure 23B.
- Figure 35
- the molecular surface of the gpl20 core is shown, from the same perspective as that in Figure 34A.
- the modeled N-terminal gpl20 core residues, V4 loop and carbohydrate structures are included.
- the variability of the molecular surface is indicated, using the shading scheme described in Figure 34B.
- the approximate locations of the V2 and V3 variable loops are indicated. Note the well-conserved surfaces near the "Phe 43" cavity and the chemokine receptor- binding site (see Figure 34A) .
- a Co tracing of the gpl20 core, oriented similarly to Figure 34A.
- the gpl20 residues within Figure 37A of the 17b CD4i antibody are shown.
- the residues implicated in the binding of CD4BS antibodyies (20) are shown. Changes in these residues significantly affect the binding of at least 25 percent of the CD4BS antibodies listed in the table from the fourth series of experiments.
- the residues implicated in 2G12 binding (19) are shown.
- the V4 variable loop, which contributes to the 2G12 epitope, (19) is indicated by dotted lines (see figure 34A) .
- Figure 35C
- Approximate locations of the faces of the gpl20 core defined by the interaction of gpl20 and antibodies.
- the molecular surface accessible to neutralizing ligands (CD4 and CD4BS, CD4i and 2G12 antibodies) is shown in white.
- the neutralizing face of the complete gpl20 glycoprotein includes the V2 and V3 loops, which reside adjacent to the surface shown (see Figure 35A) .
- the approximate location of the gpl20 face that is poorly accessible on the assembled envelope glycoprotein trimer and therefore elicits only non-neutralizing antibodies (5 , 6) is shown.
- the approximate location of an immulogically "silent" face of gpl20 which roughly corresponds to the highly glycosylated outer domain surface, is also shown.
- a likely arragement of the HIV-1 gpl20 glycoproteins in a trimeric complex The gpl20 core was organized into a trimeric array, based on the criteria discussed in the text.
- the perspective if from the target cell membrane, similar to that shown in Figure 34C.
- the CD4 binding pockets are indicated by black arrows, and the chemokine receptor-binding regions are darkly shaded.
- the lightly shaded areas indicate the more variable, glycosylated surface of the gpl20 core.
- the approximate locations of the 2G12 epitopes are indicated by open arrows .
- the approximate locations for the V3 loops and V4 regions are shown.
- the positions of the V5 regions and some complex carbohydrate addition sites are shown.
- the approximate locations of the large V1/V2 loops, centered on the known positions of the VI/V2 stems, are indicated.
- On one of the gpl20 subunits the positions of the L D and L E loops are indicated.
- the distance of each of the gpl20 monomers from the 3 -fold symmetry axis is arbitrary.
- the HIV gpl20 derivative used in the binding assay The wild-type gpl20 and gp41 envelope glycoproteins are shown in the upper figure. conserveed (black) and variable (white) regions (25) are indicated.
- the N-terminal and V1/V2 deletions correspond to those previously described for the HXBc2 gpl20 mutants ⁇ 82 and ⁇ 128-194, respectively (8,9).
- SIG signal peptide.
- FIG. 38A The radiolabeled wt ⁇ protein was incubated either with the parental LI .2 cells or with the L1.2-CCR5 cells. Incubations were carried out either in the absence or presence of sCD4 (lOOnM) . The wt ⁇ protein bound to the cells is shown. The two bands represent different glycoforms of gpl20.
- Figure 38B The radiolabeled wt ⁇ protein was incubated either with the parental LI .2 cells or with the L1.2-CCR5 cells. Incubations were carried out either in the absence or presence of sCD4 (lOOnM) . The wt ⁇ protein bound to the cells is shown. The two bands represent different glycoforms of gpl20.
- Figure 38B The two bands represent different glycoforms of gpl20.
- the wt ⁇ protein was incubated with both sCD4 and 17b antibody at the indicated concentrations prior to adition to the L1.2-CCR5 cells.
- the L1.2-CCR5 cells were incubated with 2D7 anti-CCR5 antibody or MIP-13 at the indicated concentrations prior to incubation with wt ⁇ -sCD4 complexes.
- the wt ⁇ protein bound to the cells is shown.
- Figure 38C The amount of radiolabeled wt ⁇ or selected mutant envelope glycoproteins precipitated by a mixture of HIV- 1-infected patient sera (Total), precipitated by sCD4 and an anti-CD4 antibody (Bound (sCD4) ) , or bound to LI.2- CCR5 cells (Bound (CCR5) ) is shown.
- Figure 39 The amount of radiolabeled wt ⁇ or selected mutant envelope glycoproteins precipitated by a mixture of HIV- 1-infected patient sera (Total), precipitated by sCD4 and an anti-CD4 antibody (B
- a ribbon drawing of the HIV-1 gpl20 glycoprotein (6) complexed with CD4 is shown. The perspective is that from the target cell membrane. The two amino-terminal domains of CD4 are shown. The gpl20 inner domain is shown, the outer domain is shown and the "bridging sheet" is shown. The gpl20 residues in which changes resulted in a >90% decrease in CCR5 binding are labeled. The VI/V2 stem and base of the V3 loop (strands / 512 and / ⁇ l3 and the associated turn) are indicated.
- FIG. 39A A molecular surface of the gpl20 glycoprotein from the same perspective as that of Figure 39A is shown. Shaded surfaces are associated with gpl20 residues in which changes resulted in either a ⁇ 75% decrease, a ⁇ 90% decrease or a > 50% increase in CCR5 binding, when CD4 binding was at least 50% of that seen for the wt ⁇ protein.
- Figure 39C The surface depicted in Figure 39B is shaded according to the degree of conservation observed among primate immunodeficiency viruses (25) .
- Figure 39D The molecular surface of the gpl20 glycoprotein is shown, indicating residues in which changes resulted in a ⁇ 70% decease in 17b antibody binding, in the absence of sCD4.
- Figure 39E The molecular surface of the gpl20 glycoprotein is shown, indicating residues in which changes resulted in a ⁇ 70% decrease in CG10 antibody binding in the presence of sCD4. Residues in which changes significantly decreased CD4 binding (and thus indirectly decreased CG10 binding) are not shown. Images were made with Midas-Plus (Computer Graphics Lab, University of California, San Francisco) and GRASP (26) . Mimcs of CD4 With Enhanced Affinity For gpl20
- cysteine 43 mutant derivatives produced with the active halogen reaction scheme.
- Figure 43A General reaction scheme for using a bifunctional reagent to modify the gpl20 -binding domain of CD4.
- Reaction scheme for using a bifunctional reagent to modify a residue in the gpl20-binding domain of CD4 as applied to a cysteine residue.
- the residues lining the hydrophobic pocket of gpl20 include: Trp (112), Leu (116), Pro (118), Phe (210), Val (255), Ser (375), Asn (377), Phe (382), He (424), Met (426), Trp (427), Asn (428), Ala (433), Gly (473), and Met (475)
- Figure 47 Computer-generated ribbon drawing of the tertiary structure of CD4 and gpl20 interacting. CD4 is toward the bottom and gpl20 is toward the top.
- Figure 48 Reaction scheme for chemically modifying tyrosine residues. RI may be selected from the group shown in Figure 44. An alterative mechanism may be achieved as shown on page 365 of Structure and Protein Chemistry by Jack Kyte (1994), in which a diazonium salt participate in electrophilic aromatic substitution with tyrosine.
- Figure 49 Schematic showing the structural domains of gpl20.
- the topology for the gpl20 ( ⁇ 82, ⁇ Vl/2, ⁇ V3 , ⁇ C5) construct .
- Figure 55 Provides a detailed list of all the contacts between gpl20 (designated here as molecule A) and the Fab 17b
- the light chain is designated here as molecule C; the heavy chain is designated here as molecule D) .
- the invention relates to a crystals of gpl20 suitable for x-ray diffraction.
- the three dimensional structure of gpl20 provides information which has a number of uses; principally related to the development of pharmaceutical compositions which mimic the action of gpl20.
- the crystals comprising a portion of gpl20.
- the portion of gpl20 may contain the CD4 binding site.
- the portion contains the chemokine receptor binding site.
- the portion of gpl20 contains both the CD4 binding site and the chemokine receptor binding site .
- the portion of gpl20 will be at least 100 amino acids long. In a preferred embodiment, the portion is at least 200 amino acid long.
- the essence of the invention resides in the obtaining of crystals of gpl20 of sufficient quality to determine the three dimensional (tertiary) structure of the protein by x-ray diffraction methods.
- This invention provides crystals of sufficient quality to obtain a determination of the three-dimensional structure of gpl20 to high resolution, preferably to the resolution of 2.5 angstroms.
- the value of crystals of gpl20 extends beyond merely being able to obtain a structure for gpl20.
- the knowledge of the structure of gpl20 provides a means of investigating the mechanism of action of these proteins in the body. For example, binding of these proteins to various receptor molecules can be predicted by various computer models. Upon discovering that such binding in fact takes place, knowledge of the protein structure then allows chemists to design and attempt to synthesize molecules which mimic the binding of gpl20 to its receptors. This is the method of "rational" drug design.
- One skilled in the art may use one of several methods to screen chemical entities for their ability to associate with gpl20. This process may begin by visual inspection of, for example, the active site on the computer screen based on the gpl20 coordinates. Docking may be accomplished using software such as Quanta and Sybyl , followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARMM and AMBER.
- Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include:
- GRID [P.J. Goodford, "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules” , J. Med. Chem. 28:849-857 (1985)]. GRID is available from Oxford Universit, Oxford, UK.
- MCSS [A. Miranker and M. Karplus, "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method", Proteins: Structure, Function and Genetics, 11:29-34 (1991)]. MCSS is available from Molecular Systems, Burlington, MA.
- AUTODOCK [D.S. Goodsell and A. J. Olsen, "Automated Docking of Substrates to Proteins by Simulated Annealing", Proteins, Structure, Function, and Genetics, 195-202 (1990)] AUTODOCK is available from Scripps Research Institute, La Jolla, CA.
- Assembly may be proceeded by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of gpl20. This would be followed by manual model building using software as Quanta or Sybyl .
- CAVEAT [P.A. Bartell et al . , "CAVEAT: A Program of Facilitate the Structure-Derived Design of Biologically Active Molecules” , in Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc. 78, pp. 182-196 (1989)]. CAVEAT is available from the University of California, Berkeley, CA.
- 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, CA) . This area is reviewed in Y. C. Martin, "3D Database Searching in Drug Design", J. Med. Chem., 35:2145-2154 (1992).
- inhibitory or other type of binding compounds may be designed as a whole or "de novo" using either an empty active site or optionally including some portion (s) of a known inhibitor (s) .
- These methods include :
- LUDI H.-J. Bohm "The Computer Program LUDI : A New Method for the De Novo Design of Enzyme Inhibitors", J. Co p. Aid. Molec . Design, 6:61-78 (1992)].
- LUDI is available from Biosym Technologies, San Diego, CA.
- LEGEND [Y. Nishibata and A. Itai, Tetrahedron, 47:8985 (1991)].
- LENGEND is available from Molecular Simulations, Burlington, MA.
- the gpl20 or CD4 antagonist may be tested for bioactivity using standard techniques.
- structure of the invention may be used in binding assays using conventional formats to screen inhibitors.
- Suitable assays for use herein include, but are not limited to, the enzyme-linked immunosorben assay (ELISA) , or a fluoresence quench assay.
- ELISA enzyme-linked immunosorben assay
- fluoresence quench assay a fluoresence quench assay.
- Other assay formats may be used; these assay formats are not a limitation on the present invention.
- the gpl20 structure of the invention permit the design and identification of synthetic compounds and/or other molecules which have a shape complimentary to the conformation of the gpl20 active site of the invention.
- the coordinates of the gpl20 structure of the invention may be provided in machine readable form, the test compounds designed and/or screened and their conformations superimposed on the structure of the invention. Subsequently, suitable candidates identified as above may be screened for the desired gpl20 inhibitory bioactivity, stability, and the like.
- inhibitors may be used therapeutically or prophylactically to block gpl20 activity.
- this invention also provides material which is the basis for the rational design of drugs which mimic the action of gpl20.
- the subject invention provides a crystal suitable for X- ray diffraction comprising a polypeptide having an amino acid sequence of a portion of a Human Immunodeficiency Virus envelope glycoprotein gpl20.
- the subject invention also provides the above-described crystals, which effectively diffract X-rays for determination of the atomic coordinates of the polypeptide to a resolution of 4 angstroms or better than 4 angstroms.
- the subject invention also provides the above-described crystals, which effectively diffract X-rays for determination of the atomic coordinates of the polypeptide to a resolution of 2.5 angstroms or better than 2.5 angstroms .
- the subject invention also provides the above-described crystals, wherein the portion of gpl20 comprises a CD4 binding site.
- the subject invention further provides the above- described crystals, further comprising a compound bound to the CD4 site.
- the subject invention also provides the above-described crystals, wherein the portion of gpl20 comprises a chemokine receptor binding site.
- the subject invention also provides the above-described crystals, further comprising a compound bound to the chemokine receptor binding site.
- the subject invention also provides the above-described crystals, wherein the portion of gpl20 comprises a CD4 binding site and a chemokine receptor binding site.
- the subject invention also provides the above-described crystals, further comprising of a first compound bound to the CD4 binding site of the polypeptide and a second compound bound to the chemokine receptor binding site of the polypeptide.
- the subject invention also provides the above-described crystals, wherein the first compound is the second compound .
- the subject invention also provides the above-described crystals, wherein the polypeptide is a variant of gpl20 lacking the VI, V2 , V3 , and C5 regions.
- the subject invention also provides the above-described crystals, wherein the gpl20 variant comprises a portion of the conserved stem of the V1/V2 stem- loop structure.
- the subject invention also provides the above-described crystals, wherein the gpl20 variant comprises a portion of the base of the V3 loop.
- the subject invention also provides the above-described crystals, wherein the gpl20 variant comprises a portion of the C5 region.
- the subject invention also provides the above-described crystals, wherein the polypeptide is a variant of gpl20 with 5% by weight of the carbohydrate residues linked to the gpl20 in substantially the same manner as they are linked to gpl20 in unmodified gpl20.
- the subject invention also provides the above-described crystals, wherein the polypeptide is a variant of gpl20 with 15% by weight of the carbohydrate residues linked to the gpl20 polypeptide in substantially the same manner as they are linked to gpl20 in unmodified gpl20.
- the subject invention also provides the above-described crystals, further comprising a Fab, a CD4 , a polypeptide having amino acid sequence of a portion of CD4 , or a combination thereof, bound to the gpl20.
- the subject invention also provides the above-described crystals, wherein the Fab is produced from an antibody to a discontinuous epitope.
- the subject invention also provides the above-described crystals, wherein the monoclonal antibody is designated 17b.
- the subject invention additionally provides a method for producing a crystal suitable for X-ray diffraction comprising: (a) deglycosylating a polypeptide having amino acid sequence of a portion of a gpl20 wherein said portion is produced by deleting or replacing part of the gpl20 to reduce the surface loop flexibility; (b) contacting the polypeptide with a ligand so as to form a complex which exhibits restricted conformational mobility; and (c) obtaining crystal from the complex so formed to produce a crystal suitable for X-ray diffraction.
- the subject invention also provides the above-described methods, wherein the VI, V2 , or V3 loop of the gpl20 contained in the polypeptide are partially truncated, deleted or replaced.
- the subject invention also provides the above-described methods, wherein the polypeptide lacks the VI, V2 , V3 and C5 loop of the gpl20.
- the subject invention also provides the above-described methods, wherein the polypeptide also lacks up to fifty N-terminal amino acids of the gpl20 or up to fifty C- terminal amino acid of gpl20.
- the subject invention also provides the above-described methods, wherein the ligand is a Fab, a CD4 , or a polypeptide having amino acid sequence of a portion of CD4.
- the subject invention also provides the above-described methods, wherein the resulting polypeptide after the deglycosylation contains at least 5% of the carbohydrate .
- the subject invention also provides the crystal produced by the above-described methods.
- the subject invention also provides a method for identifying a compound capable of binding to a portion of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining a binding site on the portion of gpl20 based on the atomic coordinates computed from X-ray diffraction data of a crystal comprising the portion of gpl20; and (b) determining whether a compound would fit into the binding site, a positive fitting indicating that the compound is capable of binding to the gpl20.
- the subject invention also provides a method for designing a compound capable of binding to a portion of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining a binding site on the portion of gpl20 based on the atomic coordinates computed from X-ray diffraction data of a crystal comprising the portion of gpl20; and (b) designing a compound to fit the binding site.
- the subject invention also provides the above-described methods, wherein the atomic coordinates are set forth in Figure 53.
- the subject invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising the compound identified by the above-described methods and a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carriers means any of the standard pharmaceutical carriers.
- suitable carriers are well known in the art and may include, but not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solutions, phosphate buffered saline containing Polysorb 80, water, emulsions such as oil/water emulsion, and various type of wetting agents.
- Other carriers may also include sterile solutions, tablets, coated tablets, and capsules.
- Such carriers typically contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium sterate, talc, vegetable fats or oils, gums, glycols, or other known excipients .
- excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium sterate, talc, vegetable fats or oils, gums, glycols, or other known excipients .
- Such carriers may also include flavor and color additives or other ingredients.
- compositions comprising such carriers are formulated by well known conventional methods.
- the subject invention also provides the above-described methods, wherein the compound is not previously known.
- the subject invention also provides the compounds identified by the above-described methods.
- the subject invention also provides the compound designed by the above-described methods.
- the subject invention also provides a composition comprising the above-described compounds and a suitable carrier .
- This invention also provides a method of inhibiting the interaction of HIV-gpl20 with CD4 which comprises administering to a mammal a compound, with the proviso that the compound is not CD4 , capable of disrupting two or more of the contacts between gpl20 and CD4 as set forth in Figure 54.
- This invention also provides a method for identifying a compound capable of binding to the CD4 binding site of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining the CD4 binding site on the gpl20 based on the atomic coordinates computed from X- ray diffraction data of a crystal comprising a polypeptide having amino acid sequence of a portion of gpl20 capable of binding to CD4 ; and (b) determining whether a compound would fit into the binding site, a positive fitting indicating that the compound is capable of binding to the CD4 binding site of the gpl20.
- the molecular interaction on HIV with CD4 is between the HIV envelope glycoprotein gpl20 and the DI domain of CD4.
- the crystal structure of the complex between the deglycosylated core of gpl20 and the D1D2 fragment of human CD4 defines this interaction in atomic detail (Nature paper) . Although there is an extensive interface between these components, the nexus of the interaction brings together those residues demonstrated by mutational analyses to those most crucial for binding.
- Phe 43 and Arg 59 from CD4 and Asp 368, Glu 370 and Trp 427 from gpl20 (Nature paper, Fig. 3j).
- This dominant sub-site comprises gpl20 residues 365-368, 370-371, 425-430 and 473.
- Phe 43 closes off a pocket on the HIV surface to form a large cavity (152A 3 ) at this interface (Nature paper, Fig. 3b) .
- Residues that line the Phe 43 pocket include Trp 112, Val 255, Thr 257, Glu 370, Phe 382, Tyr 384, Try 427, Met 475 and main-chain atoms of 256 and 375-377.
- the atomic coordinates of the crystallographic model also define the binding surface to be exploited by high- affinity compounds that will have the property to inhibit the gpl20-CD4 interaction, and thereby the attachment of HIV to CD4-positive cells.
- This definition of the surface provides practioners skilled in the art with the means to design such compounds .
- Appropriate fragments or chemicals entities for the design of such compounds can be formed through the use of specialized computer programs such as GRID, DOCK and LUDI . Computer graphical representatives of these entitles can then be composed into appropriate chemical compounds, using the crystal structure as a template. Medicinal chemists skilled in the art can then synthesize appropriate chemical compounds to implement these designs.
- a compound that will bind to the dominant sub-site of the CD4 intermolecular interface will have surface properties that are complementary to the surface properties of the sub-site itself.
- the surface of the sub-site can be characterized by the GRASP computer program with respect to curvature, electrostatic potential, and hydrophobicity.
- the complementary surface to this one i.e., convex vs. concave, positive vs. negative, etc.
- any compound that has an accessible conformation such as to match the surface that is complementary to the HIV gpl20 binding surface is one that has a high probability for inhibitory binding. Since it should be possible for skilled practitioners to design and synthesize such compounds when instructed by the template of the HIV gpl20 structure and the CD4 binding elements, these compounds defined by congruence with the complementary surface can be considered inventions by the process hereby defined.
- This invention also provides a method for designing a compound capable of binding to the CD4 binding site of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining the CD4 binding site on the gpl20 based on the atomic coordinates computed from X- ray diffraction data of a crystal comprising a polypeptide having amino acid sequence of a portion of gpl20 capable of binding to CD4 ; and (b) designing a compound to fit the CD4 binding site.
- This invention also provides the above-described methods, wherein the crystal further comprising a CD4 , a second polypeptide having amino acid sequence of a portion of CD4 , or a compound known to be able to bind to the CD4 site of the gpl20, bound to the polypeptide.
- This invention also provides the above-described methods, wherein the fitting is determined by shape complementarity or by estimated interaction energy.
- This invention also provides the above-described methods, wherein the atomic coordinates are set forth in Figure 53.
- This invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising the compound identified the by above-described methods and a pharmaceutically acceptable carrier.
- This invention also provides the above-described methods, wherein the compound is not previously known.
- This invention also provides the compound identified by the above-described methods.
- This invention also provides the compound designed by the above-described methods.
- This invention also provides a composition comprising the above-described compounds and a suitable carrier.
- This invention also provides a method of inhibiting Human Immunodeficiency Virus infection in a subject comprising adminstering effective of amount of the above-described composition to the subject.
- the above-described compounds are nonpeptidyl.
- This invention provides a method for identifying a compound capable of binding to the chemokine receptor binding site of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining the chemokine receptor binding site on the gpl20 based on the atomic coordinates computed from X-ray diffraction data of a crystal comprising a polypeptide having the amino acid sequence of a portion of gpl20 capable of binding to the chemokine receptor; and (b) determining whether a compound would fit into the binding site, a positive fit indicating that the compound is capable of binding to the chemokine receptor binding site of the gpl20.
- This invention also provides a method for designing a compound capable of binding to the chemokine receptor binding site of Human Immunodeficiency Virus envelope glycoprotein gpl20 comprising: (a) determining the chemokine receptor binding site on the gpl20 based on the atomic coordinates computed from X-ray diffraction data of a crystal comprising a polypeptide having the amino acid sequence of a portion of gpl20 capable of binding to the chemokine receptor; and (b) designing a compound to fit the chemokine receptor binding site.
- the crystal further comprises a chemokine receptor, a second polypeptide having amino acid sequence of a portion of chemokine receptor, an antibody or a Fab capable of binding to the chemokine receptor binding site or a compound known to be capable of binding to the chemokine receptor binding site, bound to the polypeptide.
- This invention also provides the above-described methods, wherein the fitting is determined by shape complementarity or by estimated interaction energy.
- This invention also provides the above-described methods, wherein the atomic coordinates are set forth in Figure 53.
- composition comprising the compound identified by the above-described methods and a pharmaceutically acceptable carrier.
- This invention also provides the above-described methods, wherein the compound is not previously known.
- This invention provides compounds identified by the above-described methods. This invention provides compounds designed by above-described methods.
- composition comprising the above-described compounds and a suitable carrier.
- this invention provides a method of inhibiting Human Immunodeficiency Virus infection in a subject comprising adminstering effective of amount of the above-described composition to the subject, thereby inhibiting Human Immunodeficiency Virus infection.
- This invention further provides a method of inhibiting the interaction of HIV-gpl20 with chemokine receptor which comprises administering to a mammal a compound capable of disrupting two or more of the contacts between gpl20 and chemokine receptor as set forth in Figure 55, thereby inhibiting the interaction of HIV- gpl20 with chemokine receptor with the proviso that the compound is not a chemokine receptor.
- the compound is nonpeptidyl.
- This invention further provides a method of inhibiting cell entry by HIV, comprising blocking or inhibiting the residues from 2 or more the sets of the CCR5 -binding residues set forth above, thereby inhibiting or preventing gpl20 from binding to CCR5 and thereby inhibiting cell entry by HIV.
- This invention also provides the above described method wherein 3 or more the sets of the CCR5-binding residues set forth above are blocked or inhibited from interacting with CCR5.
- This invention also provides the above described methods, wherein the blocking or inhibiting comprises contacting the CCR5 -binding residues with an antibody.
- This invention also provides the above-described methods, wherein the compound is nonpeptidyl .
- This invention provides a substance mimicking the human immunodeficiency virus envelope glycoprotein gpl20- binding region of CD4 wherein the size of a residue or analog thereof, corresponding to the phenylalanine at position 43 in the native CD4 , is larger than the size of phenylalanine so as to fill the pocket on gpl20 which extends beyond position 43 in the gpl20/CD4 complex and increase the affinity for gpl20.
- residue or analog thereof includes amino acids (both individually and as part of a polypeptide chain), modified amino acids, amino acid analogs, and chemical compounds that can be substituted for the amino acids that ordinarily make up the CD4 polypeptide chain. (Also see the discusion of peptidomimetics, synthetic polypeptides, and polypeptide analogs below.)
- This invention also provides the above-described substance, wherein the substance is a peptidomimetic analog, a synthetic polypeptide, a standard polypeptide, or a polypeptide analog.
- the substance mimicking the gpl20- binding domain of CD4 embraces a wide range of compounds.
- the present invention also embraces other CD4 polypeptides such as polypeptide analogs of CD4.
- Such analogs include fragments of CD4.
- modifications of cDNA and genomic genes can be readily accomplished by well-known site-directed mutagenesis techniques and employed to generate analogs and derivatives of the CD4 polypeptide. Such products share at least one of the biological properties of CD4 but may differ in others.
- products of the invention include those which are foreshortened by e.g., deletions; or those which are more stable to hydrolysis (and, therefore, may have more pronounced or longerlasting effects than naturally-occurring products) ; or which have been altered to delete or to add one or more potential sites for O-glycosylation and/or N-glycosylation or which have one or more cysteine residues deleted or replaced by e.g., alanine or serine residues and are potentially more easily isolated in active form from microbial systems; or which have one or more tyrosine residues replaced by phenylalanine and bind more or less readily to target proteins or to receptors on target cells.
- polypeptide fragments duplicating only a part of the continuous amino acid sequence or secondary conformations within gpl20 which fragments may possess one property of gpl20 and not others. It is noteworthy that activity is not necessary for any one or more of the polypeptides of the invention to have therapeutic utility or utility in other contexts, such as in assays of gpl20 antagonism.
- Competitive antagonists may be quite useful in, for example, cases of overproduction of gpl20.
- polypeptide analogs of the invention are reports of the immunological property of synthetic peptides which substantially duplicate the amino acid sequence extant in naturally-occurring proteins, glycoproteins and nucleoproteins . More specifically, relatively low molecular weight polypeptides have been shown to participate in immune reactions which are similar in duration and extent to the immune reactions of physiologically-significant proteins such as viral antigens, polypeptide hormones, and the like. Included among the immune reactions of such polypeptides is the provocation of the formation of specific antibodies in immunologically-active animals [Lerner et al . , Cell, 23, 309-310 (1981); Ross et al . , Nature, 294, 654-658
- This invention also provides the above-described substances, wherein the modification increases the hydrophobicity or size of the residue or analog thereof at position 43.
- This invention also provides the above-described substances, wherein the modification comprises directly or indirectly linking a hydrophobic compound to a residue or analog thereof at position 43 of the domain.
- This invention also provides the above-described substances, wherein the modification results in a residue or analog thereof that is bulkier than phenylalanine .
- This invention also provides the above-described substances, wherein the modification- results in a residue or analog thereof larger than 7 A across its longest dimension.
- This invention also provides the above-described substances, wherein the modification results in a residue or analog thereof larger than 10 A across its longest dimension.
- This invention also provides the above-described substances, wherein the modification results in a residue or analog thereof larger than 15 A across its longest dimension.
- This invention provides the above described substance which enhances hydrophobic interactions to residues that line the pocket. In another embodiment, this invention provides the above described substance which enhances hydrogen bonding to residues that line the pocket . In a separate embodiment, this invention provides the above described substance which enhances electrostatic interactions with residues that line the pocket. In a still separate embodiment, this invention provides the above described substance which enhances surface fit with residues that line the pocket.
- This invention also provides the above-described substances, wherein the modification results in a residue or analog thereof, wherein the residue's longest dimension is longer than phenylalanine' s longest dimension.
- This invention also provides the above-described substances, wherein the modification results in a residue or analog thereof that contains a localization of negative charge so as to render the gpl20-binding domain of CD4 able to hydrogen bond more strongly with the hydroxyl-containing side chains lining gpl20.
- This invention also provides the above-described substances, wherein the modification results in a residue or analog thereof that contains a localization of charge so as to render the gpl20 -binding domain of CD4 able to hydrogen bond more strongly with the hydroxyl-containing side chains lining gpl20.
- This invention also provides the above-described substances, wherein the modification results in a residue or analog thereof that contains at least one additional hydroxyl group.
- Placing a tyrosine residue at position 43 is an example of a modification resulting in a residue that contains a hydroxyl group.
- the oxygen of the hydroxyl group has a localization of negative charge so as to render the gpl20 -binding domain of CD4 able to hydrogen bond more strongly with the hydroxyl-containing side chains lining gpl20.
- the hydrogen of the hydroxyl group has a localization of charge so as to render the gpl20-binding domain of CD4 able to hydrogen bond more strongly with the hydroxyl-containing side chains lining gpl20.
- This invention also provides the above-described substances, wherein the modification involves replacemnet of the residue at position 43 with a cysteine. This invention further provides that the substition of the sulfhydryl group of this cysteine.
- This invention also provides the above-described substances, wherein the modification involves replacement of the residue at position 43 with a tyrosine. This invention further provides that the substition of this tyrosine
- This invention also provides the above-described substances, wherein the modification comprises directly or indirectly linking an adaptor residue or analog thereof to position 43.
- This invention also provides the above-described substances, wherein the adaptor residue or analog thereof is directly or indirectly linked to a hydrophobic compound, thus forming a complex.
- This invention also provides the above-described substances, wherein the complex is bulkier than phenylalanin .
- This invention also provides the above-described substances, wherein the complex is larger than 7 A across its longest dimension.
- This invention also provides the above-described substances, wherein the complex's longest dimension is longer than phenylalanine' s longest dimension
- This invention also provides the above-described substances, wherein the complex is larger than 10 A across its longest dimension.
- This invention further provides a pharmaceutical composition capable of inhibiting cell entry by HIV, comprising (a) an effective amount of the above- described substance; and (b) a pharmaceutically acceptable carrier.
- the actual effective amount will be based upon the size of the polypeptide, the biodegradability of the polypeptide, the bioactivity of the polypeptide and the bioavailability of the polypeptide. If the polypeptide does not degrade quickly, is bioavailable and highly active, a smaller amount will be required to be effective.
- the effective amount will be known to one of skill in the art; it will also be dependent upon the form of the polypeptide, the size of the polypeptide and the bioactivity of the polypeptide. Variants of CD4 with lower affinity for gpl20 will require higher dosages than variants of CD4 with higher affinity for gpl20.
- One of skill in the art could routinely perform empirical activity tests to determine the bioactivity in bioassays and thus determine the effective amount.
- a pharmaceutical composition for treating or preventing HIV infection comprising (a) an effective amount of the above-described substances; and (b) a pharmaceutically acceptable carrier.
- This invention further provides a composition capable of inhibiting cell entry by HIV, comprising (a) an effective amount of the above-described substances; and (b) a suitable carrier.
- This invention further provides a pharmaceutical composition for treating or preventing HIV infection, comprising (a) an effective amount of the above- described substances; and (b) a pharmaceutically acceptable carrier.
- This invention further provides a composition for treating or preventing HIV infection, comprising (a) an effective amount of the above-described substances; and (b) a suitable carrier.
- This invention further provides a method of inhibiting cell entry by HIV, comprising contacting the cells with an effective amount of the above-described substances, thereby inhibiting cell entry by HIV.
- This invention further provides a method of treating or preventing HIV infection in a subject, comprising administering to the subject an effective amount of the above-described substances, thereby treating or preventing HIV infection.
- the invention provides a variant of gpl20 which presents a hidden, conserved, neutralization epitope.
- the amino acid of the above variant at position 375 is changed from a Serine to a Trptophan.
- the variant further comprise one of the following changes: 88N to P, 102E to L, 113D to R, 117K to W, 257T to A, 266A to E, 386N to Q, 395W to S, 421K to L, 470P to G, 475M to S, 485K to V or a combination thereof .
- This invention further provides a composition comprising the above-described variant and a suitable carrier.
- composition as used herein means pharmaceutical compositions comprising therapeutically effective amounts of polypeptide products of the invention together with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers useful in therapy.
- suitable diluents preservatives, solubilizers, emulsifiers, adjuvants and/or carriers useful in therapy.
- a “therapeutically effective amount” as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen.
- compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol, polyethylene glycerol) , anti-oxidants (e.g., ascorbic acid, sodium metabisulfite) , preservatives (e.g., Thimerosal, benzyl alcohol, parabens) , bulking substances or tonicity modifiers (e.g., lactose, mannitol) , covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid
- compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of the admininstered materials .
- the choice of compositions will depend on the physical and chemical properties of the protein having the biological activity. For example, a product derived from a membrane-bound form of the protein may require a formulation containing detergent.
- Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils) .
- particulate compositions coated with polymers e.g., poloxamers or poloxamines
- the variants coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors are also comprehended by the invention.
- Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral.
- suitable carriers means any of the standard carriers used in the pharmaceutical industry.
- suitable carriers are well known in the art and may include, but not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solutions, phosphate buffered saline containing Polysorb 80, water, emulsions such as oil/water emulsion, and various type of wetting agents.
- Other carriers may also include sterile solutions, tablets, coated tablets, and capsules.
- Such carriers typically contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium sterate, talc, vegetable fats or oils, gums, glycols, or other known excipients.
- excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium sterate, talc, vegetable fats or oils, gums, glycols, or other known excipients.
- Such carriers may also include flavor and color additives or other ingredients.
- Compositions comprising such carriers are formulated by well known conventional methods.
- This invention also provides a vaccine comprising the above-described variant.
- a vaccine may further comprise a suitable adjuvant.
- Vaccines and adjuvants are well-known to those skilled in the art. Using a vaccine, comprising adjuvants or not, one may induce or stimulate the immune response of an individual.
- the immune response may vary, e.g. a humoral or cell-mediated immune response.
- Adjuvants are chemical compounds that enhance the immunogenicity of the vaccine so as to enhance the stimulation and induction of the immune response.
- the vaccine is administered to a subject.
- subject means any animal or artificially modified animal capable of becoming HIV- infected. Artificially modified animals include, but are not limited to, SCID mice with human immune systems.
- the subject is a human. In another embodiment, the subject is a human infected with HIV.
- a "human infected with HIV” means an individual having at least one of his own cells infected by HIV.
- an HIV-infected cell is a cell wherein HIV has been produced.
- a non-HIV-infected subject means a subject not having any cells infected by HIV.
- a non-HIV- infected subject is an HIV-exposed subject.
- an HIV-exposed subject is a subject who has HIV present in his body, but has not yet become HIV-infected. For example, a subject may become HIV-exposed upon receiving a needle stick injury with an HIV-contaminated needle.
- the value of crystals of gpl20 extends beyond merely being able to obtain a structure for gpl20.
- the knowledge of the structure of gpl20 provides a means of investigating the mechanism of action of these proteins in the body. For example, binding of these proteins to various receptor molecules can be predicted by various computer models. Upon discovering that such binding in fact takes place, knowledge of the protein structure then allows chemists to design and attempt to synthesize molecules which mimic the binding of gpl20 to its receptors. This is the method of "rational" drug design. Using such methods, one may determine a variant of gpl20 which presents a hidden, conserved, neutralization epitope.
- This invention further provides an antibody induced by the above-described vaccine.
- the antibody may be a polyclonal antibody or a monoclonal antibody.
- an antibody comprises intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F(ab') 2 and F(v), which portions are preferred for use in the therapeutic methods described herein.
- the antibody is a single-chain antibody.
- polyclonal antibodies may comprise different sera whereas “monoclonal antibody” comprises antibodies, each of which will reconize one single epitope. Methods for production of monoclonal antibodies are well-known in the art.
- the gpl20 structure of the invention permit the design and identification of synthetic compounds and/or other molecules which have a shape complimentary to the conformation of the gpl20 active site of the invention.
- the coordinates of the gpl20 structure of the invention may be provided in machine readable form, the test compounds designed and/or screened and their conformations superimposed on the structure of the invention. Subsequently, suitable candidates identified as above may be screened for the desired gpl20 inhibitory bioactivity, stability, and the like.
- inhibitors may be used therapeutically or prophylactically to block gpl20 activity. ' Such compounds may prove useful as vaccines .
- This invention provides a vaccine comprising a polypeptide having 6 or more amino acids in the same spatial proximity to each other as the amino acids from the Phe 43 cavity of naturally occurring gpl20.
- This invention also provides the above-described vaccine, wherein the 6 or more amino acids are identical to the amino acids of naturally occurring gpl20.
- This invention further provides the above-described vaccines, wherein the amino acids are within 1 angstrom of their distances in naturally occurring gpl20.
- This invention also provides the above-described vaccines, wherein the amino acids are within 3 angstroms of their distances in naturally occurring gpl20.
- This invention provides the above-described vaccines, wherein the amino acids are within 5 angstroms of their distances in naturally occurring gpl20.
- This invention also provides the above-described vaccines, wherein the polypeptide is or is part of a conserved neutralization epitope.
- This invention further provides the above-described vaccines, further comprising a carrier.
- This invention also provides the above-described vaccines, further comprising an adjuvant.
- This invention provides a vaccine comprising a polypeptide having 6 or more continuous amino acids from the Phe 43 cavity of gpl20.
- This invention provides the above-described vaccines, wherein the polypeptide is or is part of an epitope a conserved neutralization epitope.
- This invention also provides ' the above-described vaccines, further comprising a carrier.
- This invention further provides the above-described vaccines, further comprising an adjuvant.
- This invention further provides a vaccine comprising a polypeptide having 6 or more amino acids in the same spatial proximity to each other as the surface accessible amino acids adjacent to the Phe 43 cavity of naturally occurring gpl20.
- This invention also provides the above-described vaccines, wherein the 6 or more amino acids are identical to the amino acids of naturally occurring gpl20.
- This invention provides the above-described vaccines, wherein the amino acids are within 1 angstrom of their distances in naturally occurring gpl20.
- This invention also provides the above-described vaccines, wherein the amino acids are within 3 angstroms of their distances in naturally occurring gpl20.
- This invention further provides the above-described vaccines, wherein the amino acids are within 5 angstroms of their distances in naturally occurring gpl20.
- This invention also provides the above-described vaccines, wherein the polypeptide is or is part of a conserved neutralization epitope.
- This invention further provides the " above-described vaccines, further comprising a carrier.
- This invention also provides the above-described vaccines, further comprising an adjuvant.
- This invention also provides the above-described vaccines, wherein the surface accessible amino acids comprise Lysine 432, Proline 369, and Threonine 373.
- This invention further provides a vaccine comprising a polypeptide having 6 or more continuous surface accessible amino acids adjacent to the Phe 43 cavity of gpl20.
- This invention also provides the above-described vaccines, wherein the polypeptide is or is part of a conserved neutralization epitope.
- This invention further provides the above-described vaccines, further comprising a carrier. This invention also provides the above-described vaccines, further comprising an adjuvant.
- virus viruses target specific host cells for infection by attachment to cell surface receptor molecules unique to these cells . These viral receptors have particular roles in the normal functioning of these cells. The virus simply subverts these functions in order to effect entry into the cell . Certain molecules on the viral surface can in turn be the target of antibodies raised by the host in defense against this parasitic attack. Viruses can evade such antibody immunity by mutating their surface proteins. The receptor binding site, however, must remain constant. It therefore evolves to be protected from antibody surveillance .
- the viral surface protein, gpl20 (which appears to be a trimer on the surface of the virion) , -plays a central role in immune evasion.
- the precise mechanism of gpl20 immune evasion thus far remains unknown, but the structure of the gpl20 - CD4 - Fab 17b complex reveals several crucial features:
- the CD4 binding site is very large (larger than the typical antibody footprint) .
- the Vl/2 variable loop is oriented to mask the CD4 binding site.
- the V3 variable loop is not near the CD4 binding site (on a monomer) , but the tip of this loop could interact with Fab 17b, which marks the second receptor binding site. 4.
- the CD4 binding site undergoes conformational changes upon CD4 binding.
- the Vl/2 loop occludes the CD4 binding site and allow CD4 binding. With most viruses, which bind to rare cellular receptors, such a mechanism of immune evasion would not work; the virus would not find the proper receptor at high enough frequency to ensure viral propagation. It is the clustering of CD4 positive cells in such places as the thymus which allows this mechanism to function in the particular case of HIV. 2. The virus masks constant regions involved in both CD4 and second receptor binding; the act of CD4 binding induces conformational changes in gpl20 which unmask these regions . 3. The V3 loop, which forms part of the conserved second receptor binding site, is one of the regions unmasked by CD4 binding.
- This invention uses an antigen which mimics the conformation of gpl20 on the surface of the HIV-virion, with deletions in the variable loop regions to expose the conserved CD4 binding site. It is already known that CD4 -binding site antibodies are widely neutralizing, and moreover, are found in virtually all patients (although they tend to only be found late in the course of infection -- the initial antibodies produced early in the course of infection have the Vl/2 or V3 loop as epitopes) .
- This invention provides a vaccine composed of a stabilized oligomer of gpl20, with truncations in the variable loop regions to expose the conserved CD4 binding site, would elicit widely neutralizing antibodies against HIV.
- a gpl40 construct (the extracellular portion of gpl20 + gp41) with a mutation at the gpl20/gp41 consensus cut site. 4. Trimers of GCN4 have been shown to enhance oligomerization. These oligomerization stabilizers could be added t the C-terminal tail of gpl20.
- Stabilization of kinetically hidden epitopes of gpl20 uses gpl20 which has been stabilized to elicit an immune response. gpl20 may undergo conformational changes. However, only very few expose a conserved, neutralization epitope. This invention aims at using the information from the structure of gpl20 to stabilize the hidden neutralization epitope of gpl20. Specifically, the epitope may be stabilized by mutating the gpl20 or alternatively, some epitope may be stabilized by ligand/drug interaction.
- Example 1 Specific examples are illustrated below: Example 1 :
- the pocket of gpl20 ( Figure 51) only forms upon CD4 binding. If the residues along the pocket are mutated and was filled up, making it "stuck" in the CD4 conformation even without the binding CD4.
- Such mutation may include changing the Ser375 to Trp375, Val255 to Phe255 and Thr257 to Trp257.
- the residues which lines the pocket include: Trp 112 Leu 116 Pro 118 Phe 210 Val 255 Thr 257 Ser 375 Asn 377 Phe 382 He 424 Met 426 Trp 427 Asn 428 Ala 433 Gly 473 Met 475
- disulfide bridges which tie protein domains .
- the Topology for gpl20 ( ⁇ 82, ⁇ Vl/2, ⁇ V3 , ⁇ C5 ) is shown in Figure 52. One can see the two domains, the N/C termini including ⁇ l, and a barrel around c-2. A disulfide formed between S2 and S21 will tie the protein domains. As another example, disulfide bridge may be formed between ⁇ 5 and ⁇ 6 connection and top of the barrel (e.g. ⁇ lO) .
- Example 3 Cavities internal to the gpl20 may be determined after knowing the three-dimensional structure of gpl20. Analysis of all atoms are within 4 Angstroms of the surface defining each cavity allows mutations to be designed to determine if any large substitutions are allowed. Below shows some example of the analysis:
- Val225 to Trp - not as good as 375 (below) -modeling shows some clashes with Met 475, although 475 should be able to move .
- Ser375 to Trp - good fit (Note: the ser 375 mutation is incompatible with the Val225 mutation so only one can be made at a time) .
- Wild-type phenotype is 1.00 and decreases in recognition of below 1.
- the cavity filling mutant 375S/W clearly exhibits reduction in binding of CD4-BS antibody binding. While the data look good, two of the CD4-BS antibodies (15e and 21 h) still bind with reasonable affinity.
- the basic idea behind the cavity filling mutants is to stabilize the CD4-bound conformation of gpl20 at the expense of the CD4-free conformation. Additional substitutions may then be made in combination with the 375S/W. For example, taking the known mutations which exhibit similar phenotypes to 375S/W (See Thali et al (1995), J.Virol. 67, 3978-3988).
- 113D/R The aspartic acid stabilizes the bridging sheet residues Gln428 and Lys429, which are important for maintaining the CD4-bound conformation of gpl20.
- 117K/W The lysine helps stabilize the bridging sheet conformation, but this substitutions may also affects CCR5 binding so it may not be so good a choice .
- 257T/A Since this Thr is basically buried in the CD4- bound conformation, and indeed provides stabilizing hydrogen bonds, the only way to explain the phenotype of the T/A substitution is that Thr257 must be a critical element in maintaining the CD4 -minus conformation. The residue is quite close 375W, so there may be some complications. If one places 375W in its preferred rotamer conformation, it clashes with Thr257-the mutation is accommodated by a slight change in rotamer conformation or by movement of the 375 backbone) . So the T/A may actually help to accommodate the 375W change .
- 470P/G and 475M/S Both of these are close to the CD4- binding region, although both are buried and do not interact directly with CD4. Both retain good CD4 binding so the effect may be conformational.
- This invention further provides vaccine design based upon confromational stabilization using the three- dimensional structure. See e.g. Malakauskas and Mayo Nature Structure Vol .5 , p.470-475, entitled “Design, Structure and Stability of a hyperthermophilic protein variant,” the content of which is incorporated into this application by reference.
- HIV-1 human immunodeficiency virus
- Sources of likely conformational heterogeneity such as N-linked carbohydrates, flexible or mobile N- and C-termini, and variable internal loops were reduced or eliminated, and ligands such as CD4 and antigen-binding fragments (Fabs) of monoclonal antibodies were used to restrict conformational mobility as well as to alter the crystallization surface.
- ligands such as CD4 and antigen-binding fragments (Fabs) of monoclonal antibodies were used to restrict conformational mobility as well as to alter the crystallization surface.
- CD4 and Fabs antigen-binding fragments
- Crystallization by variation and modification For the more difficult crystallization challenges, which can be defined as those for which conventional screening fails, one typically tries to vary or modify the protein while maintaining biologically important properties. Meaningful results obtain since the integrity of internal structure and functional properties can often tolerate variation at the molecular surface where lattice contacts are made. The probability for success in crystallization is enhanced because flexible or heterogeneous surface features may be removed or because of the fortuitous introduction of lattice interactions.
- a prescient example that pre-dates the powerful methods of modern molecular biology was John Kendrew' s screening of myoglobins from many different organisms until he found one, from sperm whale, that crystallized well (5) .
- human myoglobin requires a Lys to Arg substitution in order to produce crystals suitable for structural analysis (6).
- crambin forms exceptionally well-ordered crystals despite being a mixture of two isoforms with sequence variation at internal residues (7) .
- HIV-1 induces acquired immunodeficiency syndrome (AIDS) in humans (17 , 18) .
- the gpl20 glycoprotein helps to mediate virus entry into cells through sequential recognition of two cellular receptors, the surface glycoprotein CD4 (19,20) and a chemokine receptor (primarily CXCR4 or CCR5 , depending on viral strain) (21-26) .
- CD4 surface glycoprotein
- chemokine receptor primarily CXCR4 or CCR5 , depending on viral strain
- gpl20 As the major virus-specific antigen accessible to neutralizing antibodies, knowledge of the gpl20 structure could also impact considerably on vaccine design. Despite this interest and considerable effort for several years with pure soluble protein, available in quantities as a byproduct in part from vaccine trials, gpl20 has resisted crystallographic analysis .
- the mature gpl20 glycoproteins of different HIV-1 strains typically have 470-490 amino acid residues (32) .
- Extensive N-linked glycosylation at 20-25 sites accounts for roughly half of the gpl20 mass(32,33).
- Sequences from many different viral isolates show that gpl20 has five variable regions (V1-V5) interspersed between relatively conserved regions (C1-C5) (32,34) and nine conserved disulfide bridges (33) .
- V1-V5 variable regions
- C1-C5 relatively conserved regions
- proteolytic digestion does not reveal a sub-domain structure. Indeed, even after extensive proteolytic cleavage, the unreduced protein runs near its native molecular weight on SDS-PAGE (PDK, unpublished data) .
- the gpl20 glycoprotein likely exhibits conformational flexibility. Some of the variable regions, the V2 and V3 loops in particular, are known to be exposed on the surface of the native protein and probably assume multiple conformations. The potential of gpl20 to undergo conformational change is also evidenced by shedding, the CD4-induced dissociation of gpl20 from the surface of the virus, by ligand-induced variations in monoclonal antibody binding (35 , 36) , and by complex CD4-gpl20 binding kinetics (37) . These changes may be related to the functional role of gpl20 in virus entry.
- protein ligands namely CD4 and the Fab fragments of several monoclonal antibodies
- CD4 and the Fab fragments of several monoclonal antibodies were used to restrict conformational mobility.
- Progressive trials of 18 different gpl20 crystallization variants yielded six different crystals, at least one of which is suitable for structural analysis.
- This paradigm of crystallization with a focus on protein modification rather than on crystallization screening, may aid in the structural analysis of other conformationally complex proteins.
- H is defined as the homogeneous fraction of the surface.
- the probability that both are homogeneous is related to [(H- ⁇ j ) 2 x (H- 3 2 ) 2 ] where "H” is the homogeneous fraction of the surface which may form lattice contacts and " ⁇ n " is a function of the relative size and total number of lattice contacts other than contact n and the degree and distribution of surface homogeneity -- related to the occlusion of available surface area upon formation of each lattice contact as well as the spatial distribution of homogeneous surface over the molecular surface.
- the probability associated with homogeneous lattice formation is related to:
- C C ave
- the observed average value of "C" (C ave ) is -4.5(39), with a minimum theoretical value for the most common space groups of 2 or 3(39). Since C may be relatively small, lattice contacts may make up only a small proportion of a macromolecule surface, with considerable surface heterogeneity tolerated. Thus, for example, many proteins that pack into well-ordered crystal lattices have disordered regions, with N- and C- termini as well as internal loops being unresolved.
- Equation 4 is still not very useful, however, since M (homogeneous) is unknown and molecule-specific. In reducing heterogeneity, however, it seems reasonable to assume that the removed portion, if it were a highly branched carbohydrate or a proteolytically exposed region, is completely heterogeneous. In such cases,
- Equation 4 Another variant of Equation 4 can be used to estimate the impact of adding a ligand of fixed structure to a molecule that contains heterogeneous portions. This expands the surface available for lattice contacts and effectively dilutes the heterogeneous component . It may be an approach of choice when the heterogeneity is essentially unremovable, such as at the lipid interface of detergent solubilized membrane proteins.
- the heterogeneity is essentially unremovable, such as at the lipid interface of detergent solubilized membrane proteins.
- the enhancement in overall probability for successful crystallization from a set of n variants can then be calculated relative to the probability for a single variant. If we assume that the probability for crystallization of this individual variant, i, is typified by the average for all variants, Pi « P ave / the enhancement factor is
- the enhancement is inversely related to the average probability of crystallizing a single variant: £ - ( Pave ) - l O )
- Fab fragments were produced by papain digestion of monoclonal antibodies. Briefly, the antibody was reduced in 100 mM DTT, 100 mM NaCl , 50 mM Tris pH 8.0 for 1 hr at 37°C, and dialyzed (4'C), first in phosphate-buffered saline (PBS) to reduce the DTT concentration to ⁇ 1 mM, then in alkylating solution (PBS titrated to pH 7.5 with 2 mM iodoacetamide, 48 hr) , and subsequently in PBS without iodoacetamide.
- PBS phosphate-buffered saline
- the reduced and alkylated antibody was concentrated to at least 2 mg/ml and digested with papain using a commercial protocol (Pierce) .
- the gpl20 proteins were subjected to digestion with papain, elastase, and subtilisin (Boehringer Mannheim) to assay for proteolytic susceptibility.
- papain elastase
- subtilisin Boehringer Mannheim
- the gpl20 concentration was kept constant and the protease diluted serially (3.3x) from a ratio of 1:10 to 1:1000.
- the digestion mix was incubated for 1 hr at 37°C and quenched by addition of 1% SDS (1:10 ratio) with immediate heating in boiling water for 2 minutes. Digestion products were analyzed with SDS-polyacrylamide gel electrophoresis (PAGE) with and without DTT reduction.
- PAGE SDS-polyacrylamide gel electrophoresis
- Carboxypeptidase Y digestion was used to analyze the C-terminus of gpl20. A 1:10 ratio of carboxylpeptidase Y (Boehringer Mannheim) to gpl20 was incubated for 1 hr at 37 "C, pH 7.0. Even though digestion could not be easily seen by SDS-PAGE, the C-terminus of gpl20, HXBc2 strain, contains a number of positively charged amino acids, and the extent of the reaction could be monitored by native-PAGE.
- Drosophila-produced gpl20 proteins were deglycosylated enzymatically . Briefly, 0.5 mg/ml of gpl20 was incubated with various deglycosylating enzymes (singly or in combination) in 0.5 M NaCl, 100 mM Na acetate, pH 5.7, for 10 hr at 37 "C.
- Endoglycosidase D was used at a concentration of 0.1 U/ml, Endoglycosidase F at 0.25 U/ml, Endoglycosidase H at 0.25 U/ml, and Glycopeptidase F at 0.1 U/ml (all from Boehringer Mannheim) .
- Monoclonal antibody binding assay The various gpl20 glycoproteins were assessed for recognition by a variety of monoclonal antibodies directed against both linear and discontinuous gpl20 epitopes by either immunoprecipitation (46) or by ELISA(47) .
- the ELISA was performed with both fully glycosylated and deglycosylated ⁇ V1/2 ⁇ V3 glycoproteins immobilized on ELISA plates using a capture antibody specific for the gpl20 carboxyl -terminus, 6205 (International Enzymes) (47) .
- Crystallization The vapor-diffusion hanging-droplet technique was used for all crystallizations. Small volumes, 0.5 ⁇ l protein solution + 0.5 ⁇ l reservoir solution, were used for most crystallizations, screenings and final optimizations.
- Crystal Screen I (Hampton Research) was used, augmented by approximately 20 conditions which tested high protein concentrations (vapor diffusion concentration of the protein at various pH ⁇ ) as well as mixtures of organic additives (2-5% MPD, PEG 400, or PEG 4000) combined with high ionic strength (2-4 M NaCl, (NH 4 ) 2 S0 4 or Na/KP0 4 ) at pH 5.5-9.5.
- high ionic strength 2-4 M NaCl, (NH 4 ) 2 S0 4 or Na/KP0 4
- a subset of 12 different conditions was analyzed in depth to establish the approximate precipitation point of the protein for a variety of different precipitants .
- the factorial solutions were then individually adjusted to target the observed precipitation point and a full screen of -70 conditions was set up at 20 °C.
- Type E crystals were grown from the following conditions: Protein ( ⁇ 82 ⁇ Vl/2* ⁇ V3 ⁇ C5 gpl20, two-domain CD4 (D1D2) , Fab 17b purified as a ternary complex on the Superdex S-200); Droplet (0.5 ⁇ l protein solution consisting of -10 mg/ml protein in gel filtration buffer + 0.4 ⁇ l droplet mix containing 0.1 M NaCitrate, 0.02 M NaHepes, 10% isopropanol, 10.5% PEG 5000 monomethylether (Fluka) , 0.0075% SeaPrep Agarose (FMC BioProducts) , pH 6.4; Reservoir: (0.35 M NaCl, 0.1 M NaCitrate, 0.02 M Hepes, 10% isopropanol, 10.5% PEG 5000 monomethylether, pH 6.4) .
- Variant constructs of the gpl20 protein Variants of gpl20 were developed through an iterative cycle which strove to eliminate heterogeneity.
- the cycle involved recombinant production of gpl20 variants, deglycosylation, and then assessment of heterogeneity and flexibility by examinations of glycosylation status, monoclonal antibody binding, and protease sensitivity, leading to the design of new constructs. For example, protease digestion monitored by PAGE indicated susceptibility at the C-terminus, and a form with 15-20 residues removed by carboxypeptidase Y retained CD4 binding activity.
- a homogeneous product was difficult to make by this method, and primer-based PCR mutagenesis and recombinant expression were used to generate a homogeneous gpl20 derivative with a 19-residue C-terminal deletion.
- sequencing of the initial constructs showed the expected signal cleavage at +31, with four additional amino acids, Gly-Ala-Arg-Ser, added from the signal peptide (a consequence of different processing of the cloning vector signal peptide with gpl20) .
- Protease digestion gave a product at +40, indicating flexibility in the N-terminus.
- variable loops VI, V2 , and V3
- VI, V2 , and V3 were deleted and replaced with shorter segments, as reported earlier (52,53). Little effect was found on CD4 binding activity (47, 53) .
- Three constructs were made which contained deletions of the VI, V2 , and V3 loops (Table 2) .
- the ⁇ V1/2 ⁇ V3 construct the entire base and stem of the variable loops VI, V2 and V3 were excised.
- the conserved stem of the VI/V2 stem-loop structure was retained, restoring the CD4-induced antibody epitopes in the presence of soluble CD4.
- the base of the V3 loop was retained as well, fully restoring CD4-induced antibody epitopes, even in the absence of soluble CD4.
- Mass spectroscopy of the deglycosylated ⁇ 82 ⁇ Vl/2* ⁇ V3 ⁇ C5 gpl20 showed a molecular mass of 39,000 + 50 Da, consistent with a mass of 35.4 kDa for the protein (based on the DNA sequence) and 3.6 kDa for the remaining carbohydrate.
- Carbohydrate analysis showed only fucose and N-acetyl-glucosamine sugars to be present, in a ratio of 1:3.05 ⁇ 0.02, respectively.
- CD4 has a flexible juncture between the second and third extracellular domains (54), and Fabs have a conformationally mobile "elbow bend" between their variable and constant domains (55) .
- Fabs have a conformationally mobile "elbow bend" between their variable and constant domains (55) .
- Crystallization was originally devised as a method for deducing the essential crystallization factors from combinations of different conditions (1) .
- a high probability of success has been reported with as few as 6 different conditions at 4 different concentrations (56) , and commercial kits are available with 50-100 conditions (Hampton Research) .
- small volume droplets were used, typically 0.5 ⁇ l of protein per crystallization trial. With small volumes, 1-2 mg of protein was sufficient to evaluate each gpl20 crystallization variant. Smaller volumes were also more efficient at nucleation than larger droplets, perhaps due to higher surface tension effects which may result in a greater range of precipitant concentrations for each droplet to sample. Indeed, droplets that were "spread-out" also showed enhanced nucleation. This explanation may also account for the well-known observation that crystals frequently nucleate from the edges of crystallization droplets.
- the initial crystallization screens produced six different types of crystals (Fig. 1, Table 5) .
- crystal types A-D extensive optimization was unable to produce single crystals large enough to be characterized.
- crystal types E and F single crystals of needle morphology could be grown.
- the growth of single crystals of type E required the addition of agarose, which was identified during optimization by the additive screening process.
- further crystallization optimization failed to produce large single crystals, and the best typical crystals were rods with a cross-section of only 30 x 40 ⁇ m.
- a closely related crystallization variant which retained 10 additional amino acids in the stem of the V3 loop, failed to crystallize (Table 4) .
- Characteristics of gpl20 crystals Single crystals of type E and F were analyzed for diffraction in capillary mounts. Only type E crystals showed diffraction. The needle axis of type E crystals proved to coincide with the a axis, and the rhombohedral cross-section perpendicular to the needle axis proved to be bounded by faces of the form (0 1 1) . These could be distinguished from type F crystals, where the cross-section was hexagonal. Gel electrophoresis of type E crystals demonstrated that they contained all the elements of the ternary complex: gpl20, D1D2 , and Fab 17b (Fig. 2) .
- the resistance of gpl20 to crystallization may be related in part to its functional role in eluding the immune system; the mechanisms evolved to prevent the formation of specific immune system : gpl20 contacts, might also thwart formation of the homogeneous gpl20 : gpl20 contacts needed for crystallization.
- the protein modifications that most greatly reduced heterogeneity (and thus enhanced the crystallization probability) , removal of carbohydrate and substitution of the variable loops (Table 3) have been shown in vivo to enhance the generation of neutralizing antibodies (58 , 59) . It is difficult to evaluate the predictions of the crystallization algorithms derived here in a statistically significant manner.
- crystallization literature is replete with examples of protein manipulation, from proteolytic digestion, to variation in solvating detergent, to screening of DNA oligonucleotides (38) . What distinguishes our efforts is the derivation of a theoretical foundation, which allows the probabilistic assessment of the most effective crystallization approach. Because of the conformational complexity of gpl20, we focused on surface modification to eliminate heterogeneity and to present new crystallization variants -- coupled to a limited screen of crystallization conditions. The types of crystallization problems embodied in gpl20 (Table 3) are not so different from many of the typical problems facing present day crystallographers ; both from a theoretical or from a practical perspective, the strategy of probability analysis coupled to variational crystallization may be broadly applicable.
- solubility requires -300 mM NaCl for solubility ++
- (+) refers to almost no change in probability after optimization, whereas (+++++) refers to a large change in probability.
- the scale used here is a qualitative estimate; for more quantitative results, see Table 3.
- optimization refers to the effect on crystallization of making the protein more chemically homogeneous.
- optimization refers to the effect of removing or circumventing the particular source of heterogeneity. Table 2.
- ⁇ V1/2 ⁇ V3 and ⁇ V1/2 ⁇ V3 ⁇ C5 constructs were chimeras of strains BH10 and HXBc2, ⁇ Sequence numbers refer to the translated gpl60, with the mature gpl20 beginning at +31. N-terminal sequencing showed that all constructs contained 4 additional amino acids, Gly- Ala-Arg-Ser, an artifact of the signal peptide cleavage. GAG here refers to the tripeptide, Gly-Ala-Gly, which was substituted for the removed amino acids.
- Conformational Conformation restriction with protein ligands heterogeneity such as CD4 and Fabs from conformationally ( p-we ) " 1 sensitive monoclonal antibodies
- the molecular weight for the glycosylated gpl20 is approximately 90 kDa; the deglycosylated gpl20, 60 kDa; and the deglycosylated
- i D1D2 sCD4 refers to two-domain soluble CD4. Antibody epitopes are described in the text..
- D1D2 sCD4 refers to the two domain soluble CD4.
- the protein concentration is given as the absoettee (280 nm) of the complex per ml of solution.
- Crystallization reagents are conditions from Crystal Screen 1 (Hampton Research); the reagent numbers given here refer to the crystallization reagent from this commercial kit.
- Hanging droplets were 0.5 ⁇ l protein (in 0.35 M NaCl, 5 mM Tris pH 7.0, 0.02% NaN 3 ) + 0.5 ⁇ l reservoir, except for crystal type B, which used 0.5 ul of 3-fold diluted reservoir.
- Crystallization reservoirs were 500 ⁇ l; an additional 35 ul of 5 M NaCl was added after the droplet was mixed to compensate for the NaCl in the protein solution. All dilutions used H2O, except for crystal type F, where 22.5% isopropanol was used. Crystallizations were setup at room temperature and incubated at 20°C.
- PROTEIN 4 CONC RESERVOIR SOLUTION" ulP ⁇ lR FK-ORE
- PS Factorial #28 0.5 0.5 14 200 ⁇ l factorial/ 400 ⁇ l total volume (2.0 dilution) 1.36
- PS Factorial #35 0.5 0.5 15 200 ⁇ l factorial/ 700 ⁇ l total volume
- PS Factorial #12 .05 0.5 23 200 ⁇ l factorial/ 300 ⁇ l total volume
- PS Factorial #29 .05 0.5 24 200 ⁇ l factorial/ 500 ⁇ l total volume
- the final volume was made up by water , if there is a volume discrepancy .
- the human immunodeficiency viruses HIV-1 and HIV- 2 and simian immunodeficiency viruses (SIVs) are the etiologic agents of acquired immunodeficiency syndrome (AIDS) in their respective human and simian host (1) .
- infection with primate immunodeficiency viruses is characterized by an initial phase of high-level viremia, followed by a long period of persistent virus replication at a lower level (2) .
- Viral persistence occurs despite specific antiviral immune responses, which include the generation of neutralizing antibodies.
- the primate immunodeficiency viruses like all retroviruses, are surrounded by an envelope consisting of a host cell-derived lipid bilayer and virus-encoded envelope glycoproteins (3) .
- the viral membrane must be fused with the plasma membrane of the cell, a process mediated by the envelope glycoproteins.
- the exposed location of these proteins on the virus allows them to carry out their function but also renders them uniquely accessible to neutralizing antibodies.
- dual selective forces, virus replication and immune pressure have shaped the evolution of the envelope glycoproteins and continue to do so within each infected host.
- the envelope glycoproteins are synthesized as approximately 845-870 amino acid precursor in the rough endoplasmic reticulum. (N) - linked, high-mannose sugar chains are added to form the gpl60 glycoprotein, which assembles into oligomers (4- 6) . The preponderance of evidence suggests that these oligomeric complexes are trimers (4,5) .
- the gpl60 trimers are transported to the Golgi apparatus, where cleavage by a cellular protease generates mature envelope glycoproteins: gpl20, the exterior envelope glycoprotein, and gp41, the transmembrane glycoprotein (3) .
- the gp41 glycoprotein possesses an ectodomain that is largely responsible for trimerization (7) , a membrane -spanning anchor, and a long cytoplasmic tail. Most of the surface-exposed elements of the mature, oligomeric envelope glycoprotein complex are contained on the gpl20 glycoprotein. Selected, presumably well- exposed, carbohydrates on the gpl20 glycoprotein are modified in the Golgi apparatus by the addition of complex sugar (6) . The gpl20 and gp41 glycoproteins are maintained in the assembled trimer by non-covalent, somewhat labile interactions between the gp41 ectodomain and discontinuous structures composed of N- and C- terminal gpl20 sequences (8) .
- Virus attachment also involves the interaction of the gpl20 envelope glycoproteins with specific receptors, the CD4 glycoprotein (11) and members of the chemokine receptor family (12, 13) (Fig. 26).
- the CD4 glycoprotein is expressed on the surface of T lymphocytes, monocytes, -Ill- dendritic cells, and brain microglia, the main target cells for primate immunodeficiency virus in vivo.
- the requirement for CD4 binding exhibited by most primate immunodeficiency viruses for efficient entry is consistent with this observed in vivo tropism.
- CD4 binding A major function of CD4 binding is to induce conformational changes in the gpl20 glycoprotein that contribute to the formation and/or exposure of the binding site for the chemokine receptor (13, 14).
- feline immunodeficiency viruses use chemokine receptors but not CD4 for entry (16) raise the distinct possibility that the chemokine receptors represent the primordial, obligate receptors for this retroviral lineage.
- the use of CD4 as a receptor may have evolved subsequently, allowing the high-affinity chemokine receptor-binding site of primate immunodeficiency viruses to be sequestered from host immune surveillance.
- the more conserved regions fold into a gpl20 core which has been recently crystallized in a complex with fragments of CD4 and a neutralizing antibody (20) .
- the gpl20 core is composed of two domains, an inner domain and an outer domain, and a ⁇ sheet (the "bridging sheet") that does not properly belong to either domain (Fig. 27a) .
- These names reflect the likely orientation of gpl20 in the assembled envelope glycoprotein trimer: the inner domain faces the trimer axis and, presumably, gp41, while the outer domain is mostly exposed on the surface of the trimer. Elements of both domains contribute to CD4 binding.
- CD4 binds in a recessed pocket on gpl20, making extensive contact over approximately 800 A° 2 of the gpl20 surface. Two cavities are evident in the gpl20-CD4 interface. A shallow cavity is filled with water molecules, while a deep cavity extends 10-15 A° into the interior of gpl20. The opening of this deep cavity is occupied by phenylalanine 43 of CD4 , which has been shown by mutagenic analysis to be critical for gpl20 binding
- gpl20 residues previously identified as important for CD4 binding (22,23) surround the opening of the deep cavity and contribute to interactions with phenylalanine 43 of CD4.
- aspartic acid 368 of gpl20 forms a salt bridge with arginine 59 of CD4 , also shown by mutagenesis to be important for gpl20 binding (21) .
- mainchain atoms on gpl20 and CD4 form hydrogen bonds bridging the two proteins.
- the formation of the deep cavity in gpl20 likely contributes to the transmission of CD4-induced conformational changes to gpl20 elements involved in the interaction with chemokine receptors and/or gp41.
- the deep cavity may be a useful target for intervention by small molecular weight compounds.
- CCR5 for entry (12) .
- CCR5 is an obligate coreceptor, and rare individuals that are genetically deficient in CCR5 expression are relatively resistant to HIV-1 infection (24) .
- HIV-1 isolates arising later in the course of infection often-use other chemokine receptors, frequently CXCR4 , in addition to CCR5 (12,24).
- Studies of chimeric envelope glycoproteins demonstrated that the third variable (V3) loop of gpl20 is a major determinant of chemokine receptor choice (12,25) .
- V3-deleted versions of gpl20 do not bind CCR5 , even though CD4 binding occurs at wild-type levels (14) .
- Antibodies against the V3 loop interfere with gpl20-CCR5 binding
- CD4i epitopes are discussed further below. Recent mutagenic and structural analysis have revealed the existence of a highly conserved gpl20 structure that is important for CCR5 binding (20,27) (Fig. 27, a and b) . This structure is adjacent to the V3 loop and the CD4i epitopes, and is oriented to face the target cell upon gpl20-CD4 binding.
- the gp41 ectodomain structures reveal an extended, trimeric coiled coil that could potentially bridge the viral and target cell membranes (5) .
- Interactions of other gp41 helical segments near the membrane-spanning region with the interhelical grooves of the internal coiled coil are important for fusion-related conformational changes in gp41. This interaction can be inhibited by helical peptides that mimic either of the involved gp41 helices (30) and is a potential target for future intervention with small molecular weight compounds.
- HIV-1 envelope glycoproteins as antigens.
- the success of these viruses in achieving persistent infections implies that the viral envelope glycoproteins have evolved to be less-than-ideal immunogens and antigens.
- Structures on the viral envelope glycoproteins that are conserved among diverse viral strains are, in general, poorly exposed to the humoral immune system.
- the crystal structure of the gpl20 core reveals a third, immunologically silent face of gpl20 (Fig 6D) .
- HIV-1 viruses that have been passaged in immortalized cell lines are typically more sensitive to neutralization by antibodies or soluble CD4 than are primary, clinical isolates (34) .
- a major determinant is the structure of the gpl20 major variable loops, V1/V2 and V3 (35) .
- V1/V2 and V3 variable loops of a laboratory-adapted virus with those of a neutralization- resistant primary isolate creates a virus similar to the parental primary virus (35) .
- the basis for the decreased sensitivity of primary HIV-1 isolates to neutralization appears to involve a decreased exposure of the relevant gpl20 epitopes to soluble CD4 or antibody.
- gpl20 and gp41 contributes to the lability of the functional envelope glycoprotein trimer (8,9) .
- the interactive regions of gpl20 and gp41 are particular immunogenic (37) .
- neutralizing antibodies can be detected in the sera of infected animals or humans (38) . These antibodies neutralize the infecting virus but often exhibit little of no activity against other stains of virus . A subset of these strain-restricted antibodies recognize the HIV-1 V3 loop (38) . These antibodies can block chemokine receptor binding (14) .
- Other variable gpl20 elements can contribute to the epitopes recognized by the strain- restricted neutralizing antibodies. It is known, for example, that antibodies directed against the gpl20 V2 loop can also exhibit neutralizing activity (39) . The V2 loop-associated neutralization epitopes are typically conformation-dependent.
- V2-or V3- directed antibodies to recognize more than one HIV-1 strain (39,40) suggests that these major variable loops assume a finite number of conformations. This is consistent with the functional consequences on virus entry of some changes in these variable structures (41) , and with the observation that amino acid substitutions in the variable loops are not random (42) .
- the requirement for chemokine receptor binding probably constrains V3 loop variation.
- the V2 loop although dispensible for the replication of some HIV-1 viruses in culture (33), helps protect the V3 loop and the conserved epitopes near the chemokine receptor binding site from neutralizing antibodies.
- the V2 and V3 loops reside proximal to the chemokine receptor binding site (Fig. 27), masking more conserved gpl20 elements and presenting potentially variable epitopes to the immune system.
- the gpl20 residues important for antibody binding are all located within the CD4 -binding pocket on gpl20 (Fig. 27b) , and several of the most important residues are near the opening of the deep cavity (20) . Therefore, some broadly neutralizing antibodies can apparently access the more recessed elements of the CD4 binding pocket. This is consistent with the observation that the gpl20-CD4 interface is as large as that of a typical antibody-antigen complex (20) .
- CD4i CD4- induced epitopes
- the CD4i epitopes are located near conserved gpl20 structures important for chemokine receptor interaction (14) (Fig. 27b) .
- CD4 binding has been shown to cause a change in the V2 loop conformation that allows better CD4i epitope exposure (33).
- the antibodies recognizing the CD4i epitopes must bypass the overlapping V2 and V3 loops (33) . Indeed, as is evident in the current crystal structure (20) , this is accomplished by the protrusion of the CDR3 loop of the antibody heavy chain.
- Antibodies against CD4i epitopes need to bind viruses before CD4 binding occurs to achieve neutralization (47) .
- the reason is that once the envelope glycoprotein complex binds cell surface CD4 , there are severe steric constraints on the binding of an antibody to the gpl20 surface facing the target cell (Fig. 26) .
- Another fairly conserved gpl20 neutralization epitope is recognized by the 2G12 antibody (48) .
- the 2G12 antibody Unlike the other characterized HIV-1 neutralizing antibodies, which recognize gpl20 structures near or within the receptor- binding sites, the 2G12 antibody apparently binds an epitope in the outer domain (Fig. 27b) . Given the variability in this outer domain, the ability of the 2G12 antibody to neutralize a fair number of HIV-1 strains (48) seems paradoxical. The marked sensitivity of 2G12 binding to alterations in gpl20 glycosylation provides a clue to this puzzle.
- the 2G12 antibody may recognize more conserved carbohydrate structures formed as a result of the heavy concentration of N-linked glycosylation in the gpl20 outer domain.
- the apparent rarity with which 2G12-like antibodies are elicited attests to the success of the viral strategy of employing a heavily glycosylated outer domain surface in immune evasion.
- the HIV-1 envelope glycoproteins as vaccine components. That the human and simian immunodeficiency virus envelope glycoproteins are not ideal immunogens is an expected consequence of the immunological selective forces that drove the evolution of these viruses.
- the same features of the envelope glycoproteins that dictate poor immunogenicity in natural infections have hampered vaccine development.
- the lability of envelope glycoprotein complex has frustrated attempts to present oligomers mimicking the functional spike to the immune system.
- the disintegration of envelope glycoprotein oligomers contributes to the preferential elicitation of non-neutralizing antibodies by the newly exposed gpl20 N- and C-termini.
- variable loops elicit the majority of neutralizing antibodies, probably due to the exposed nature of these epitopes. It is still unclear whether conserved features in the V2 and V3 variable loops exist that can be exploited in vaccine design, or whether all possible functional configurations of these variable structures need to be represented in a cocktail of immunogens .
- the discontinuous gpl20 structures surrounding the receptor binding sites exhibit a relatively high degree of conservation (20), in keeping with the minimal polymorphism in the host cell receptors.
- the CD4 binding site contributes a particularly attractive target. It appears to be accessible to antibodies, more so than the conserved elements of the chemokine receptor-binding region. A large fraction of the broadly neutralizing antibodies that eventually appear in HIV-1-infected individuals is directed against the CD4 binding site (43), indicating that ability of the human immune system to recognize this gpl20 region and to generate an appropriate response. Nonetheless, these antibodies have been difficult to elicit in animals and vaccinated humans (49) . The reasons for the relatively poor immunogenicity of the CD4 binding site are not yet understood, although several possibilities can be envisioned.
- Interdomain flexibility may disrupt the CD4BS epitopes and decrease their representation in the pool of immunogens.
- Masking by variable loops (19,33) and glycosylation may contribute to the recessed nature of the CD4BS epitopes which, even on the crystallized gpl20 core, occupy a 20 A° deep canyon (20) .
- Within the CD4 -binding pocket not all of the gpl20 surface is conserved among HIV-1 strains. Therefore, even when elicited, some CD4BS-directed antibodies may lack the breadth and affinity to be optimal neutralization agents. While many monoclonal antibodies against the CD4 binding site exhibit reasonable potency and breadth (44) , whether a polyclonal response against the envelope glycoprotein can be focused to preferentially contain these types of antibodies remains to be seen.
- HIV-1 envelope glycoproteins have evolved to be inefficient at eliciting effective antiviral antibody responses.
- the availability of structural information on the conserved HIV-1 gpl20 neutralization epitopes should facilitate the modification of this important antigen and allow the rational testing of hypotheses regarding its poor immunogenic properties. These efforts should complement ongoing efforts to improve antigen presentation to the immune system and to create suitable animal models for the screening of vaccine candidates .
- the structure reveals a cavity- laden CD4-gpl20 interface, a conserved binding site for the chemokine receptor, evidence for conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion.
- Our results provide a framework for understanding the complex biology of HIV entry into cells and will guide efforts to intervene.
- the crystallized gpl20 is from the HXBc2 strain of HIV-1. It has deletions of 52 and 19 residues from the N- and C- termini, respectively; Gly-Ala-Gly tripeptide substitutions for 67 Vl/V2-loop residues and 32 V3-loop residues; and the removal of all sugar groups beyond the linkages between the two core N-acetylglucosamine residues.
- This deglycosylated core gpl20 eliminates over 90% of the carbohydrate but retains over 80% of the non-variable-loop protein. Its capacity to interact with CD4 and relevant antibodies is preserved at or near wild-type levels26.
- the final model, composed of 7877 atoms comprises residues 90-396 and 410-492 of gpl20 (excepting loop substitutions), residues 1-181 of CD4 , and residues 1-213 of the light chain and 1-229 of the heavy chain of the 17b monoclonal antibody.
- 11 N-acetylglucosamine and 4 fucose residues, and 602 water molecules have been placed.
- the overall structure of the complex of gpl20 with D1D2 of CD4 and Fab 17b is as depicted in Fig. 28.
- the polypeptide chain of gpl20 is folded into two major domains plus certain excursions that emanate from this body.
- the inner domain (inner with respect to the N- and C-termini) features a two-helix, two-strand bundle with a small five-stranded 3-sandwich at its termini -proximal end and a projection at the distal end from which the V1/V2 stem emanates.
- the outer domain is a stacked double barrel that lies alongside the inner domain such that the outer barrel and inner bundle axes are approximately parallel .
- the proximal barrel of the outer-domain stack is composed from a 6-stranded, mixed-directional /3-sheet that is twisted to embrace helix ⁇ 2 as a 7th barrel stave.
- the distal barrel of the stack is a 7-stranded antiparallel ⁇ barrel.
- the two barrels share one contiguous hydrophobic core, and the staves also continue from one barrel to the next except at the domain interface. This interruption is centered at a side between barrels where the chain enters the outer domain with loop ⁇ B insinuated as a tongue between strands 316 and 523.
- the extended segment just preceding ⁇ B is like an 8th stave of the distal barrel, but it is slightly out of reach for hydrogen bonding with its ⁇ !6 and 319 neighbors.
- the chain returns to complete the inner domain after ⁇ 24 .
- the proximal end of the outer domain includes variable loops V4 and V5 and loops ⁇ D and ⁇ E, which are variable in sequence as well. Loop ⁇ C is also at this end, close in space to loop ⁇ A of the inner domain, although by topology it is at the other end of this domain.
- the distal end does include the stem of the excised variable loop V3 and also an excursion via loop ⁇ F into a ⁇ hairpin, (320- / 321, which in turn hydrogen bonds with the V1/V2 stem emanating from the inner domain.
- Direct interatomic contacts are made between 22 CD4 residues and 26 gpl20 amino-acid residues. These include 219 van der Waals contacts and 12 hydrogen bonds . Residues in contact are concentrated in the span from 25 to 64 of CD4 , but they are distributed over six segments of gpl20 (Figs. 29d & 30i) : 1 residue from the V1/V2 stem, loop LD, the beta-15-alpha-3 excursion, the beta-20-beta-21 hairpin, strand beta-23 and the beta- 24-alpha-5 connection. These interactions are compatible with previous analyses of mutational data on both CD4(11, 12, 29) and gpl20(3, 13, 14).
- gpl20 residues that are covered by CD4 are variable in sequence. This variation is accommodated in part by the large interfacial cavity (Fig. 30e) .
- the gpl20 residues in contact with this water-filled cavity are especially variable (Fig. 30g) .
- half of the gpl20 residues that make contacts with CD4 do so only through main-chain atoms (including C ⁇ ) of gpl20, and 60% of CD4 contacts are made by gpl20 main-chain atoms (Fig. 3 Of ) . Included among these are 5 of the 12 hydrogen bonds in the interface.
- One such contributing element is an antiparallel ⁇ - sheet alignment of CD4 strand C" with gpl20 strand beta-15 (Figs. 30a & i) .
- Phe 43 interacts with residues Glu 370, He 371, Asn 425, Met 426, Trp 427 and Gly 473 as well as Asp 368, but only the contacts with He 371 have a conventional hydrophobic character. Those to 425-427 and 473, including Trp 427, are only to backbone atoms. A surprisingly large fraction of the Phe 43 contacts (28%) are to polar groups. The phenyl group is stacked on the carboxylate group of Glu 370, and there are contacts with the carbonyl oxygen atoms of residues 425, 426 and 473 and the NH group of Trp 427.
- the larger cavity is lined by mostly hydrophilic residues, half derived from gpl20 and half from CD4. It is not deeply buried; while formally a cavity in the crystal structure, minor changes in sidechain orientation would make it solvent accessible.
- the observed electron density and predicted hydrogen bonding are consistent with at least 8 water molecules in the cavity.
- Residues from gpl20 that actually line the cavity include Ala 281, Ser 364, Ser 365, Thr 455, Arg 469) exhibit sequence variability, whereas surrounding this variable patch are conserved residues, the substitution of which affect CD4 binding. These include the critical contact residues Asp 368, Glu 370 and Trp 427, which flank one end of the..cavity, and Asp 457 at the other end (Fig. 30e) .
- CD4 residues that line the cavity can be mutated with only moderate effect on gpl20 binding, whereas Arg 59 suffers less loss of solvent accessible surface upon gpl20 binding but is highly sensitive to mutation.
- This cavity thus serves as a water buffer between gpl20 and CD4 (Fig. 30e) .
- the tolerance for variation in the gpl20 surface associated with this cavity produces a variational island (Fig. 30g) , or "anti-hot spot", which is centrally located between regions required for CD4 binding, and may help the virus escape from antibodies directed against the CD4 binding site.
- the "Phe 43" cavity (Fig. 30b & h) is very different in character from the larger binding- interface cavity. It is roughly spherical, with a diameter of ⁇ 8 A (atom center to atom center) across the center of the cavity. It is positioned just beyond Phe 43 of CD4 , at the intersection of the inner domain, the outer domain and the bridging sheet. It is relatively deeply buried, extending into the hydrophobic interior of gpl20. The phenyl ring of Phe 43 is the only non-gpl20 residue contacting this cavity, forming a lid which covers the bottom of the cavity (Fig. 30b) .
- mutations at Thr 257 (no contacts) and Trp 427 (only main-chain contacts) can substantially reduce binding.
- Changes in cavity-lining residues also affect the binding of antibodies directed against the CD4 binding site.
- many of the residues that line the cavity interact with elements of the chemokine receptor binding region (see below) . It may be that the Phe 43 cavity and the other interdomain cavities form as a consequence of a CD4- induced conformational change (see below) .
- the 17b antibody is a broadly neutralizing human monoclonal isolated from the blood of an HIV-infected individual. It binds to a CD4-induced (CD4i) gpl20 epitope that overlaps the chemokine receptor-binding site (20) .
- the interface between Fab 17b and core gpl20 in the ternary complex involves a small area of interaction.
- the solvent accessible area excluded upon binding is only 455 A 2 from gpl20 and 445 2 A from 17b, which is largely from the heavy chain (371A 2 ) .
- the long (15 residue) complementarity-determining region 3 (CDR3) of the heavy chain dominates, but the heavy-chain CDR2 and the light-chain CDR3 also contribute.
- the 17b contact surface is very acidic (3 Asp, 3 Glu, no Arg or Lys) although hydrophobic contacts (notably a cis proline and tryptophan from the light chain) predominate at the center.
- the 17b epitope lies across the base of the four-stranded bridging sheet (Fig. 31c & e) . All four strands make substantial contact with 17b, suggesting that the integrity of the bridging sheet is necessary for 17b binding.
- the gpl20 surface that contacts 17b consists of a hydrophobic center surrounded by a highly basic periphery (3 Lys, 1 Arg, and no Asp or Glu) (Fig. 31d) . Although this basic gpl20 surface complements the acidic 17b surface, only one salt bridge is observed (between Arg 419 of gpl20 and Glu 106 of the 17b heavy chain) . The rest of the specific contacts occur between hydrophobic and polar residues.
- the interaction between 17b and gpl20 involves a hydrophobic central region flanked on the periphery by charged regions, predominately acidic on 17b and basic on gpl20.
- CD4-17b contacts There are no direct CD4-17b contacts and none of the gpl20 residues contacts both 17b and CD4. Rather, CD4 binds on the opposite face of the bridging sheet, providing specific contacts that appear to stabilize its conformation (Fig. 30i and 30j) and may explain in part the CD4- induction of 17b binding.
- the 17b epitope is well conserved among HIV-1 isolates. Of the 18 residues that show loss in solvent accessible surface upon contact with 17b, 12 residues (67%) are conserved among all HIV-1 viruses. By contrast, only 19 of the 37 gpl20 residues (51%) that show loss of solvent accessible surface upon CD4 binding are similarly conserved.
- CD4i epitopes tend to be masked from immune surveillance by the adjacent V2 and V3 loops (see accompanying paper) . Indeed, in the complex structure, a large gap is seen between gpl20 and tips of the light-chain CDR1 and CDR2 loops. Pointing directly at this gap is the base of the V3 loop.
- variable loops may need to be bypassed for access to the conserved structures in the bridging sheet .
- the 17b epitope may be further protected from the immune system by a CD4- induced conformational change (see below) .
- gpl20 Although monomeric in isolation, gpl20 likely exists as a trimeric complex with gp41 on the virion surface.
- the large electroneutral surface on the inner domain (Fig. 30c) is the probable site of trimer packing based on its lack of glycosylation, its conservation in sequence, the location of CD4 and CCR5 binding sites, and the immune response to this region.
- the Phe 43 cavity (now a pocket) would present a perplexing structural dilemma.
- the cavity-lining residues have few structural restrictions, with ample room for larger substitutions into the cavity, yet these residues are highly conserved and inexplicably hydrophobic if exposed in a pocket.
- This pocket structure is in turn intimately connected to the bridging sheet, itself peculiar in absence of CD4.
- the backbone amide of bridging- sheet residue 425 is hydrogen-bonded to Glu 370, a critical CD4 contact residue (Fig.
- core gpl20 may differ in the absence of CD4 comes from comparison with theory.
- the evolutionary algorithm of PHD37 gives secondary-structure predictions with 90% estimated reliability for roughly 45% of the core gpl20 sequence. Compared to our structure, it is accurate except at three places where it is markedly wrong (four consecutive residues with reliability index greater than 90%) . All of these are at the Phe 43 cavity or in contacts with CD4 : loop ⁇ B, strand 315, and the segment of 320 into the turn to ,321. (Fig. 30h) . Most significantly, the latter segment (residues 422-429) entering the bridging sheet is predicted to be helical.
- CD4 binds efficiently to a gpl20 derivative with both 32 and 33 truncated (38 ) . Since the bridging sheet is most likely not stable in the absence of half its strands, CD4 binding must possess the ability to properly orient strands 320 and ,321 from a very different prior conformation .
- the Phe 43 cavity is at the nexus of the CD4 interface, between the inner domain, the outer domain, and the bridging sheet. As such, Phe 43 itself seems to serve as a keystone without which the structure might collapse. If so, to what state and, in reverse, how does CD4 binding lead to the state seen in this ternary complex? Certainly, it is clear that CD4-gpl20 binding kinetics are complex(39), and microcalorimetric analysis reveals unusually large ⁇ H and compensating T ⁇ S values for soluble CD4 binding to gpl20 (M. L. Doyle, personal communication) .
- Fig. 30c stabilizes a nascent complex state, and inserts the Phe 43 to induce formation of the Phe 43 cavity.
- gpl20 Analysis of the antigenic structure of gpl20 shows that most of the envelope protein surface is hidden from humoral immune responses by glycosylation and oligomeric occlusion (accompanying paper) . Most broadly neutralizing antibodies generally access only two surfaces, one which overlaps the CD4 binding site (shielded by the V1/V2 loop) and the other which overlaps the chemokine receptor binding site (shielded by the V3 loop) . Conformational changes in core gpl20 provide additional mechanisms for evasion from immune surveillance. In the case of the CD4-binding surface, which contains a high proportion of mainchain atoms in the complex (Fig. 30f ) , the conformation without CD4 bound may expose underlying sidechain variability (Fig. 30g) .
- Escape may also be provided by the recessed nature of the binding pocket (steric occlusion) (Fig. 30a) and by a topographical surface mismatch, which encloses a variational island or "anti-hot spot" (described above, Fig. 30d) .
- Similar mechanisms may be found in the chemokine receptor region: conformational change may hide the conserved epitope (unformed prior to CD4 binding) ; steric occlusion may take place between the CD4 anchored viral spike and the proximal target membrane; and an "anti-hot spot” equivalent may camouflage chemokine-receptor binding residues on the V3 loop in surrounding variability.
- Some of the defenses used to elude antibody-based responses may also help HIV avoid cellular immunity. Understanding the specific gpl20 mechanisms of immune evasion may be prerequisite to the design of effective prophylaxis.
- the HIV surface proteins function to fuse the viral membrane with the target cell membrane.
- the gpl20 glycoprotein plays roles crucial to the control and initiation of fusion.
- One set of roles concerns positioning: locating a cell capable of productive viral infection, anchoring the virus to the cell surface, and orienting the viral spike next to the target membrane.
- Another set concerns timing: holding the gp41 in a metastable conformation and triggering the coordinate release of the three N-terminal fusion peptides of the trimeric gp41. While it is clear that this is a complex multi-conformational process, the simplicity of the system, composed only of two membranes, the viral oligomer, and two host receptors, raises the possibility that we may be able to understand the entire mechanism.
- Crystallography has now provided two snapshots: an intermediate state in which gpl20 is bound to CD4 , described herein; and a probably final, "fusion-active" state of the gp41 ectodomain (40,41) .
- an intermediate state in which gpl20 is bound to CD4 described herein
- a probably final, "fusion-active" state of the gp41 ectodomain 40,41
- the vast biochemical data concerning the membrane fusion process mediated by the HIV-1 envelope glycoproteins allow us to extend our understanding from these two states .
- CD4 binding also induces conformational changes in gpl20, which result in the creation of a metastable oligomer. Although some of the more flexible gpl20 regions and gp41 are missing, the structure of the core gpl20-CD4 complex presented here describes this state in atomic detail . CD4 binding results in movement of the V2 loop, which numerous experiments suggest partially occludes the V3 loop and CD4i epitopes (18, 36) . It also creates, or at least stabilizes, the bridging sheet on which these epitopes are located (described above for the core) .
- CD4 binding results in changes in the conformation of the V3 region, with the tip of the loop becoming more accessible, as judged by enhanced proteolytic susceptibility and altered exposure of V3 epitopes (19) .
- the V3 loop together with the uncovered epitopes comprise the chemokine-receptor binding site.
- CD4 binding not only orients the gpl20 surface implicated in chemokine receptor binding to face the target cell, but it also forms and exposes the site itself.
- these changes may all result from a single, concerted shift in the relative orientation of the inner and outer domains.
- This conformational shift may alter the orientation of the N- and C- termini, at the proximal end of the inner domain, perhaps partially destabilizing the oligomeric gpl20/gp41 interface (21) .
- Such a shift would also alter the relative placement of the V1/V2 stem (in the CD4i site) , which emanates from the inner domain, and the V3 loop, which emanates from the outer domain.
- mutations that permit an adaptation of HIV-1 to CD4-independent entry using CXCR4 involve sequence changes in both the V1/V2 stem and the V3 loop (42) .
- the next step in HIV-1 entry is the interaction of the gpl20-CD4 complex with the chemokine receptor (Fig. 32, step 2) .
- Fig. 32, step 2 interactions between CD4 and chemokine receptor may occur, mutagenic analyses (H.
- the structure of the gpl20/CD4/l7b antibody ternary complex described here reveals some of the molecular aspects of HIV-1 entry, including the atomic structure of gpl20, the explicit interactions with CD4 , and the conserved site of binding for the chemokine receptor. Still unknown are details of the apo state of core gpl20, the oligomeric structure, the interaction with the chemokine receptor, the conformational changes that trigger the reorganization of the gp41 ectodomain and the structural basis for insertion of the fusion peptide of gp41 into the target membrane. Further understanding will require snapshots of other intermediates.
- the conformational complexity and observed intricate domain associations of gpl20 may reflect genome restrictions at the protein level akin to those that lead to overlapping reading frames at the transcription level. Multiply protected infection machinery is contained in these condensed intricacies. Its mechanisms frustrate host defenses ; understanding them may inspire medical intervention.
- the two-domain CD4 (D1D2, residues 1-182) was produced in Chinese hamster ovarian cells (8), the monoclonal antibody 17b in an Epstein-Barr virus immortalized B-cell clone isolated from an HIV-1 infected individual and fused with a murine B-cell fusion partner(18), and the core gpl20 from Drosophila Schneider 2 lines under control of an inducible metallothionein promoter (20) .
- the various biochemical manipulations e.g. deglycosylation for the gpl20 and papain digestion to produced the Fab 17b
- protein purification e.g. deglycosylation for the gpl20 and papain digestion to produced the Fab 17b
- ternary complex crystallization e.g. deglycosylation for the gpl20 and papain digestion to produced the Fab 17b
- cryoprotectant containing stabilizer 10% ethylene glycol with 10.5% monomethyl-PEG 5,000, 10% isopropanol, 50 mM NaCl, 100 mM Citrate/HEPES buffer pH 6.3
- cryoprotectant containing stabilizer (10% ethylene glycol with 10.5% monomethyl-PEG 5,000, 10% isopropanol, 50 mM NaCl, 100 mM Citrate/HEPES buffer pH 6.3)
- immiscible oil Paratone-N; Exxon
- Diffraction data were collected at beamline X4A, Brookhaven National Laboratory, using phosphor image plates and a Fuji BAS2000 scanner. To avoid overlap problems from the relatively high mosaicity (-1.0°), oscillation data were collected using a rotation axis that was off-set at least 30° from the 197A c axis. Although crystals initially diffracted to Bragg spacing of greater than 2A, ⁇ axis mosaicity and substantial radiation damage despite cryogenic cooling reduced the overall resolution to -2.5A. Data processing and reduction were performed using DENZO and SCALEPACK (45) (Table 1) .
- crystals were soaked in over 20 different heavy atom solutions and screened for isomorphous replacement using the statistical ⁇ chi>2 test in SCALEPACK (45) .
- Derivatives were identified from two heavy atom compounds : 10 mM K3IrCl6 (10 hr equilibration in heavy atom containing cryoprotectant stabilizer; 2.8A) and 5 mM K20sCl6 (24 hr soak; 3.5A) .
- K3IrCl6 derivative was modeled as 9 partially occupied sites; two sites of occupancy 0.158 and 0.142, and 7 of less than 0.07. While relatively isomorphous, poor data quality (Rsym of greater than 20% past 3.0A) combined with relatively small isomorphous differences (Riso of 12.0%) reduced the quality of phasing. In contrast, the K20sCl6 derivative had an Riso of 15.6%, but was only isomorphous to roughly 5A. It was modeled as 4 sites of occupancy 0.321, 0.207, 0.194 and 0.128, with the highest site at the same position as the second highest site from K 3 IrCl 6 .
- Deviations of the CD4 structure in the complex from the free state were measured by the procedure of Wu et al. (10) . Deviations were taken as significant when the root mean square (rms) residue deviation was greater than the overall value and also more than 0.5 ⁇ greater than variation among the free structures. Interatomic contacts were defined as in Zhu et . al . (48) . Structural alignments were made by visual comparison of the SCOP databas, and automatic searches were performed with PrISM (A.-S. Yang and B. Honig) .
- HIV-1 entry co- factor functional cDNA cloning of a seven- transmembrane, G protein-coupled receptor. Science 272, 872-877 (1996) .
- chemokine receptors as human immunodeficiency virus type 1 coreceptors determined by individual amino acids in the envelope V3 loop. J. Virol. 71, 7136-7139 (1997) .
- HIV-1 Human immunodeficiency virus
- AIDS acquired immune deficiency syndrome
- the HIV-1 envelope glycoproteins, gpl20 and gp41 are assembled into a trimeric complex that mediates virus entry into target cells (1) . HIV-1 entry depends upon the sequential interaction of the gpl20 exterior envelope glycoprotein with the receptors on the cell, CD4 and members of the chemokine receptor family (2-4).
- the gpl20 glycoprotein which can be shed from the envelope complex, elicits both virus-neutralizing and non-neutralizing antibodies during natural infection. Antibodies that lack neutralizing activity are often directed against the gpl20 regions occluded on the assembled trimer and exposed only upon shedding (5,6) .
- Neutralizing antibodies by contrast, must access the functional envelope glycoprotein complex (7) and typically recognize conserved or variable epitopes near the receptor-binding regions (8-11) .
- conserved neutralization epitopes on gpl20, utilizing epitope maps in conjunction with the X-ray crystal structure of a ternary complex that includes a gpl20 core, CD4 and a neutralizing antibody (12) .
- a large fraction of the predicted accessible surface of gpl20 in the trimer is composed of variable, heavily glycosylated core and loop structures that surround the receptor-binding regions. Understanding the structural basis for the ability of HIV-1 to evade the humoral immune response should assist vaccine design.
- human and simian immunodeficiency virus gpl20 glycoproteins consist of five variable regions (VI-V5) interposed among mor e conserved regions
- Variable regions V1-V4 form exposed loops anchored at their bases by disulfide bonds (14) .
- Neutralizing antibodies recognize both variable and conserved gpl20 structures.
- the V2 and V3 loops contain epitopes for strain-restricted neutralizing antibodies (15-17) . More broadly neutralizing antibodies recognize discontinuous, conserved epitopes in three regions of the gpl20 glycoprotein (Table 1) . In HIV-1 infected humans, the most abundant of these are directed against the CD4 binding site (CD4BS) and block gpl20-CD4 interaction (8,9) . Less common are antibodies against epitopes induced or exposed upon CD4 binding (CD4i) (18) . Both CD4i and V3 antibodies disrupt the binding of gpl20-CD4 complexes to chemokine receptors (10, 11) .
- a third gpl20 neutralization epitope is defined by a unique monoclonal antibody, 2G12, (19) which does not efficiently block receptor binding (11) .
- (12) we report the X-ray crystal structure of an HIV-1 gpl20 core in a ternary complex with two-domain soluble CD4 and the Fab fragment of the CD4i antibody, 17b.
- the gpl20 core lacks the V1/V2 and V3 variable loops, as well as N- and C- terminal sequences, which interact with the gp41 glycoprotein, (6) and is enzymatically deglycosylated
- the gpl20 core binds CD4 and antibodies against CD4BS and CD4i epitopes
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- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Crystallography & Structural Chemistry (AREA)
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- General Physics & Mathematics (AREA)
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- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Virology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
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Abstract
Description
Claims
Applications Claiming Priority (24)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US96714897A | 1997-11-10 | 1997-11-10 | |
US96698797A | 1997-11-10 | 1997-11-10 | |
US96740397A | 1997-11-10 | 1997-11-10 | |
US96693297A | 1997-11-10 | 1997-11-10 | |
US966987 | 1997-11-10 | ||
US967148 | 1997-11-10 | ||
US966932 | 1997-11-10 | ||
US967403 | 1997-11-10 | ||
US97674197A | 1997-11-24 | 1997-11-24 | |
US976741 | 1997-11-24 | ||
US8958098P | 1998-06-17 | 1998-06-17 | |
US8958198P | 1998-06-17 | 1998-06-17 | |
US89580 | 1998-06-17 | ||
US10052198A | 1998-06-18 | 1998-06-18 | |
US10063198A | 1998-06-18 | 1998-06-18 | |
US10052998A | 1998-06-18 | 1998-06-18 | |
US10076298A | 1998-06-18 | 1998-06-18 | |
US10076398A | 1998-06-18 | 1998-06-18 | |
US100529 | 1998-06-18 | ||
US100521 | 1998-06-18 | ||
US100762 | 1998-06-18 | ||
US100763 | 1998-06-18 | ||
US100631 | 1998-06-18 | ||
PCT/US1998/023905 WO1999024553A2 (en) | 1997-11-10 | 1998-11-10 | X-ray crystal comprising hiv-1 gp120 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP1037963A1 true EP1037963A1 (en) | 2000-09-27 |
EP1037963A4 EP1037963A4 (en) | 2004-09-22 |
Family
ID=27583731
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP98959406A Withdrawn EP1037963A4 (en) | 1997-11-10 | 1998-11-10 | X-ray crystal comprising hiv-1 gp120 |
Country Status (2)
Country | Link |
---|---|
EP (1) | EP1037963A4 (en) |
WO (1) | WO1999024553A2 (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6743594B1 (en) | 1995-06-06 | 2004-06-01 | Human Genome Sciences, Inc. | Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5) |
US6025154A (en) | 1995-06-06 | 2000-02-15 | Human Genome Sciences, Inc. | Polynucleotides encoding human G-protein chemokine receptor HDGNR10 |
US20030125518A1 (en) * | 2001-12-01 | 2003-07-03 | Crevecoeur Harry F. | Surface simulation synthetic peptides useful in the treatment of hyper-variable viral pathogens |
EP1940867A2 (en) | 2005-09-06 | 2008-07-09 | The Government of the United States of America as represented by the Secretary of the Department of Health and Human Services | Hiv gp120 crystal structure and its use to identify immunogens |
US20130101617A1 (en) * | 2010-06-30 | 2013-04-25 | Torrey Pines Institute For Molecular Studies | Env trimer immunogens |
CN114609392A (en) * | 2022-03-08 | 2022-06-10 | 武汉科技大学 | Screening method and application of HIV (human immunodeficiency virus) fully-humanized broad-spectrum neutralizing antibody |
-
1998
- 1998-11-10 EP EP98959406A patent/EP1037963A4/en not_active Withdrawn
- 1998-11-10 WO PCT/US1998/023905 patent/WO1999024553A2/en not_active Application Discontinuation
Non-Patent Citations (8)
Title |
---|
GHIARA J B ET AL: "Structure-based design of a constrained peptide mimic of the HIV-1 V3 loop neutralization site" JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 266, no. 1, 14 February 1997 (1997-02-14), pages 31-39, XP004462201 ISSN: 0022-2836 * |
KWONG P D ET AL: "STRUCTURE OF AN HIV gp120 ENVELOPE GLYCOPROTEIN IN COMPLEX WITH THE CD4 RECEPTOR AND A NEUTRALIZING HUMAN ANTIBODY" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 393, 18 June 1998 (1998-06-18), pages 648-659, XP002093502 ISSN: 0028-0836 * |
OXFORD J S ET AL: "New scientific developments towards an AIDS vaccine: report on a workshop organized by EU programme EVA entitled Novel approaches to AIDS vaccine development held at the Institut Pasteur, Paris" VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 14, no. 17, 1 December 1996 (1996-12-01), pages 1712-1717, XP004016836 ISSN: 0264-410X * |
RINI JAMES M ET AL: "Crystal structure of a human immunodeficiency virus type 1 neutralizing antibody, 50.1, in complex with its V3 loop peptide antigen" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 90, no. 13, 1993, pages 6325-6329, XP002276067 1993 ISSN: 0027-8424 * |
SANEJOUAND Y-H: "On the role of CD4 conformational change in the HIV-cell fusion process" COMPTES RENDUS DES SEANCES DE L'ACADEMIE DES SCIENCES. SERIE III: SCIENCES DE LA VIE, ELSEVIER, AMSTERDAM, NL, vol. 320, no. 2, February 1997 (1997-02), pages 163-170, XP004349275 ISSN: 0764-4469 * |
See also references of WO9924553A2 * |
STURA E A ET AL: "CRYSTALLIZATION, SEQUENCE, AND PRELIMINARY CRYSTALLOGRAPHIC DATA FOR AN ANTIPEPTIDE FAB 50.1 AND PEPTIDE COMPLEXES WITH THE PRINCIPAL NEUTRALIZING DETERMINANT OF HIV-1 GP120" PROTEINS: STRUCTURE, FUNCTION AND GENETICS, ALAN R. LISS, US, vol. 14, no. 4, December 1992 (1992-12), pages 499-508, XP001069692 ISSN: 0887-3585 * |
WYATT R ET AL: "THE ANTIGENIC STRUCTURE OF THE HIV gp120 ENVELOPE GLYCOPROTEIN" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 393, 18 June 1998 (1998-06-18), pages 705-711, XP002093503 ISSN: 0028-0836 * |
Also Published As
Publication number | Publication date |
---|---|
WO1999024553A9 (en) | 2001-05-31 |
WO1999024553A8 (en) | 1999-07-22 |
WO1999024553A3 (en) | 2003-12-11 |
WO1999024553A2 (en) | 1999-05-20 |
EP1037963A4 (en) | 2004-09-22 |
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