WO1998031703A9 - Procedes de modification d'une structure proteique tridimensionnelle et compositions produites par un tel procede - Google Patents

Procedes de modification d'une structure proteique tridimensionnelle et compositions produites par un tel procede

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WO1998031703A9
WO1998031703A9 PCT/US1998/000853 US9800853W WO9831703A9 WO 1998031703 A9 WO1998031703 A9 WO 1998031703A9 US 9800853 W US9800853 W US 9800853W WO 9831703 A9 WO9831703 A9 WO 9831703A9
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protein
leu
altered
val
pro
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PCT/US1998/000853
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  • This relates generally to the field of protein structure and protein design.
  • a second important determinant of structure is amino acid helical propensity, which reflects the entropic cost of incorporating a residue into an ordered secondary structure element [P. C. Lyu et al, Science. 250: 669 (1990); K. T. O'Neil and W. F. DeGrado, Science. 250: 646 (1990); S. Padmanabhan et al, Nature. 344: 268 (1990); T. P. Creamer and G. D. Rose, Proc. Natl. Acad. Sci. USA. 89: 5937 (1992)].
  • these designed proteins may have novel functions, or a change in their functional properties relative to the native protein from which they were derived.
  • these designed proteins retain the function of the native protein from which they are derived, but have some other advantage, such as enhanced stability, improved binding, lower molecular mass, or the like.
  • the present invention provides a method for altering the three-dimensional structure of proteins.
  • the method involves the steps of: identifying a native protein with three-dimensional structure to be altered; identifying the hydrophobic residues within this protein; distinguishing the hydrophobic residues on the basis of side chain size into large and small hydrophobic residues; providing mutants of the protein having substitutions in the hydrophobic residues distinguished above; and assaying the mutant proteins for a switch in three-dimensional structure.
  • the method of the invention further involves the steps of generating mutants of the selected protein having at least one of the large hydrophobic residues within its hydrophobic core substituted by hydrophobic residues with small side chains and assaying the mutant proteins for ability to at least maintain the biological function of the native protein.
  • the method of the invention involves substituting at least one of the small hydrophobic residues within the hydrophobic core of the protein with large hydrophobic residues.
  • the present invention provides an altered protein comprising p53 oligomerization domain altered according to the above method, wherein the side chain size of the hydrophobic amino acid with the largest side chain in the p53 protein hydrophobic core (Phe341) has been decreased and the side chain of another hydrophobic amino acid in the hydrophobic core (Leu344) has been increased.
  • the invention provides an altered p53 protein oligomerization domain designed as described above, which has further been modified to contain a Lys at amino acid position 340.
  • the invention provides a p53 protein containing the altered p53 oligomerization domains as described above in place of the native p53 oligomerization domain.
  • the invention provides p53 fusion proteins comprising an altered p53 oligomerization domain fused to a heterologous protein.
  • the present invention provides nucleic acid sequences encoding the altered proteins according to the present invention.
  • the invention provides vectors comprising nucleic acid sequences of the invention under the control of suitable regulatory sequences.
  • the invention provides host cells transformed with the vectors of the invention. Also provided are pharmaceutical compositions containing the nucleic acid sequence of the invention and method of administering same. Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.
  • Fig. 1 illustrates the three-dimensional structure of the p53wt oligomerization domain corresponding to residues 325-355 of SEQ ID NO: 2.
  • Residue type is indicated by the single letter code: F, Phe; L, Leu.
  • Fig. 2 illustrates the three-dimensional structure of the p53KIY oligomerization domain corresponding to residues 327-353 of human p53 [SEQ ID NO: 2]. Residue type is indicated by the single letter code: Y, Tyr; I, He.
  • Fig. 3 A illustrates the packing of residues with large hydrophobic side chains, specifically, Phe341 in p53wt [SEQ ID NO: 2].
  • the ⁇ -helix of one of the subunits is not shown for clarity, also only two of the four subunits are shown.
  • Residue type is abbreviated: L, Leu; F, Phe; N, Asn.
  • Fig. 3B illustrates the packing of Tyr344 in p53KIY [SEQ ID NO: 4].
  • Residue type is abbreviated: L, Leu; Y, Tyr; N, Asn.
  • This invention provides a general method for changing the three-dimensional structure of proteins.
  • the invention further provides modified proteins generated by this method and nucleic acid sequences encoding them. These modified proteins and nucleic acid sequences are particularly useful in pharmaceutical compositions and therapeutic regimens, and also in biotechnology and other industrial applications.
  • the altered proteins of the invention have an altered three-dimensional structure and are further characterized by maintaining substantially the same level of a desired biological function of the native protein.
  • the altered proteins of the invention advantageously can be designed or selected such that they are also characterized by improved functional properties relative to the native (wild-type) domain, e.g., improved binding ability, improved stability, or the like.
  • the present invention provides a method for altering the three-dimensional structure of proteins without denaturing the proteins.
  • the examples provided herein demonstrate alteration of the three-dimensional structure of the oligomerization domain of wild-type p53.
  • other proteins may be readily altered.
  • the method of the invention is performed upon a protein that has a hydrophobic core (i.e., upon essentially all proteins). Most preferably, however, the protein is useful for industrial, therapeutic or diagnostic purposes.
  • non-linear proteins including, without limitation, industrial enzymes, such as proteases, lipases, chymases, etc., and therapeutically useful proteins, such as the members of the globin family, in which the side chain sizes of specific hydrophobic residues are highly conserved [D. Bashford et al, J. Mol. Biol.. 196: 199 (1987)].
  • the three-dimensional (or crystalline) structure of the protein to be altered is known.
  • the three-dimensional structure may be determined using known techniques, e.g., NMR spectroscopy, x-ray crystallography and the like.
  • hydrophobic residues within this protein are identified and distinguished into large and small hydrophobic residues on the basis of side chain size.
  • amino acid residues Phe, Tyr and Trp are considered large and amino acid residues Ala, Val and He are considered small.
  • Leu is intermediate in size and may be considered either large or small.
  • the total surface area not only is the total surface area, but also the surface area buried upon folding with is relevant, as the latter determines the strength of the hydrophobic effect [J.R. Livingstone et al, Biochemistry. 30:4237-4244 (1991)].
  • the total surface areas of residues [G.D. Rose et al, Science. 229:834-838 (1985)] and the surface areas buried upon folding are provided in the table below. All values are in square Angstroms
  • mutant proteins are generated by increasing the size of one or more of the small residues and/or decreasing the size of one or more of the large residues. Since Leu cannot be unambiguously classified as large or small, mutants are generated which replace Leu with both larger and smaller amino acids.
  • it is preferable to decrease the size of the largest residue in the hydrophobic core e.g., Phe is substituted with He, Val or Ala
  • increase the size of one of the smaller residues in the hydrophobic core e.g., Val is substituted with Phe or Trp. All substitutions are made in such a manner as to preserve the hydrophobic character of the native residue.
  • one or two substitutions are made for each secondary structure element whose packing in the three-dimensional structure of the protein is to be altered.
  • two substitutions are performed, one decreases the side chain size of a large hydrophobic amino acid and the other increases the side chain size of a small hydrophobic amino acid.
  • the mutant proteins may be generated using conventional techniques.
  • the peptides may be synthesized using a commercially available automatic synthesizer according to standard procedures.
  • other standard techniques may be utilized. See, e.g., Merrifield, J. Amer. Chem. Soc. 85:2149-2154 (1963).
  • the mutant proteins of the invention are generated recombinantly, making use of a variety of well-known techniques (such as site- directed mutagenesis [see, Gillman & Smith, Gene. 8:81-97 (1979) and S. Roberts et al, Nature. 328:731-734 (1987)]) and, desirably, the nucleic acid sequences of the invention. See, e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual.. 2d Edit., Cold Spring Harbor Laboratory, New York (1989).
  • mutant proteins generated by the method of the invention may be assayed for biological function as a preliminary screening step. In this manner, mutant proteins having the desired biological function (e.g., binding ability) may be selected.
  • the assays for examining the function of the protein will depend on the particular function that needs to be assayed. Such assays are well known to those of skilled in the art and are not a limitation on this invention. For example, if the protein altered according to the invention is a DNA binding protein, then one could use a DNA binding assay to examine the function of the mutant proteins. If engineering involves a protein that forms oligomers, e.g., the oligomerization domain of p53, then one could use a glutaraldehyde crosslinking assay [W.
  • the mutant proteins generated according to the method of the invention may then be screened for a change in the three-dimensional structure.
  • Biophysical methods to probe protein structure include NMR spectroscopy, X-ray crystallography, [G. M. Clore et al, Science. 265: 386 (1994); W. Lee et al, Nature Structural Biol.. ⁇ : 877 (1994); G. M. Clore et al, Nature Structural Biol.. 2: 321 (1995); P. D. Jeffrey et al, Science, 267: 1498 (1995)], among other techniques.
  • the applications of a DNA binding assay, a glutaraldehyde crosslinking assay and NMR spectroscopy are illustrated in the examples of engineered p53 oligomerization domains appropriate for determining three-dimensional conformation.
  • the p53 oligomerization domain has been altered according to the method of the invention.
  • the method of the invention may involve generating mutant of fragments of a useful protein which is responsible for biological activity. As described above, similar mutants may be generated using the hydrophobic core of other selected proteins, or full-length proteins, as desired.
  • the inventor has altered the oligomerization domain of p53 to provide a p53 protein with an altered three-dimensional structure and oligomerization stoichiometry relative to the native (wild-type) domain.
  • All references to p53 residue numbers herein refer to the numbering scheme provided by Zakut-Houri et al, EMBO J, 4: 1251-1255 (1985) [GenBank Code Hsp53] for human p53.
  • the nucleotide and amino acid sequences of human p53 are reproduced as SEQ ID NOS: 1 and 2, respectively.
  • SEQ ID NOS: 1 and 2 respectively.
  • the p53 tumor suppressor protein is a sequence-specific transcription factor with fundamental significance to the pathogenesis and therapy of human cancer [C. C. Harris, Science. 262: 1980 (1993); L. J. Ko and C. Prives, Genes Dev.. 10: 1054 (1996)].
  • the tumor suppressor activity of p53 requires homo-oligomerization [M. J. F. Waterman et al, Cancer Res., 56: 158 (1996)], which is mediated by a thirty residue domain at the C-terminus of the protein [H. Sakamoto et al, Proc. Natl. Acad. Sci. USA. 91 : 8974 (1994); P. Wang et al, Mol. Cell. Biol.. 14: 5182 (1994); J. L.
  • the native thirty residue p53 oligomerization domain has a ⁇ -strand, a tight turn and an ⁇ -helix in each subunit. [Clore et al, cited above (1994); Lee et al, cited above; Clore et al, cited above, (1995); Jeffrey et al, cited above]. This oligomerization domain folds independently with antiparallel packing of its ⁇ -helices.
  • the altered p53 generated according to the invention contains substitutions of residues Phe341 and Leu344 [SEQ ID NO: 2] in the ⁇ -helix by other hydrophobic amino acids, that decreased the side chain size at position 341 and increased the side chain size at position 344 [corresponding to SEQ ID NO: 2], resulting in an altered p53 domain that assembles as a dimer instead of a tetramer.
  • the three-dimensional structure of a mutant dimeric domain determined in solution by NMR spectroscopy differs substantially from the wild-type structure, since the ⁇ -helices are packed parallel, rather than antiparallel, and are rotated significantly relative to each other and to the ⁇ -strands.
  • the engineered p53 oligomerization domain was found to drive the sequence-specific DNA binding function of the modified p53 protein.
  • the present invention provides altered p53 oligomerization domains that assemble as dimers, rather than tetramers, and which have a different three-dimensional structure relative to wild-type p53.
  • These altered p53 oligomerization domains of the invention desirably contain the following residues, which differ from the residues in native human p53 [SEQ ID NO: 2].
  • the altered p53 oligomerization domains described above may be further modified to contain Lys at position 340 [SEQ ID NO: 4].
  • the inventor has found that the latter modification increases the solubility of the mutant p53 oligomerization domains with altered three-dimensional structure. For example, at a 1-2 mM concentration a mutant p53 domain with He at position 341 and Tyr at position 344 [SEQ ID NO: 3] precipitates within one hour when heated to 40°C. In contrast, a mutant p53 domain [SEQ ID NO: 4] with Lys at position 340, He at position 341 and Tyr at position 344 remains soluble under the same conditions. Based on the functional activities of other dimeric p53 proteins
  • the altered p53 oligomerization domain has a different three-dimensional structure than the wild-type p53 domain, the two types of domains will not hetero-oligomerize. Accordingly, a p53 tumor suppressor protein will not be sequestered into inactive hetero-oligomers with tumor-derived p53 mutant proteins.
  • a p53 protein of the invention can be delivered by gene therapy vectors and not be suppressed by the mutant p53 protein present in tumor cells.
  • the altered p53 oligomerization domains of the invention may be engineered on an otherwise unmodified p53wt protein.
  • the altered p53 oligomerization domain may also be fused to a selected heterologous protein.
  • the altered p53 oligomerization domains may be engineered on p53 proteins bearing additional modifications.
  • One suitable modification is substitution of residue threonine 284 [SEQ ID NO: 2] with Arginine. This substitution enhances the tumor suppressor function of wild-type p53 5- to 7-fold [Wieczorek et al, Nature Medicine. 2: 1143 (1996)].
  • Suitable heterologous proteins include those which in the past have been fused to a leucine zipper.
  • Leucine zippers have the disadvantage that they may interact with host leucine-zipper bearing proteins, which interaction may compromise the biological activity of the chimeric protein bearing the leucine zipper.
  • the altered p53 oligomerization domains described in this application do not exist in nature, and do not form oligomers with any host proteins.
  • both leucine zippers and the altered p53 oligomerization domain of the invention have similar topologies, i.e., both types of domains have parallel ⁇ -helices, they may be used in many similar applications.
  • heterologous proteins include single chain antibody variable chains (scFv antibodies).
  • the prior art has described scFv antibodies fused to Jun and Fos leucine zippers to produce dimeric antibodies that have higher affinity for their ligands, because they are bivalent [Kruif and Logtenberg, J. Biol. Chem., 271 :7630 (1996)].
  • scFv antibodies can be fused to the altered p53 oligomerization domain of the invention by using the sequences encoding the altered p53 oligomerization domain in place of the leucine zipper sequences of the prior art. This will lead to homodimeric (hence monospecific), bivalent (high affinity) antibodies.
  • a suitable heterologous protein includes a soluble interleukin-2 (IL-2) receptor.
  • IL-2 soluble interleukin-2
  • Wu et al, J. Biol. Chem.. 270: 16039 (1995) has described a soluble IL-2 receptor complex formed by attaching leucine zippers to the C-terminus of the extracellular domain of the receptor.
  • the leucine zipper domain of the prior art is replaced by the altered p53 oligomerization domain of the invention.
  • Such soluble domains are useful for screening ligands (drugs) that bind to the native receptors.
  • Such soluble domains are also useful therapeutically as decoys competing for ligand binding with the endogenous receptors of patients.
  • the extracellular domains of the T-cell receptor can be isolated in a soluble form, then fused to the modified p53 domains, using a modified version of the technique described in Chang et al., Proc. Natl. Acad. Sci. USA. 91: 11408 (1994).
  • heterodimers can be isolated from homodimers by conventional protein chromatography or other suitable techniques.
  • the altered p53 oligomerization domains of the invention may be fused to a transmembrane receptor. Many such receptors become physiologically activated by ligand-induced dimerization. Fusion of a dimerization domain to such receptors can therefore constitutively activate them.
  • a transmembrane receptor such as the Trp-Met fusion receptor, in which the Trp protein provides a leucine zipper which induces dimerization and activation of the Met receptor [Rodriques and Park, Mol. Cell Biol. 13:6711 (1993)].
  • the altered p53 oligomerization domains of the invention may be used to activate a receptor of choice. Depending upon the type of receptor to which the modified p53 oligomerization domain of the invention is fused, the outcome could be cell proliferation or cell death.
  • the altered p53 oligomerization domain of the invention can also be used to induce dimerization of DNA binding proteins.
  • Many DNA binding proteins for example c-Myc, bind DNA as dimers.
  • c-Myc will not homodimerize, but will bind DNA as a hetero-dimer with a protein called Max.
  • c-Myc will homo- dimerize if its native leucine zipper is replaced by the leucine zipper of GCN4, since the latter zipper has a high tendency to homo-oligomerize.
  • a c-Myc fusion bearing a GCN4 leucine zipper binds DNA with a very high affinity [Halazonetis and Kandil, Science. 255:464 (1992)].
  • a c-Myc protein whose native C-terminus is fused to the modified p53 oligomerization domain of this invention, would bind to DNA with high affinity and could compete for DNA binding of the native Myc/Max heterodimer without interfering with native proteins which contain leucine zippers. Furthermore, if the chimeric Myc protein lacked the N-terminal domain of p53, which is required for carcinogenic transformation of cells overexpressing c-Myc, then it can be used to revert the tumorigenic phenotype of cells overexpressing c-Myc, such as many leukemia and lymphoma cells.
  • EWS-ATF-1 proteins that bind DNA and are implicated in cancer development, such as EWS-ATF-1 [Fujimura et al., Oncogene. 12: 159 (1996)] and the E2A-HLF [Yoshihara et al., Mol. Cell. Biol.. 15:3247 (1995)], can be similarly modified.
  • altered p53 produced according to the method of the invention is used by way of example only.
  • Other proteins e.g., enzymes, antibodies and members of the globin family
  • the present invention further provides nucleic acid sequences encoding the altered proteins of this invention.
  • the nucleic acid sequences of the invention include the complementary DNA sequence representing the non-coding strand, the messenger RNA sequence, the corresponding cDNA sequence and the RNA sequence complementary to the messenger RNA sequence.
  • Variants of these nucleic acids of the invention include variations due to the degeneracy of the genetic code and are encompassed by this invention. Such variants may be readily identified and/or constructed by one of skill in the art. In certain cases specific codon usage may be employed to optimize expression.
  • the above nucleotide sequences can be included within larger DNA or RNA fragments, or may be interrupted by introns.
  • the nucleic acids encoding the proteins of the invention are present in the context of vectors suitable for amplification in prokaryotic or eukaryotic cells.
  • vectors suitable for amplification in prokaryotic or eukaryotic cells Many such vectors are known and many of these are commercially available.
  • plasmids with bacterial or yeast replication origins allow amplification in bacteria or yeast, respectively.
  • Such vectors allow the production of large quantities of nucleic acids encoding the proteins of the invention, which nucleic acids can be used for gene therapy or for expression of the proteins of the invention, e.g., p53.
  • the nucleic acids encoding the proteins of the invention are present in the context of vectors suitable for expression in cell-free extracts or lysates or in prokaryotic or eukaryotic cells.
  • vectors are known [Ausubel et al, Current Protocols in Molecular Biology. Greene Publishing Associates and John Wiley & Sons, NY (1997)] and many of these are commercially available.
  • the vector pGEM4 Promega, Madison, WI
  • the vector pSV2 [ATCC] is suitable for expression in mammalian cells.
  • Such vectors allow the production of the proteins of the invention in vitro for analysis of their functional properties or for delivery to patients.
  • the nucleic acid sequences of the invention may be inserted into a vector capable of targeting and infecting a desired cell, either in vivo or ex vivo for gene therapy, and causing the encoded modified protein of this invention to be expressed by that cell.
  • viral vectors are useful for this purpose, e.g., adenoviruses, retroviruses and adeno-associated viruses (AAV) [Schreiber et al. , Biotechniques. 14: 818-823 (1993); Davidson et al, Nature Genetics. 3: 219-223 (1993); Roessler et al, J. Clin. Invest.. 92: 1085-1092 (1993); Smythe et al, Ann. Thorac.
  • AAV adeno-associated viruses
  • these viral vectors containing nucleic acid sequences encoding a protein of the invention are prepared by one of skill in the art with resort to conventional techniques (see references mentioned above).
  • a recombinant viral vector, e.g. an adenovirus, of the present invention comprises DNA of at least that portion of the viral genome which is capable of infecting the target cells operatively linked to the nucleic acid sequences of the invention.
  • infection is generally meant the process by which a virus transfers genetic material to its host or target cell.
  • the virus used in the construction of a vector of the invention is rendered replication-defective to remove the effects of viral replication on the target cells.
  • the replication-defective viral genome can be packaged by a helper virus in association with conventional techniques.
  • the vector(s) containing the nucleic acids encoding an altered protein of the invention is suspended in a pharmaceutically acceptable carrier, such as saline, and administered parenterally (or by other suitable means) in sufficient amounts to infect the desired cells and provide sufficient levels of modified protein to achieve the desired therapeutic or prophylactic effect, e.g., sufficient p53 activity to arrest abnormal cellular proliferation.
  • a pharmaceutically acceptable carrier such as saline
  • Other pharmaceutically acceptable carriers are well known to those of skill in the art.
  • a suitable amount of the vector containing the chimeric nucleic acid sequences is between about 10 6 to 10 9 infectious particles per mL carrier.
  • the delivery of the vector may be repeated as needed to sustain satisfactory levels of biological activity. For example, where modified p53 is administered, activity may be determined by monitoring clinical symptoms.
  • this therapy may be combined with other therapies for the disease or condition being treated.
  • therapy involving the administration of a vector capable of expressing an altered p53 protein of the invention is well suited for use in conjunction with conventional cancer therapies, including surgery, radiation and chemotherapy.
  • Nucleic acid sequences driving expression of a protein of the invention may also be introduced by "carriers" other than viral vectors, such as liposomes, nucleic acid-coated gold beads or can simply be injected in situ [Fujiwara et al (1994b), cited above; Fynan et al, Proc. Natl. Acad. Sci. USA. 90: 11478-11482 (1993); Cohen, Science. 259: 1691-1692 (1993); Wolff et al, Biotechniques. U : 474-485 (1991)].
  • carriers other than viral vectors, such as liposomes, nucleic acid-coated gold beads
  • compositions The altered proteins and nucleic acid sequences of this invention may also be formulated into pharmaceutical compositions and administered using a therapeutic regimen compatible with the particular formulation.
  • the composition may contain "naked” DNA, or a vector containing the nucleic acid sequences.
  • naked DNA means substantially pure DNA which is not associated with a protein, lipid, carbohydrate or contained within a cell or an artificial delivery system such as a liposome.
  • compositions within the scope of the present invention include compositions containing an altered protein of the invention (or a nucleic acid sequence encoding a modified protein) in an effective amount to have the desired physiological effect, e.g. to arrest the growth of cancer cells without causing unacceptable toxicity for the patient.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form, e.g. saline.
  • suspensions of the active compounds may be administered in suitable conventional lipophilic carriers or in liposomes.
  • compositions may be supplemented by active pharmaceutical ingredients, where desired.
  • Optional antibacterial, antiseptic, and antioxidant agents in the compositions can perform their ordinary functions.
  • the pharmaceutical compositions of the invention may further contain any of a number of suitable viscosity enhancers, stabilizers, excipients and auxiliaries which facilitate processing of the active compounds into preparations that can be used pharmaceutically.
  • these preparations, as well as those preparations discussed below, are designed for parenteral administration.
  • compositions designed for oral or rectal administration are also considered to fall within the scope of the present invention.
  • suitable amount or “effective amount” means an amount which is effective to treat the conditions referred to below.
  • a preferred dose of a pharmaceutical composition containing a protein of this invention is generally effective above about 0.1 mg modified protein per kg of body weight (mg/kg), and preferably from about 1 mg/kg to about 100 mg/kg. These doses may be administered with a frequency necessary to achieve and maintain satisfactory activity levels. Although a preferred range has been described above, determination of the effective amounts for treatment of each type of tumor or other condition may be determined by those of skill in the art.
  • Dosage units of such pharmaceutical compositions containing the proteins of this invention preferably contain about 1 mg to 5 g of the protein.
  • nucleic acids encoding altered p53 proteins and the altered p53 proteins themselves can be introduced into human patients for therapeutic benefits in conditions characterized by insufficient wild-type p53 activity. Such conditions have been described in the art. See, e.g., PCT/US95/15353 (June 6, 1996).
  • the pharmaceutical compositions of the invention including the gene therapy vectors, may be employed to induce the cellular defense to DNA damaging agents such as sunlight UV irradiation, as well as radiation and chemotherapeutics used for cancer treatment.
  • the therapeutic indications include inducing apoptosis of specific cells, such as proliferating lymphocytes, the prevention of transplant rejection, and the treatment of autoimmune diseases, e.g., systemic lupus erythrematosis, rheumatoid arthritis and the like.
  • compositions of this invention may also be employed to restore p53 function in tumor cells and to suppress cell proliferation in diseases other than cancers, which are characterized by aberrant cell proliferation.
  • diseases include psoriasis, atherosclerosis and arterial restenosis.
  • Pharmaceutical compositions containing other altered proteins of the invention (or nucleic acids encoding them) may also be readily prepared and used for a variety of indications which will be readily apparent to one of skill in the art.
  • altered proteins of the invention are useful for generating antibodies, which may be used as diagnostic reagents, for example, to monitor the presence of modified protein or protein domain.
  • Specific antisera may be generated using known techniques. See,
  • antibodies of the invention may be produced by conventional methods, including the Kohler and Milstein hybridoma technique, recombinant techniques, such as described by Huse et al, Science, 246: 1275-1281 (1 88), or any other techniques known to the art.
  • the invention further encompasses functional fragments of the antibodies of the invention, including, Fab, F v , and F(ab') 2 fragments, the binding site of the antibodies, and the complementarity determining regions (CDRs).
  • the binding site and/or CDRs may be contained in a synthetic molecule which provides antibody framework regions.
  • these functional fragments may be used in the production of recombinant antibodies, including bifunctional antibodies, chimeric antibodies, and humanized antibodies, which preferably retain the antigen binding specificity of the antibodies of the invention.
  • Such recombinant antibodies may be constructed and produced according to known techniques [see, e.g., S. D. Gillies et al, J. Immunol. Meth..
  • the altered proteins of the invention may be used therapeutically or as diagnostic reagents. These reagents may optionally be labeled using diagnostic labels, such as radioactive labels, colorimetric enzyme label systems and the like conventionally used in diagnostic or therapeutic methods. Alternatively, the N- or C- terminus of an altered protein of the invention may be tagged with a detectable label which can be recognized by a specific antisera.
  • diagnostic labels such as radioactive labels, colorimetric enzyme label systems and the like conventionally used in diagnostic or therapeutic methods.
  • diagnostic labels such as radioactive labels, colorimetric enzyme label systems and the like conventionally used in diagnostic or therapeutic methods.
  • the N- or C- terminus of an altered protein of the invention may be tagged with a detectable label which can be recognized by a specific antisera.
  • the reagents derived from p53 may be used in diagnosis of a variety of conditions associated with p53 and/or aberrant cell proliferation, including autoimmune diseases, e.g., systemic lupus erythrematosis, rheumatoid arthritis and the like, cancers, psoriasis, atherosclerosis and arterial restenosis.
  • autoimmune diseases e.g., systemic lupus erythrematosis, rheumatoid arthritis and the like
  • cancers psoriasis
  • atherosclerosis e.g., atherosclerosis and arterial restenosis
  • reagents produced from other altered proteins of the invention e.g., antibodies and the like, may similarly be utilized as diagnostic reagents.
  • the selection of the appropriate assay format and label system is within the skill of the art and may readily be chosen without requiring additional explanation by resort to the wealth of art in the diagnostic area.
  • Example 1 Method for altering the three-dimensional structure of wild-type p53 Wild-type p53 was identified as the native protein with three-dimensional structure to be altered, according to the method of the invention.
  • wtp53 [SEQ ID NO: 2] was found to have a structure which consists of a ⁇ -strand, a tight turn and an ⁇ -helix.
  • Four identical subunits assemble as a dimer of dimers (Fig. 1).
  • the ⁇ -strands form an antiparallel ⁇ -sheet and the ⁇ -helices also pack antiparallel.
  • Two primary dimers form a tetramer by packing their ⁇ -helices at an 81 ° angle.
  • the residues of the hydrophobic core were distinguished into large and small hydrophobic residues.
  • the center of the hydrophobic core of the domain is formed primarily by residues Phe341 and Leu344, Phe341 is positioned at the interface of the two p53 monomers that form the primary dimers, whereas Leu344 forms the interface between the primary dimers
  • Mutants of p53 were then generated according to the method of the invention and assayed for a switch in conformation. Plasmids encoding mutant p53 proteins were generated by PCR-directed mutagenesis of pGEMhump53wtB, as described in Waterman et al, EMBO J.. 14:512-519 (1995) which is incorporated by reference herein. The names of the mutants indicate the hydrophobic residues at positions 341 and 344 [SEQ ID NO: 3], respectively, using the single letter amino acid code. For example, p53FL is wild-type p53 [SEQ ID NO: 2]. Additional mutants made include: p53A344, Ala344 [SEQ ID NO: 3]; p53IF, Ile341 and Phe344
  • DNA binding of the mutants was assayed using 32 P-labeled oligonucleotide BC.S10 and in vitro translated p53 (Waterman et al, cited above)]. DNA binding is an indirect measure of the function of the p53 oligomerization domain. This assay was performed to screen the mutants whose function is similar to wtp53, as a preliminary to assaying for conformational switch. Table 1 shows the effect of amino acid substitutions targeting residues 341 and 344 of human p53 on the subunit stoichiometry of p53 as assayed by the electrophoretic migration of its complexes with DNA. The names of the mutants [SEQ ID NO: 3] indicate the hydrophobic residues at positions 341 and 344, respectively, using the single letter amino acid code.
  • p53FL is wild-type p53 [SEQ ID NO: 2].
  • Wild-type p53 (p53wt; SEQ ID NO: 2) and these mutant p53 oligomerization domains [SEQ ID NOS: 3 and 4] were expressed in E. c ⁇ li, purified to homogeneity and assayed for subunit stoichiometry by glutaraldehyde crosslinking using the techniques described below. 1. Expression and Purification
  • the cells were pelleted, stirred on ice for 20 min. in glycerol, 0.7% v/v Triton-X and 0.4% v/v ⁇ -mercaptoethanol, and then for another 15 min. in lysis buffer (10 mM Tris [pH 8.0], 500 mM NaCl, 5 mM EDTA, 1 mM DTT, 0.6 mg/ml lysozyme) containing Pefabloc SC (Pentapharm, Basel, Switzerland) and pepstatin (Sigma, St. Louis, MO). 120 U/ml DNase I (Sigma) was added and stirring continued for another 30 min.
  • lysis buffer 10 mM Tris [pH 8.0], 500 mM NaCl, 5 mM EDTA, 1 mM DTT, 0.6 mg/ml lysozyme
  • Pefabloc SC Pentapharm, Basel, Switzerland
  • pepstatin Sigma, St. Louis, MO
  • p53 was purified in three steps. In a first step, the p53 was run on a 50 ml Phenyl-Sepharose column (Pharmacia, Piscataway, NJ) equilibrated with 1.5 M ammonium sulfate, 50 mM sodium phosphate [pH 7.0], 5 mM EDTA and eluted by decreasing salt concentration on a GradiFrac system (Pharmacia).
  • a second step the eluate from the first step was run on an 8 ml Phenyl-Superose column (Pharmacia) equilibrated with 1.7 M ammonium sulfate, 50 mM sodium phosphate [pH 7.0], 5mM EDTA and eluted by decreasing salt concentration on a SMART system (Pharmacia).
  • the eluate from the second step was run on a 1 ml Resource S column (Pharmacia) equilibrated with 50 mM sodium phosphate [pH 7.0], 50 mM NaCl, 0.01 mM EDTA and eluted by increasing NaCl concentration on the SMART system.
  • 0.1% v/v glutaraldehyde Sigma, St. Louis, MO
  • Table 2 illustrates subunit stoichiometry of p53wt [SEQ ID NO: 2], p53IY (Ile341 and Tyr344) [SEQ ID NO: 2] and p53KIY (He341 and Tyr344) [SEQ ID NO: 4] oligomerization domains as determined by glutaraldehyde (Gl.) crosslinking and SDS-gel electrophoresis.
  • Multidimensional solution NMR spectroscopy was performed on uniformly 15 N- and 15 N, 13 C-labeled samples to determine the structural basis for the switch in oligomerization stoichiometry observed in the mutant proteins as follows.
  • p53IY SEQ ID NO: 3
  • p53KIY SEQ ID NO: 4
  • p53wt SEQ ID NO: 2
  • NOE restraints were obtained from 2D NOESY (unlabeled sample in D20), 3D 15 N-edited NOESY-HMQC ( 15 N-labeled sample) and 13 C-edited HSQC-NOESY ( 15 N/ 13 C-labeled sample) experiments [A. Majumdar and E. R. P. Zuiderweg, J. Magn. Reson.. 102B: 242 (1993)].
  • Inter- and intrasubunit NOEs were differentiated with 12 C-filtered/ 13 C-edited and 13 C-filtered/ 13 C- edited HMQC-NOESY-HMQC experiments performed on an equilibrated 1 : 1 mixture of 12 C- and 13 C-labeled samples [W.
  • Glu346, Ala347 and Lys351 [corresponding to SEQ ID NO: 2] differed by more than 0.4 ppm in the proton frequency and/or by more than 1.2 ppm in the nitrogen frequency between p53KIY [SEQ ID NO: 4] and p53wt [SEQ ID NO: 2] (Table 3).
  • Such large chemical shift differences are suggestive of changes in three-dimensional structure [K. Wuthrich, cited above], especially since some residues, such as Gln331 and Arg333 are 10 and 7 A, respectively, from the nearest substituted residue in the established p53wt structure [G. M. Clore et al, Science. 265: 386 (1994); W. Lee et al, Nature Structural Biol..
  • Table 3 illustrates shifts in the amide resonance frequencies of Gln331 and Arg333 by amino acid substitutions targeting Phe341 and Leu344 related to p53wt [SEQ ID NO: 2]. Proteins are labelled as in Tables 1 and 2.
  • P53K340 [SEQ ID NO: 4] has Lys at position 340.
  • P53KIF [SEQ ID NO: 4] has Lys340, Ile341 and Phe344.
  • p53FF [SEQ ID NO: 3] and p53wt [SEQ ID NO: 2] are tetramers
  • p53KIY [SEQ ID NO: 4] are dimers (Table 2).
  • NOE intensities were classified as strong, medium and weak, corresponding to distance restraints of 1.8-3.2, 1.8-4.0 and 1.8-5.0 A, respectively.
  • Dihedral angles were restrained to -140 ⁇ 60° or to 60 ⁇ 55° for 3J HNH ⁇ measurements of >8.5 or ⁇ 5 Hz, respectively.
  • Hydrogen bond restraints were incorporated as two NOEs restraining O-NH to 1.7-2.3 A and N-O to 2.8-3.3 A. Pseudo atom restraints were used whenever stereospecific assignments could not be made.
  • the average structure was calculated from thirty simulated annealing structures and was refined using restrained minimization and a repulsive term to stimulate the van der Waal's potential [Br ⁇ nger, cited above].
  • Geometry was evaluated with PROCHECK and PROMOTIF [R.A. Laskowski et al, J. Appl. Cryst.. 26:283 (1993)].
  • Intraresidue (122) 0.001 ⁇ 0.0003 0.000
  • the p53KIY oligomerization domain [SEQ ID NO: 4] is a dimer with two-fold cyclic symmetry.
  • Each subunit consists of three secondary structure elements: a ⁇ -strand forms an antiparallel ⁇ -sheet and the two ⁇ -helices pack parallel to each other (Fig. 2).
  • Parallel packing of the ⁇ -helices is stabilized by hydrophobic interactions substantially involving Tyr344, which interacts with Ile341 of the same subunit and Tyr344 of the other (Fig. 2).
  • the structural switch between p53KIY [SEQ ID NO: 4] and p53wt [SEQ ID NO: 2] can be evaluated by comparing their structures (Figs. 1 and 2). Such comparison reveals differences in the orientation of the secondary structure elements.
  • the interhelical angle changes from 155° in the primary dimer of p53wt to 83° in p53KIY [SEQ ID NO: 4], similar to the 81 ° angle with which the ⁇ -helices pack across primary dimers in p53wt.
  • p53wt [SEQ ID NO: 2] and p53KIY [SEQ ID NO: 4] differ by only three amino acid substitutions.
  • the changes in the sizes of the side chains at positions 341 and 344 appear to be necessary and sufficient for the structural switch.
  • p53KIY In p53KIY [SEQ ID NO: 3], the decrease in side chain size of residue 341 results in fewer interactions being required to shield its surface from solvent, while the increase in side chain size of residue 344 requires new interactions.
  • the switch in three dimensional structure between p53wt [SEQ ID NO: 2] and p53KIY [SEQ ID NO: 4] can be explained in terms of the loss of contacts that bury the side chain of residue 341 and gain in contacts that bury the side chain of residue 344.
  • the tip of the Phe341 ring lies in a hydrophobic pocket formed by the side chains of Leu344, Asn345 and Leu348, all from the other subunit (Fig. 3A).
  • p53KIY [SEQ ID NO: 4] oligomerization domains is probably secondary to the altered packing of the ⁇ -helices.
  • residues Leu344 form a hydrophobic patch for assembly of two primary dimers into a tetramer (Fig. 1).
  • Tyr 344 is involved in parallel packing of the ⁇ -helices and does not allow two dimers to form a tetramer.
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE
  • CAC TCC AGC CAC CTG AAG TCC AAA AAG GGT CAG TCT ACC TCC CGC CAT 1275 His Ser Ser His Leu Lys Ser Lys Lys Gly Gin Ser Thr Ser Arg His 365 370 375 380

Abstract

Cette invention se rapporte à des procédés permettant de modifier la structure tridimensionnelle d'une protéine sélectionnée sans dénaturer la protéine. Ledit procédé consiste à identifier les restes hydrophobes au sein de la protéine à modifier, à établir une distinction entre les restes hydrophobes de petite taille et ceux de grande taille. On génère ensuite des mutants de la protéine et on les analyse en vue d'une permutation avec des éléments de la structure tridimensionnelle. L'invention se rapporte également à des domaines d'oligomérisation p53 modifiés, produits conformément au procédé de l'invention, et à des protéines de fusion contenant ces domaines d'oligomérisation modifiés. L'invention se rapporte enfin à des séquences d'acides nucléiques codant ces protéines et à des compositions contenant lesdites protéines et lesdites séquences d'acides nucléiques.
PCT/US1998/000853 1997-01-17 1998-01-15 Procedes de modification d'une structure proteique tridimensionnelle et compositions produites par un tel procede WO1998031703A1 (fr)

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US6388062B1 (en) 1998-05-08 2002-05-14 The Wistar Institute Of Anatomy And Biology Modified p53 tetramerization domains having hydrophobic amino acid substitutions
CA2372881A1 (fr) * 1999-05-12 2000-11-16 Xencor, Inc. Nouveaux acides nucleiques et proteines ayant une activite p53 et comportant des domaines de tetramerisation modifies

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