WO2013100855A1

WO2013100855A1 - A novel phosphotyrosine-binding structure

Info

Publication number: WO2013100855A1
Application number: PCT/SG2012/000487
Authority: WO
Inventors: Jayaraman SIVARAMAN; Manjeet MUKHERJEE; Graeme R GUY; Soah Yee CHOW; Permeen YUSOFF
Original assignee: Agency for Science Technology and Research Singapore; National University of Singapore
Current assignee: Agency for Science Technology and Research Singapore; National University of Singapore
Priority date: 2011-12-27
Filing date: 2012-12-26
Publication date: 2013-07-04
Anticipated expiration: 2014-06-27

Description

A NOVEL PHOSPHOTYROSINE-BINDING STRUCTURE

The invention relates to at least one isolated novel phosphotyrosine-binding structure typically, but not exclusively, found in Hakai protein, termed herein the Hakai pTyr-binding HYB) domain, and its use in a screening assay to identify drugs for treating diseases or conditions characterised by migration or metastasis or invasion or a lack of cell-cell contact, such as cancer.

Background of Invention

In eukaryotic cells, phosphorylation events regulate cell signalling by providing docking sites for protein domains, such as the Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains. The SH2 was the first signalling domain to be identified and has been extensively characterized. The SH2 is a dedicated phosphotyrosine-binding domain and plays a critical role in signal transduction, hence making it a target for drug development. Binding specificity of SH2 domains is generally conferred by the sequences flanking the C-terminus of the phosphotyrosine (pTyr), and motif recognition is usually relatively inflexible. The other major class of pTyr-binding domain is the PTB domain. The specificity of binding to the PTB domain is conferred typically by residues on the target that are N-terminal to the pTyr. However, the PTB domain also recognizes non-pTyr motifs. Atypical phosphotyrosine- binding domains have also been detected in PKCd and the human M2 pyruvate kinase (PKM2).

In 2002, Fujita et al (2002) discovered a novel ubiquitin E3 ligase protein that targeted pTyr sites on E-cadherin. The E3 ligase, Hakai protein, possesses three domains: a RING domain, a short pTyr recognition sequence and a proline-rich domain (Fujita et al, 2002). Hakai is involved in the regulation of cell adhesion, cell migration and embryogenesis (Figueroa et al, 2009; Kaido et al, 2009; Gong et al, 2010). Among the reported protein interactions of Hakai, its association with and ubiquitination of E- cadherin upon Src activation is the best characterized (Fujita et al, 2002). Based on molecular modelling, Fujita et al (2002) assumed the pTyr-binding domain of Hakai to be a derivative SH2 domain.

In this study, we report that the Hakai pTyr-binding (HYB) domain consists of a homodimer formed at a structurally novel interface. Each monomer consists of two zinc-finger domains: a RING domain and a minimum pTyr- binding domain that incorporates a novel, atypical zinc coordination motif. Both domains play key roles in dimerization. The HYB domain is therefore composed of four zinc-binding domains co-operating to bind pTyr residues surrounded by acidic amino acids. Whereas the RING domain appears in other proteins, the atypical zinc-binding domain component is a novel protein fold that incorporates an intertwined configuration. In order to obtain its consensus target sequence, we have characterized the recognition motif of the HYB domain and identified several Src substrates that are also targeted. In addition, we have shown the HYB domain can also be found in a testis-specific ubiquitin E3 ligase, ZNF645, and the Ligand-of-Numb protein X1 and 2 (LNX1 and LNX2).

Given the biological role of Hakai, the novel structural features of the HYB domain and its infrequent distribution among proteins the HYB domain represents a highly suitable drug target because any compound designed to target the HYB domain would be unlikely to react with other proteins, suggesting a naturally inherent specificity.

Statements of Invention

According to a first aspect of the invention there is provided a drug screening method comprising:

a)contacting an isolated polypeptide with a compound wherein the polypeptide comprises or consists of the following phosphotyrosine- binding domain:

TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENV

H (a.a. 159-206) SEQ ID NO:1 or a sequence at least 31% homologous thereto wherein the following amino acids are conserved C166, C172, H185, and H190; b) determining whether binding occurs between the polypeptide and the compound; and

c) where said binding occurs concluding said compound may be useful in preventing the degradation of proteins that bind with said polypeptide.

In the above method said sequence homology may be 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44% or 45%.

In an alternative aspect of the invention there is provided a drug screening method comprising:

a) contacting an isolated polypeptide with a compound wherein the polypeptide comprises or consists of the following phosphotyrosine- binding domain:

TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENV

H (a.a. 159-206) SEQ ID NO:1 or a sequence at least 76% homologous thereto;

b) determining whether binding occurs between the polypeptide and the compound; and

In the above method said sequence homology may be 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

In a preferred embodiment of the invention said polypeptide comprises or consists of sequence structure:

VHFCDKCGLPlklYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDPVQRIE QCTRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENV

H (a.a. 106-206) SEQ ID NO: 2 or a sequence at least 23 % homologous thereto wherein the following amino acids are conserved C109, C112, C125, H127, C130, C133, C145 C148, C166, C172, H185, and H190. In the above method said sequence homology may be 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44% or 45%.

In a preferred embodiment of the invention said polypeptide cpmprises or consists of sequence structure:

VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDPVQRIE QCTRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENV

H (a.a. 106-206) SEQ ID NO: 2 or a sequence at least 71% homologous thereto.

In the above method said sequence homology may be 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

More preferably still, said polypeptide comprises or consists of a phosphotyrosine-binding domain characterised by two of the following sequence structures:

TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENVH

(a.a. 159-206) SEQ ID NO:1 or a sequence at least 31% homologous thereto where the following amino acids are conserved C166, C172, H185, and H190, arranged as a dimer, ideally an anti-parallel dimer.

TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENVH

(a.a. 159-206) SEQ ID NO:1 or a sequence at least 76% homologous thereto, arranged as a dimer, ideally an anti-parallel dimer.

More preferably yet, said polypeptide comprises or consists of a phosphotyrosine-binding domain characterised by two of the following sequence structures: VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDPVQRIE QCTRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENV

H (a.a. 106-206) SEQ ID NO: 2 or a sequence at least 23% homologous thereto where the following amino acids are conserved C109, C112, C125, H127, C130, C133, C145 C148, C166, C172, H185, and H190, arranged as a dimer, ideally an anti-parallel dimer.

More preferably yet, said polypeptide comprises or consists of a phosphotyrosine-binding. domain characterised by two of the following sequence structures:

H (a.a. 106-206) SEQ ID NO: 2 or a sequence at least.71% homologous thereto, arranged as a dimer, ideally an anti-parallel dimer.

Yet more preferably still, said method is undertaken using a ubiquitin 3 ligase protein or a polypeptide fragment thereof which comprises a phosphotyrosine-binding domain characterised by

TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENVH

(a.a. 159-206) SEQ ID NO:1 or a sequence at least 31% homologous thereto where the following amino acids are conserved C166, C172, H185, and H190; or

H (a.a. 106-206) SEQ ID NO: 2 or a sequence at least 23% homologous thereto where the following amino acids are conserved C109, C112, C125, H127, C130, C133, C145 C148, C166, C172, H185, and H190.

TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENVH (a.a. 159-206) SEQ ID NO:1 or a sequence at least 76% homologous thereto; or

H (a.a. 106-206) SEQ ID NO: 2 or a sequence at least 71% homologous thereto.

In yet a further preferred embodiment of the invention said protein is selected from the group comprising Hakai, ZNF645, Ligand-of-Numb protein X1 and Ligand-of-Numb protein X2.

In yet a further preferred embodiment of the invention said polypeptide or protein has the following conserved target binding residues H127 and H185. Preferably said polypeptide or protein also has the following conserved target binding residues R189 and/or Y176.

In yet a further preferred embodiment of the invention said polypeptide or protein has a 1 :1 binding relationship with its target molecule. Preferably, but not exclusively, the target molecule is E-cadherin, DOK1 or cortacin.

In still yet a further preferred embodiment of the invention said polypeptide or protein has a RING domain characterized by sequence structure:

VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGC SEQ ID NO:3 (106-148 a.a.).

In still yet a further preferred embodiment of the invention said polypeptide or protein comprises two zinc-finger domains, a RING domain and a minimum pTyr-binding domain that incorporates a novel, atypical zinc coordination motif. Where two polypeptides of the invention are provided the HYB domain is therefore composed of four zinc-finger domains cooperating to bind pTyr residues, ideally, surrounded by acidic amino acids. In yet a further preferred embodiment of the invention under part c) where said binding occurs concluding said compound may be useful in preventing cell migration or metastasis or invasion or cancer or dysplasias or hyperplasias.

In yet a further preferred embodiment of the invention said method further includes providing reagents and conditions that enable ubiquitination to take place and determining whether ubiquitination of a protein of interest takes place in the presence of absence of said test compound and where it does not take place using this fact to demonstrate or confirm binding between said polypeptide and said compound. For example, and without limiting how this ubiquitin assay may be performed, selected cells, such as HEK 293 cells, are transfected with plasmids expressing a protein of interest and epitope-tagged ubiquitin E2 conjugating enzyme in the presence (or absence - control sample) of the said polypeptides of the invention and the test compound. Ideally also in the presence of the proteosomal inhibitor MG132. 24 hours post-transfection, cells lysates are collected using appropriate buffer. Target proteins are then precipitated using specific antibodies against those proteins. Typically the polypeptides of the invention will be included in the buffers during this assay. Precipitated proteins are analysed using the SDS-PAGE gel and immunoblotting is undertaken for detection of ubiquitination levels in the complex. The level of ubiquitination should be low in the complex where the polypeptides are included in the assay and the said polypeptides bind to the test compound thus showing the test compound is an inhibitor of same. Alternatively, GST- fusion proteins of the targets (E-Cadherine, DOK1 and cortactin) are produced and purified. GST-fusion proteins of the E3 ligases (Hakai, ZNF645, Ligand-of-Numb protein X1 and Ligand-of-Numb protein X2, or parts thereof including at least the HYB binding domain) are also produced and purified. Before the in vitro assay is carried out, one set of E3 ligases is incubated with said test compounds, while another set with a control solution. The ubiquitination assay is then carried out by adding the ubiquitinating buffer, E1 , E2 and ATP. Upon stopping the reaction samples are analyzed using the SDS-PAGE and western analysis. Modified proteins will be detected using the anti-Ubiquitin immunoblotting. The levels of inhibition of ubiquitination of the targets will be deduced from the control samples. The level of ubiquitination should be low in the complex where the test compound binds to the E3 ligases, or at least the HYB domain thereof, thus showing the test compound is an inhibitor of same.

In a further preferred method of the invention said binding under part c) may be determined either in vitro, in vivo or in siiico and in the latter instance having regard to the crystalline structure of the HYB domain provided in Table 1 and, ideally, the figures contained herein wherein a structure having the requisite co-ordinates and, ideally shape, is modeled for the purpose of determining binding with candidate modeled drug molecules.

According to a second aspect of the invention there is provided an isolated polypeptide selected from the group comprising: i) TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLEN VH (a.a. 159-206) SEQ ID NO:1 or a sequence at least 31% homologous thereto wherein the following amino acids are conserved C 66, C172, H185, and H190; ii) TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLEN VH (a.a. 159-206) SEQ ID NO:1 or a sequence at least 76% homologous thereto; iii) VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDPV QRIEQCTRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTR ASLENVH (a.a. 106-206) SEQ ID NO: 2 or a sequence at least 23 % homologous thereto wherein the following amino acids are conserved C109, C112, C125, H127, C130, C133, C145 C148, C166, C172, H185, and H190; iv) VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDPV QRIEQCTRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTR ASLENVH (a.a. 106-206) SEQ ID NO: 2 or a sequence at least 71% homologous thereto; v) two of the following sequence structures:

TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENV

H (a.a. 159-206) SEQ ID NO:1 or a sequence at least 31% homologous thereto, where the following amino acids are conserved C166, C172, H185, and H190, arranged as a dimer, ideally an anti-parallel dimer; vi) two of the following sequence structures:

TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENV

H (a.a. 159-206) SEQ ID NO:1 or a sequence at least 76% homologous thereto arranged as a dimer, ideally an anti-parallel dimer; vii) two of the following sequence structures:

VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDPVQ RIEQCTRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRA SLENVH (a.a. 106-206) SEQ ID NO:2 or a sequence at least 23% homologous thereto where the following amino acids are conserved C109, 0112, C125, H127, C130, C133, C145 C148, C166, C172, H185, and H190, arranged as a dimer, ideally an anti-parallel dimer; viii) two of the following sequence structures:

VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDPVQ RIEQCTRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRA SLENVH (a.a. 106-206) SEQ ID NO:2 or a sequence at least 71% homologous thereto arranged as a dimer, ideally an anti-parallel dimer; ix) an isolated polypeptide according to i),ii), v) and vi) in combination with a RING domain characterized by sequence structure: VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGC SEQ ID NO:3 (a.a. 106-148); x) IHFCDKCDLPIKIYGRIIPCKHAFCYHCANLYDKVGYKVCPRCRYPVL RIEAHKRGSVFMCSIVQQCKRTYLSQKSLQAHIKRRHKRARKQVTSAS LEKVR (a.a. 54-154 ZNF645) SEQ ID N0:4 or a sequence at least 71 % homologous thereto wherein, when aligned with Hakai a.a. 106- 206, the following amino acids are conserved C109, C112, C125, H127, C130, C133, C145 C148, C166, C172, H185, and H190; xi) DLVCHICLLQPLLQPLDTPCGHTFCYKCLRNFLQEKDFCPLDRKRLH FKLCKKSSILVHKLLDKLLVLCPFSSVCKDVMQRCDLEAHLKNRCPGA SHRRVALERRKTS (a.a. 47-153 LNX2) SEQ ID NO:5 or a sequence at least 25% homologous thereto wherein, when aligned with Hakai a.a. 106-206, the following amino acids are conserved C109, C112, C125, H127,€130, C133, C145 C148, C166, C172, H185, and H190; and xii) DLICHICLQALLDPLDTPCGHTYGTLCLTNFLVEKDFCPMDRKPLVL QHCKKSSILVNKLLNKLLVTCPFREHCT-

QVLQRCDLEHHFQTSCKGASHYGLTKDRKRRS (a.a. 38-144 LNX1 ) SEQ ID NO:6 or a sequence at least 23% homologous thereto wherein, when aligned with Hakai a.a. 106-206, the following amino acids are conserved C109, C112, C125, H127, C130, C133, C145 C148, C166, C172, H185, and H190.

According to a third aspect of the invention there is provided a crystal form of the isolated polypeptide described herein wherein said crystal is characterised by the co-ordinates and structure factors deposited at the Protein Data Bank (PDB) with the accession code 3VK6 and/or as described herein with reference to the text and figures and/or Table 3.

According to a fourth aspect of the invention there is provided a molecular target for treating a disease characterised by migration or metastasis or invasion or a lack of cell-cell adhesion such as cancer, comprising a protein selected from the group comprising E-cadherin, DOK1 or cortactin.

As mentioned above the method of the invention may be undertaken in silico, this we have done using conventional software such as the software Glide, version 5.5 (Schrodinger, LLC, New York, 2009). With this in silico method we have demonstrated that Methotraxate Hydrate is effective at binding with the polypeptide or protein of the invention and so blocking its ability to bind E-cadherin, DOK1 or cortactin.

According to a fifth aspect of the invention there is therefore provided the use of Methotraxate Hydrate, or a derivative or salt thereof, to treat a disease characterised by migration or metastasis or invasion or a lack of cell-cell adhesion, such as cancer.

According to a sixth aspect of the invention there is therefore provided the use of Methotraxate Hydrate, or a derivative or salt thereof, in the manufacture of a medicament to treat a disease characterised by migration or metastasis or invasion or a lack of cell-cell adhesion, such as cancer.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprises", or variations such as "comprises" or "comprising" is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art. Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

The invention will now be described by way of example only with reference to the following figures:

Figure 1 shows a novel protein fold in Hakai. (A) A schematic diagram of the Hakai protein. (B) The crystal structure of Hakai (aa 106-206) reveals a dimer in an anti-parallel configuration. Each monomer contains three zinc coordination sites. Sites 1 and 2 lie in the RING domain. Site 3 is shared between the two monomers. (C) The coordination of zinc ions (purple spheres) by the RING domain of Hakai is shown for one of the monomers. (D) A schematic diagram of the cross-brace arrangement of the Hakai RING domain as shown in (C). (E) The Hakai dimer forms an intertwined configuration spanning the points indicated in circles, with the entry and exit paths shown in green and brown arrows. The zinc-interacting side chains are shown as green and brown sticks. (F) The backbone of the Hakai (aa 106-206) residues involved in intermolecular main-chain H-bonding and the zinc-coordinating side chains of adjacent monomers at the dimer interface are shown in cyan and yellow. The pink dots indicate the main-chain H- bonds; the red dots indicate the zinc coordination bonds. (G) The monomers of the interlinked Hakai dimer are shown in surface representation and Ca trace, respectively. The Ca trace monomer enters and exits the other monomer at the red and black circles, respectively. Brown arrows show its entry and exit path. (H) A schematic diagram of the novel Hakai interlinked arrangement as shown in (G);

Figure 2 shows Hakai forms a dimer in solution. (A) A 3D ¹⁵N-NOESY spectrum showing the intermolecular NOE cross-peaks of amides corresponding to residues of Hakai (aa 106-206). (B) WT Hakai (aa 106- 206) and four Hakai (aa 106-206) point mutants were each separately used for gel-filtration chromatography. Their respective elution profiles were overlain and compared. (C) HA- and FLAG-tagged Hakai were overexpressed in the presence of Src in HEK293 cells. FLAG immunoprecipitates were analysed for HA-tagged Hakai. (D) A schematic representation of the point mutations made to C 66, C172, H185 and H190 in Hakai. (E) Cell lysates containing WT Hakai or Hakai mutants were used to analyse the effects of Hakai dimerization on E-cadherin recognition;

Figure 3 shows Hakai domain recognizes acidic residues. (A) Y753, Y754 and Y755 (red) of E-cadherin were mutated to phenylalanine (blue) in different combinations. (B) The WT E-cadherin and the mutants shown in (A) were overexpressed in HEK293 cells with v-Src to analyse their pTyr signals. (C) HEK293 cells were co-transfected with WT E-cadherin or its mutants together with Hakai to identify the tyrosine residues recognized by Hakai. (D) The Y754-phosphorylated and non-phosphorylated E-cadherin peptides were titrated against Hakai (aa 106-206) using ITC. The top panels show the heat release profiles after baseline correction and the lower panels indicate the binding isotherms for the interactions. The dissociation constant (K_d) and binding stoichiometry (N) are shown in the table. (E) The E-cadherin aa 747-758 were each substituted with alanine. (F) The E-cadherin mutants from (E) and Hakai were co-transfected into HEK293 cells to identify the target motif on E-cadherin. (G) E-cadherin and Hakai were co-transfected into HEK293 cells. Their interaction was analysed through immunoprecipitation of FLAG-tagged Hakai. (H) Cortactin was co-transfected into HEK293 cells with Hakai. The interaction between cortactin and Hakai was compared with that in (G). (I) Y482 and Y485 (red) were separately substituted with phenylalanine (blue). (J) WT and mutated cortactin were co-transfected into HEK293 cells with Hakai, in the absence or presence of v-Src. The pTyr signal of cortactin and its interaction with Hakai were analysed. (K) An alanine scan of cortactin aa 478-489. Each residue was substituted with alanine (blue). G484 was not mutated as glycine mutations affect the protein structure. (L) The cortactin mutants described in (K) were co-transfected into HEK293 cells with Hakai. The interaction between the cortactin mutants and Hakai was determined using immunoprecipitation;

Figure 4 shows DOK1 interacts with Hakai. (A) The sequence alignment of the different Src phosphorylation target sites in E-cadherin, cortactin and DOK1. The acidic amino-acid residues flanking the phosphorylated tyrosine are shown in blue. (B) Hakai and DOK1 were overexpressed in HEK293 cells in the absence or presence of Src. FLAG immunoprecipitates were analysed for DOK1 interaction. (C) DOK1 was co-transfected into HEK293 cells with Hakai to study its competition with endogenous cortactin for binding to Hakai. FLAG immunoprecipitates were immunoblotted for cortactin;

Figure 5 shows target-binding amino acids of Hakai. (A) An overlay of the H-¹⁵N-HSQC spectra of Hakai (aa 106-206) in the absence (green) or the presence (red) of an tyrosine-phpsphorylated E-cadherin peptide. (B) A graphical representation of the combined chemical shift perturbation (p.p.m.) plotted against all Hakai (aa 106-206) residues, with the cutoff at the combined chemical shift perturbation of 0.15 p.p.m. (C) The six potential E-cadherin-interacting residues in Hakai (aa 106-206) are highlighted as sticks in the ribbon representation of the crystal structure. (D) An electrostatic surface potential representation of Hakai (aa 106-206) shows that H127, Y176, H185 and R189 form part of the positively charged pocket. (E) The interaction between E-cadherin and the Hakai mutants of the residues identified in (C) was analysed by immunoprecipitating FLAG- tagged Hakai. (F) HEK293 cells were transfected with the identified Hakai mutants, and their interaction with endogenous cortactin was studied. (G) Immunoprecipitates of either WT Hakai or the Hakai zinc-coordinating mutants were tested for interaction with endogenous cortactin. (H) A schematic representation of the Hakai dimer and the HYB domain;

Figure 6 shows the HYB domain in other proteins. (A) A comparison of the Hakai protein from amino-acid residues 127-191 and the equivalent sequence in ZNF645. (B) E-cadherin and ZNF645 were analysed for their interaction using immunoprecipitation. Hakai was used as a positive control. The dotted arrow indicates a non-specific band; the solid arrow indicates the ZNF645 band. (C) ZNF645 was overexpressed in HEK293 cells and its interaction with endogenous cortactin was analysed using immunoprecipitation. (D) Sequence alignment of LNX1 and LNX2 with Hakai and ZNF645 based on the conserved zinc-coordinating residues from Hakai aa 106-206;

Figure 7 shows the novel structure of the HYB domain. (A) Representative structures of SH2 (PDB code 1SHB), PTB (PDB code 1SHC), PKCd C2 (PDB code 1YRK) and PKM2 (PDB code 3BJF) in ligand-free forms are compared with the HYB domain. (B) The corresponding topologies of the domains in (A);

Figure 8 shows ITC analysis Hakai (106-206) interactions with DOK1 and Cortactin Figure 8A The Y361 phosphorylated DOK1 peptide was titrated against Hakai (aa 106 - 206) using ITC. The top panels show the heat release profiles after baseline correction and the lower panels indicate the binding isotherms for the interactions. The dissociation constant (Kd) and binding stoichiometry (N) are shown in the table. Figure 8B The cortactin peptide double phosphorylated at Y482 and Y485 was titrated against Hakai (aa 106 - 206) using ITC. The top panels show the heat release profiles after baseline correction and the lower panels indicate the binding isotherms for the interactions. The dissociation constant (Kd) and binding stoichiometry (N) are shown in the table. Notably, these binding studies with novel cancer targets Cortacin and DOKIhave Kd values for binding of Cortacin and DOK1 to the HYB domain in the order of 28.4μΜ and 5.33μΜ, respectively, thus demonstrating the specificity of the HYB domain for these proteins and their role as targets in cancer therapy; and

Figure 9 shows the amino acid sequence structure of Hakai with the HYB domain highlighted.

METHODS

Plasmids

Mouse Hakai, human E-cadherin and avian v-Src were gifts from W Birchmeier (Max-Delbru ^'ck-Center for Molecular Medicine, Germany), W Hunziker (IMCB, Singapore) and XM Cao (IMCB, Singapore), respectively. Mouse cortactin was from Addgene (Cambridge, MA) (plasmid 26722, deposited by A Weaver). Human ZNF645 and DOK1 were from Origene (Rockville, MD). Where necessary, the genes were cloned into pXJ40-HA or pXJ40-FLAG. For structural studies, Hakai constructs were cloned into pGEX6P-1 (GE Healthcare, UK). Point mutants and truncates were generated using the proofreading Pfu DNA polymerase.

Antibodies and reagents

Mouse anti-FLAG M2, rabbit anti-FLAG and anti-HA and agarose- conjugated anti-FLAG M2 beads were obtained from Sigma-Aldrich (St Louis, MO). Rabbit GST, cortactin, E-cadherin and DOK1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Protein-A- conjugated agarose beads were from Roche Molecular Biochemicals (Germany). Mouse anti-E-cadherin and HRP-conjugated anti-pTyr PY20 were from BD Transduction Laboratories (Lexington, KY). Mouse anti-b- actin was obtained from Abeam (Cambridge, MA).

Cell lines and transfection

HEK293 cells were purchased from ATCC (Manassas, VA) and maintained as described (Yusoff et al, 2002). Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

Immunoprecipitation and immunoblotting

Immunoprecipitation and immunoblotting were carried out as described

(Yusoff et al, 2002) with the following modifications. HEK293 cells were harvested 24 h post-transfection with a lysis buffer containing protease inhibitors (Roche) and 1 mM Na₃V0₄. Immunoprecipitations

were performed using agarose-conjugated anti-FLAG or protein-specific antibodies followed by incubation with protein-A-conjugated agarose beads at 41C.

Liquid chromatography-mass spectrometry/mass spectrometry

Immunoprecipitates were separated by SDS-PAGE, and stained with Coomassie Blue G250. Protein bands were excised and washed with 25 mM ammonium bicarbonate (ABB) in 50% acetonitrile (ACN) buffer thrice. The proteins in the gel were reduced with 10 mM DTT in 25 mM ABB buffer, alkylated with 5 mM iodoacetamide, dehydrated and digested with trypsin overnight. After in-gel digestion, the solution was transferred to a clean tube and sonicated for 30 min in the presence of 50 ml 50% ACN and 5% acetic acid for protein extraction. This extraction procedure was repeated three times; the pooled extracts were dried with a vacuum concentrator. The samples were processed and analysed as described (Zhang et al, 2010) using a LTQ-FT ultra mass spectrometer. For each experiment, MS/MS (dta) spectra were extracted from the raw data files using the extract_msn program in Biowork 3.3 (ThermoFinnigan). The extracted dta files were combined into a single file in the Mascot generic file (mgf) format. Except for the conversion of precursor mass from MH p in dta to m/z in mgf, the fragment ion m/z and intensity values were used as determined. Proteins were identified by searching the combined data against the IPI human database (downloaded on 25 November 2009, including 86 845 sequences and 35122 444 residues) via an in-house Mascot server (version 2.2.07). Two missing cleavages were allowed. Precursor ion and MS/MS fragment ion error tolerances were set to o10 p. p.m. and o0.8 Da, respectively. A protein was accepted as a true positive if it had a significant score (Po0.05) and at least two unique peptides.

Protein purification and gel-filtration chromatography

The GST-tagged Hakai (aa 106-206) constructs were expressed in Escherichia coli BL21 (DE3) and purified using glutathione-conjugated sepharose (GE Healthcare). The GST-tag was cleaved using GST- PreScission Protease (GE Healthcare) and the proteins were applied to a Superdex 75 size-exclusion column (GE Healthcare) equilibrated using 10 mM Bis-Tris, pH 6.5, 250 mM NaCI and 5 mM DTT and pre-calibrated using a gel-filtration standard (Bio-Rad).

NMR spectroscopy and chemical shift perturbation analysis

¹⁵N/ ³C-labelled Hakai (aa 106-206) was obtained from cultures grown in M9 media supplemented with ¹⁵N-labelled ammonium chloride and ^Relabelled glucose as the sole nitrogen and carbon sources, respectively. The labelled proteins were purified as described above. NMR spectra were acquired at 298 K in an 800-MHz NMR spectrometer (Bruker, Karlsruhe, DE). The backbone assignment was obtained using standard ¹⁵N-edited HSQC, HNCACB and CBCA (CO)NH experiments; side chains were assigned using standard 3D-T0CSY, 3D-N0ESY and HCCH-TOCSY experiments. N MR data were processed using NMRPipe (Delaglio et al, 1995) and analysed by NMRView (Johnson and Blevins, 1994).

For the chemical shift perturbation analysis, the 2D H-¹⁵N-HSQC spectra for the ¹⁵N-labelled Hakai (aa 106-206) were acquired in the absence or presence of the phosphorylated E-cadherin peptide. Perturbed residues on Hakai were assigned by super-imposing the two HSQC spectra.

Crystallization and structure determination

SelMet-substituted Hakai (aa 106-206) was expressed in a methionine auxotroph (Doublie, 1997) and purified as described above. SelMet incorporation was verified by MALDI-TOF.

SelMet Hakai (aa 106-206) crystals were grown at 289 K by the hanging drop vapour diffusion method. The protein (30 mg/ml) was mixed with an equal volume of reservoir solution (140 mM Li₂S0₄; 100 mM Tris, pH 7.8; 15 mM Na₂S₂0₃; 20-22% PEG 5000 MME; 1% isopropanol and 20-25% ethylene glycol). A complete SAD data set was collected to 1.9 A resolution at the synchrotron beamlines (NSLS, Brookhaven National Laboratory and the National Synchro-tron Radiation Research Center [NSRRC], Taiwan) using a Quantum4-CCD detector (Area Detector Systems Corp., Poway, CA). The datasets were processed and scaled using HKL2000 (Otwinowski and Minor, 1997). The crystals belonged to space group P6₂22 with a = 64.66 A, b = 64.66 A, c = 121.04 A, and contained one molecule in the asymmetric unit.

All four expected selenium sites in the asymmetric unit were located by SOLVE (Terwilliger and Berendzen, 1999). Initial phases were developed by RESOLVE (Terwilliger, 2003); the overall figure of merit was improved to 0.83, and over 90% of the molecule was built automatically. The remaining parts of the model were built manually using COOT (Emsley and Cowtan, 2004) and alternatively refined by CNS (Brunger et al, 1998) and PHENIX (Adams et al, 2002). The final model was refined to a 1.9-A resolution with an R-factor of 0.2175 (Rf_ree = 0.2396) and analysed using PROCHECK (Laskowski et al, 1993). AILstructure-related figures were prepared using PyMOL (DeLano, 2002).

Circular dichroism spectrometry

Far UV spectra (260-190 nm) of Hakai (aa 148-206) and Hakai (aa 106- 206) and its mutants were measured using a Jasco J-810 spectropolarimeter in phosphate buffer (pH 7.5) at room tempera-ture using a 0.1 -cm path length and stoppered cuvettes. Six scans were recorded, averaged and the baseline subtracted.

Dynamic light scattering

Dynamic light scattering studies were carried out on a DynaPro Light Scattering instrument (Protein Solutions, USA) at a protein concentration of 2 mg/ml, in a buffer containing 10 mM Bis-Tris pH 6.5, 250 mM NaCI and 5 mM DTT.

Isothermal titration calorimetry

Phosphorylated and non-phosphorylated peptides of E-cadherin (residues 749-761); phosphorylated peptides of DOK1 (residues 356-366) and Cortactin (residues 477-489) were titrated at a molar concentration of 800 mM against 100 mM of Hakai (aa 106- 206) dimer in a VP-ITC microcalorimeter (Microcal, Northhampton, UK) at 293 K. The titrations were carried out using 30 10-ml injections of the appropriate peptide into the sample cell containing Hakai (aa 106-206) and the data were analysed with a one-site binding model using the Origin software package v7.0 supplied by Microcal. All measurements were repeated twice. Accession number

The coordinates and structure factors have been deposited at the Protein Data Bank (PDB) with the accession code 3VK6.

RESULTS

A novel protein fold in Hakai

We experimentally established that the minimum E-cadherin phosphotyrosine-binding sequence in Hakai was contained within amino acids 148-206 (aa 148-206) (Supplementary Figure S1A-G). Circular dichroism analysis however revealed that purified Hakai (aa 148-206) was unstructured (Supplementary Figure S1 H), and was not suitable for crystallization. Therefore, the Hakai sequence spanning amino-acid residues 106- 206 (aa 106-206) that contained both the RING domain and the minimum pTyr-binding domain, as represented schematically in Figure 1 A, was purified and crystallized.

The crystal structure of Hakai (aa 106-206) was solved at 1.9 A resolution (Figure 1B). The striking feature of the crystal structure was the formation of a dimer from paired, anti-parallel Hakai (aa 106-206) monomers. Each monomer consisted of an N-terminal RING domain, followed by the C- terminal atypical zinc-binding domain that is contained within the experimentally derived minimum pTyr-binding domain. Furthermore, each monomer contained three zinc ions at three distinct sites. One zinc ion coordinated with the atypical zinc-binding domains of both monomers (Figure 1 B). The Hakai RING domain (residues 106-148) adopted a typical RING domain fold stabilized by co-ordinating with two zinc ions, forming a cross-brace arrangement (Figure 1C and D). The zinc-coordinating residues in the RING domain are also indicated in Figure 1 D.

The uniqueness of the Hakai (aa 106-206) region was revealed when the structure was compared with other proteins in the PDB (Protein Data Bank) using the DALI server (http://ekhidna.biocenter.helsinki.fi/dali_server/). The results show that only the RING domain of Hakai is structurally similar to RING domains of other proteins. There is, however, no similarity beyond amino-acid residue 159 of the Hakai minimum pTyr-binding domain, which is located on the dimerization interface. The minimal pTyr-binding domain of Hakai adopts a novel, three-dimensional fold and contains three b-strands (b4, b5 and b6) and a C-terminal a-helix. The b-strands b4, b5 and b6 were in an extended configuration and formed anti-parallel b-sheets with the corresponding b-strands of the monomeric partner during homodimerization (Figure 1 E). The atypical zinc-finger motif within this region is formed by two histidine residues (H185 and H190) and one cysteine residue (C172) from one monomer and a second cysteine residue (C166) from the adjacent monomer (Figure 1 F; Supplementary Figure S2), unlike a classical C2H2 zinc finger (ZnF). Although the Hakai (aa 106-206) region in each monomer fulfils the required criteria of the zinc coordination consensus pattern of cysteine and histidine residues [C-x(5)-C-x(12)-H-x(4)-H], it is not capable of forming the zinc coordination sphere by itself, since a -second cysteine residue is required of its anti-parallel monomeric partner (Figure 1 F). Therefore, each dimer contains two atypical zinc-finger motifs.

The crystal structure of Hakai also shows that the two Hakai monomers intertwine through a stretch of residues ranging from F164 to Y176 during dimerization, resulting in the formation of three β-sheets based on 12 main- chain hydrogen bonds (Figure 1 E-H). This novel interlinked con-figuration and the two atypical zinc ion interactions at the dimer interface are unique features of this distinctive homo-dimeric assembly. A surface area of approximately 1650A² of each monomer (or -21% of each monomer surface) was formed at the dimer interface of Hakai (aa 106-206), with 34 hydrogen bond contacts between the monomers, as analyzed by the PISA server (Krissinel and Henrick, 2007). Hakai forms a dimer in solution

A previous study alluded to the similarity between the Hakai (aa 106-206) polypeptide with the dimerization domain of the V(D)J recombination- activating protein RAG (Fujita et al, 2002). In addition to the crystal structure, the inter-molecular NOE cross-peaks corresponding to the amides of residues at the dimer interface also indicates that Hakai (aa 106- 206) forms a dimer in solution (Figure 2A). Significantly, the structures of the dimerization interface of the RAG1 domain (Bellon et al, 1997) and Hakai (aa 106-206), as described in this study, are completely dissimilar. The formation of Hakai (aa 106-206) dimers in solution is also supported by the results obtained through dynamic light scattering, which show an apparent molecular weight of 24.1 kDa, twice than that of the monomer.

We next examined whether zinc coordination is necessary for the dimerization of Hakai and its ability to interact with its target. We first investigated whether mutations of the zinc-coordinating residues within the minimum pTyr-binding do-main (C166, C172, H185 and H190) would affect the proposed Hakai dimerization. The Hakai (a. a. 106-206) polypeptides containing point mutations at these residues were separated on a calibrated gel-filtration column. Their gel-filtration elution profiles show that each point mutant had an apparent molecular weight equivalent to a monomeric unit of wild-type (WT) Hakai (a.a. 106-206) protein (B_.12 kDa), whereas WT Hakai eluted as a dimer (Figure 2B). Furthermore, circular dichroism performed using all the Hakai (a.a. 106-206) mutants ascertained that each one has maintained a well-defined secondary structure. These findings suggest that each zinc-coordinating residue is instrumental in forming the dimer interface.

Having determined that Hakai (a.a. 106-206) dimerizes in solution, we next examined if this occurs with full-length proteins. Full-length FLAG-tagged Hakai was observed to bind to its HA-tagged counterpart (Figure 2C), indicating that dimerization also occurs between the full-length proteins. To further determine whether Hakai dimerization is required for its function in binding its targets, the full-length proteins containing alanine point mutations of the zinc-coordinating residues (Figure 2D) were tested for their ability to bind to tyrosine-phosphorylated E-cadherin. HEK293 cells were used for such studies as they did not express detectable levels of endogenous E-cadherin, which could have interfered with the mammalian cellular assays used. The evidence presented in Figure 2E indicates that none of the four Hakai point mutants interacted with tyrosine- phosphorylated E-cadherin. The collective results therefore show that zinc coordination is necessary for both dimerization of Hakai and its subsequent function in interacting with its target.

Hakai domain recognizes acidic residues

Having established the novel characteristics of the Hakai zinc-coordinated homodimer, we sought to identify the target motif of this new domain. At this point, the only described target motif was in Src-phosphorylated E-cadherin. Within this motif, two (Y755 and Y756 in mouse; Y753 and Y754 in humans) of three consecutive tyrosine residues were reported to be involved in the interaction with Hakai (Fujita et al, 2002). To analyse the relative contributions of the three tyrosine residues in binding to Hakai, mutations were made to the tyrosine residues (Figure 3A). To determine which of the tyrosines were phosphorylated, we analysed the patterns of the tyrosine phosphorylation of the point mutants after v-Src activation. The pTyr signals shown in Figure 3B indicate that all three adjacent tyrosines were phosphorylated.

We next examined the importance of the three tyrosine residues in E- cadherin for its interaction with Hakai. The E-cadherin mutants used in the earlier experiment were analysed for, their potential to bind to WT Hakai. The results shown in Figure 3C indicate that Y754 is the only tyrosine significantly involved in binding; each mutant containing a substitution in this position did not bind Hakai, whereas all other mutants showed significant binding. These combined results also show that while Src binds to and phosphorylates most of the E-cadherin mutants, all the mutants with a Y754F substitution do not bind to Hakai, even when phosphorylated. This implies that the interaction between Hakai and E-cadherin depends on the direct recognition of specific pTyr residues on E-cadherin by Hakai. To verify the necessity of Y754 phosphorylation for the interaction between E- cadherin and Hakai, isothermal titration calorimetry (ITC) was performed using phosphorylated and non-phosphorylated E-cadherin peptides corresponding to a.a. 749-761 with Hakai (aa 106-206). The results in Figure 3D show that binding occurred only with the phosphorylated peptide. Furthermore, the results also indicate that only one E-cadherin peptide binds to the Hakai (aa 106-206) dimer at any one time.

In a similar manner, we analysed the importance of the amino-acid residues flanking the tyrosines for the interaction between the two proteins. via an alanine scan (Figure 3E). The immunoprecipitation results in Figure 3F show that there were profound contributions from the aspartic acid D756 and glutamic acid E757, and significant contributions from valine V752 and aspartic acid D750. Consequently, a cluster of negative charges from the acidic amino acids is formed around the centrally binding tyrosine 754 of E- cadherin.

To verify the motif recognized by Hakai, additional target proteins were identified. This identification was accomplished by analysing Hakai-binding proteins phosphorylated by Src using mass spectrometry. A list of the proteins obtained from a typical experiment is appended in Supplementary Figure S4A and B, in which cortactin was identified as a potential target. Mouse cortactin is phosphorylated by Src primarily on Y482 and Y485. Interestingly, these two tyrosines are also surrounded by several acidic residues. We investigated whether Hakai binds to Src-phosphorylated cortactin, as well as the importance of Y482, Y485 and their flanking residues in this interaction. In addition to the WT proteins, phenylalanine substitution and alanine scan experiments were also per-formed on cortactin as described for E-cadherin. The results for cortactin mirror those obtained for E-cadherin. Like E-cadherin, cortactin interacts with Hakai only when phosphorylated by Src (Figure 3G and H). Phenylalanine substitutions of the Src-phosphorylated tyrosine residues revealed that Y482 is the main tyrosine residue involved in Hakai binding (Figure 31 and J). Furthermore, the acidic residues (E478, D480 and E486) surrounding Y482 contributed profoundly to the interaction between Hakai and E- cadherin, and significant contribution was also observed from S487 (Figure 3K and L).

To further verify that Hakai targets pTyrs of Src substrates with surrounding acidic residues, another Src substrate was selected: DOK1 , which also contains pTyrs with adjacent acidic groups. One such particular tyrosine residue was found to be a primary phosphorylation site of Src (Figure 4A). The results show that DOK1 interacts with Hakai. Furthermore, DOK1 competed with endogenous cortactin for Hakai, implying that DOK1 and cortactin bind Hakai on the same site (Figure 4A-C).

Target-binding amino acids of Hakai

To identify the residues in Hakai within a.a. 106-206 necessary for its interaction with E-cadherin, 2D ¹H-¹⁵N-HSQC spectra of ¹⁵N-labelled Hakai (aa 106-206) were acquired in the absence and presence of a pTyr peptide derived from amino-acid residues 749-761 of E-cadherin. Based on the changes in the NMR spectrum of Hakai (aa 106-206), several residues underwent perturbations in chemical shift (Figure 5A and B). A minimum criterion of a chemical shift difference Ddp._p._m .40.15 p. p.m. was applied. Five residues were identi-fied (Y176, H185, N187, H188 and R189), which resided in the Hakai minimum pTyr-binding domain, whereas a sixth residue (H127) was from the RING domain (Figure 5C). However, the crystal structure shows that only four of these six residues reside in a close three-dimensional spatial proximity. While H127, Y176, H185 and R189 face the interior of the E-cadherin-binding site (Figure 5C) and form a positively charged pocket, N 187 and H188 face outward and are not part of the pocket (Figure 5D).

These residues identified through in vitro peptide-domain binding assays were then further tested by expressing full-length point mutants in HEK293 cells. The immunoprecipitation results shown in Figure 5E indicate that the residues identified in the NMR analysis also abrogated binding when mutated, with the exception of residues N 87 and H188. As expected, the required residues were situated on the interior of the target-binding domain, whereas the non-binding residues, N187 and H188, were on the exterior. Similar results were obtained with experiments using cortactin (Figure 5F), demonstrating the importance of these Hakai residues. Furthermore, dimerization of Hakai is also required, as with E-cadherin, as cortactin was unable to bind to Hakai containing mutations to its zinc-coordinating residues (Figure 5G).

Based on the evidence obtained, it can be concluded that two Hakai monomers interact in an anti-parallel manner to form a dimer via the interlinked zinc-coordinating domain. This domain binds pTyrs flanked by acidic amino acids in Src substrates. The target-binding domain resulting from this dimerization process represents the functional Hakai phosphotyrosine-binding domain, henceforth referred to as the HYB (Hakai pY-binding) domain (Figure 5H).

The HYB domain in other proteins

We next investigated whether the HYB domain is found in other proteins. Literature and database searches revealed that the testis-specific ubiquitin E3 ligase ZNF645 exhibited high-sequence homology (71 %) with Hakai, as shown in Figure 6D. The sequence homology between Hakai and LNX2 and 1 were 25% and 23%, respectively. When the homology was investigated for only the novel binding sequence (a.a. 159-206) the testis- specific ubiquitin E3 ligase ZNF645 exhibited high-sequence homology (76%) with Hakai, as shown in Figure 6D. The sequence homology between the binding sequence Hakai (a.a. 159-206) and LNX2 and 1 were 31% and 37%, respectively. We therefore questioned whether ZNF645 could also interact with E-cadherin and cortactin. The results in Figure 6B show that ZNF645 bound to v-Src-phosphorylated E-cadherin but not to cortactin (Figure 6C). This result implies that although there is significant homology between Hakai and ZNF645, they are likely to have their own sets of targets due to the differences in their sequences between the key zinc-coordinating residues.

Based on the key amino-acid residues involved in zinc coordination and binding in HYB, we searched the NCBI database to analyse gene origins and protein homologies (Table 2). Two interesting results emerged. First, a comparison of the species distribution of the Hakai and ZNF645 gene products indicated that the latter, found only in primates, is most likely a recent copy of the former. Second, ZNF645 is an intronless gene, implying that it is a retrotransposed copy of Hakai.

Further database searches based on the conserved zinc-coordinating cysteine and histidine residues within the HYB domain showed that a similar series of residues is present in LNX1 and LNX2 (Figure 6D). This implies that the HYB domain may be distributed in other proteins, although the latter observation requires experimental confirmation.

Novel structure of the HYB domain

The structures of the five pTyr-binding domains that have been discovered to date are illustrated in Figure 7A and B. All of the domains, except for the HYB domain, are contained within one monomer. The HYB domain consists of a pair of monomers arranged in an anti-parallel configuration and is composed of two RING and two atypical zinc-coordinating domains. From this comparison, it is apparent that all five of these pTyr domains have completely different structures, with different strategies to recognize tyrosine phosphorylation. References

Fujita Y, Krause G, Scheffner M, Zechner D, Leddy HE, Behrens J, Sommer T, Birchmeier W (2002) Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex. Nat Cell Biol 4: 222- 231

Bellon SF, Rodgers KK, Schatz DG, Coleman JE, Steitz TA (1997) Crystal structure of the RAG1 dimerization domain reveals multi-ple zinc-binding motifs including a novel zinc binuclear cluster.Nat Struct Biol 4: 586-59.

Table 1 Crystallographic data and refinement statistics

Data collection

Space group P6₂22

Cell dimensions

a, b, c (A) 64.66, 64.66, 121.04

90.00, 90.00, 120.00

Wavelength (A) 0.979

Resolution (A) 50-1.9 (1.97-1.90) Observed reflections 509428

Unique reflections 22371

R ·* sym ^a 0.052 (0.428)

/ / ^'■ / 21.3 (12.6)

Completeness ( ) 99.7 (100)

Redundancy 22.8 (22.6)

Refinement

Resolution (A) 25.2-1.9

Reflections (working set / test set) 19327/2153

R_work» / R_freec 0.2175/0.2396 No. atoms

Protein 756

Zn²⁺ 3

Water 73

β-factors (A²)

Protein 38.75

Zn²⁺ 30.46

Water 44.06

Ramachandran statistics

Most favorable regions (%) 90.2

Additional allowed regions (%) 9.8

Generously allowed regions ( ) 0.00

Disallowed regions (%) 0.00

R.m.s deviations

Bond lengths (A) 0.008

Bond angles ( ) 1.202 Values in parentheses are for highest-resolution shell.

a/¾ym =∑|li ~ <l>|/∑|li|, where I, is the intensity of the i-th measurement, and <l> . is the mean intensity for that reflection.

ftwork =∑| I Fo sl - 1 Fcaicl |/∑| F_obs| , where F_caic and F_obs are the calculated and observed structure factor amplitudes, respectively.

cRf_ree = as for R_WOrk, but was calculated using 10% of data excluded from refinement.

Table 2

List of proteins with sequences that potentially match the region of Hakai from amino acid residues 127 to 189.

HVFCYDCAILHEKKGDKMCPGCSDPVQRIEQCTRGSLFMCSIVQGCKRT

Species YLSQRDLQ AHI HRH

HVFCYDCAILHEKKGDKMCPGCSDPVQRIEQCTRGSLFMCSIVQGCKRT

Homo sapiens (Human) YLSQRDLQ AHINHRH

HVFCYDCAILHEKKGDKMCPGCSDPVQRIEQCTRGSLFMCSIVQGCKRT

Bos taurus (Cattle) YLSQRDLQ AHINHRH

Macaca mulatta (Rhesus HVFCYDCAILHEKKGDKMCPGCSDPVQRIEQCTRGS.LFMCSIVQGCKRT monkey) YLSQRDLQ AHINHRH

Rattus norvegicus (Norway HVFCYDCAILHEKKGD MCPGCSDPVQRIEQCTRGSLFMCSIVQGCKRT rat) YLSQRDLQ AHINHRH

Ailuropoda melanoleuca HVFCYDCAILHEKKGDKMCPGCSDPVQRIEQCTRGSLFMCSIVQGCKRT

(Giant panda) YLSQRDLQ AHINHRH

Mus musculus (House HVFCYDCAILHEKKGDKMCPGCSDPVQRIEQCTRGSLFMCSIVQGCKRT mouse) YLSQRDLQ AHINHRH

HVFCYDCAILHEKKGDKMCPGCSDPVQRIEQCTRGSLFMCSIVQGCKRT

Equus caballus (Horse) YLSQRDLQ AHINHRH

Canis lupus HVFCYDCAILHEKKGDKMCPGCSDPVQRIEQCTRGSLFMCSI QGCKRT familiaris (Dog) YLSQRDLQ AHINHRH

Pan HVFCYDCAILHEKKGDKMCPGCSDPVQRIEQCTRGSLFMCSIVQGCKRT troglodytes (Chimpanzee) YLSQRDLQ AHINHRH

HVFCYDCAILHEKKGDKMCPGCNEPVQRIEQCVRGSLF CSIVQGCKRT

Gallus gallus (Chicken) YLSQRDLQ AHINHRH

Xenopus laevis HVFCYDCALMHEKKADKLCPGTLVEESTDTFKRMSCNDPVQRIEQCARG

(African clawed frog) SLFMCSIV QGCKRTYLSQRDLQAHINHRH

HVFCYDCAVYYEKKCDKMCPGLSLYSCTDPVQRIEQCQRGSLFMC IVQ

Danio rerio (Zebrafish) GC RTYLS QRDLQAHINHRH

HAKAI

Salmo salar (Atlantic HVFCYDCALLHEKKMEKMCPGLTLYSCTDPVQRIEQCLRGLLYMCSIVP salmon) GCKRTYLS QRDLQAHVNHRH

HVFCLSCA

Harpegnathos saltator KREDKVCPRCMEKVSRVEQTGLGTVFMCTHGGTRYGNTGCRRTYLSQRD

(Jerdon's jumping ant) LQAHINHR H

HVFCLSCA—

Camponotus floridanus KREDKVCPRCMEKVSRVEQTGLGTVFMCTHGGTRYGNAGCRRTYLSQRD

(Carpenter ant) LQAHINHR H

Tetraodon nigroviridis HVFCYDCALLHEKKGEKMCPGLTLYNCTDPVQRIEQCQRGSLYMCSVVP

(Green pufferfish) GCKRTYLS QRDLQAHVNHRH

HVFCLRCA

Anopheles darlingi RSETLKMCPRCKEKVVRVEQTALGTVFMCTHGGTRYGNTGCRRTYLSQR

(Mosquito) DLQAHINH' RH

HVFCLKCA

Drosophila grimshawi RAEPIKCCPRCNDKVLRVEQSGLGTVFMCTHGGSRYGSTGCRRTYLSQR

(Fruit fly) DLQAHINH RH ^'

HVFCLSCG

Tribolium castaneum KQEQKQCPRCLEKVSRVEQTGLGTVFMCTHGGTRYGSSGCRRTYLSHRD

(Red flour beetle) LQAHINHR H

Papilio xuthus HVFCLSCA

(Asian swallowtail RSDHTHCPRCREKVLRVEQTGLGTVFMCTHSGTRYGNTGCRRTYLSQRD butterfly) LQAHINHR H

HVFCLSCA

Hydra magnipapillata ENSNGECVRCEERIDRIEPATIGQIFVCSFGGNRNITSGCRRSYLSQRD

(Freshwater hydrozoan) LIAHIRHR H HAFCYHCANLYDKVGYKVCPRCRYPVLRIEAHKRGSVFMCSIVQQCKRT

Species YLSQKSLQ AHIKRRH

ZNF645 HAFCYHCANLYDKVGYKVCPRCRYPVLRIEAHKRGSVFMCSIVQQCKRT

Homo sapiens (Human) YLSQKSLQ AHIKRRH

Macaca fascicularis HAFCYNCANLYDKIGYKICPRCSYPVLRIEEHKRGSVFMCSVVQGCKRT (Cynomolgus Monkey) YLSQKSLQ AHIKRRH

Macaca mulatta HAFCYNCANLYDKIGYKICPRCSYPVLRIEEHKRGSVFMCSVVQGCKRT (Rhesus monkey) .YLSQKSLQ AHIKRRH

Pongo abelii HAFCYDCANLDDKIGYKICPRCRYPVLRIEEHKRGSVFMCSVVPQCKRT (Sumatran orangutan) YLSQKSLQ AHIKRRH .

Table 3

HEADER ^{^} LIGASE -^'09-NOV-ll 3VK6

TITLE CRYSTAL STRUCTURE OF A PHOSPHOTYROSINE BINDING DOMAI

COMPND MOL_ID: 1;

COMPND MOLECULE E3 UBIQUITIN-PROTEIN LIGASE HAKAI ;

COMPND CHAIN: A

COMPND FRAGMENT : PHOSPHOTYROSINE BINDING DOMAIN, UNP RESIDUES 106-

206;

COMPND 5 SYNONYM: CASITAS B-LINEAGE LYMPHOMA-TRANSFORMING SEQUENCE-

LIKE

COMPND 6 PROTEIN 1, . E-CADHERIN BINDING PROTEIN E7, C-CBL-LIKE

PROTEIN 1;

COMPND 7 EC : 6.3.2.- COMPND 8 ENGINEERED: YES

SOURCE MOL_ID: 1;

SOURCE 2 ORGANISM_SCIENTIFIC: MUS .MUSCULUS;

SOURCE ■ 3 ORGANISM_COMMON: MOUSE;

SOURCE 4 ORGANISM_TAXID: 10090;

SOURCE 5 GENE: CBLLl, HAKAI;

SOURCE 6 EXPRESSION_SYSTEM: ESCHERICHIA COLI;

SOURCE 7 EXPRESSION_SYSTEM_TAXID: 562;

SOURCE 8 EXPRESSION_SYSTEM__STRAIN: BL21 (DE3);

SOURCE 9 EXPRESSION_SYSTEM_VECTOR_TYPE: PLASMID;

SOURCE 10 EXPRESSION_SYSTEM_PLASMID: PGEX-6P-1

KEYWDS HYB, PHOSPHOTYROSINE BINDING DOMAIN, LIGASE

EXPDTA X-RAY DIFFRACTION

AUTHOR ^' J.SI ARAMAN, .MUKHERJEE

REVDAT 1 25-JAN-12 3VK6 0

JRNL AUTH

M.MUKHERJEE, S. Y.CHOW, P. YUSOFF, J.SEETHARAMAN, C.NG, S. SINNIAH,

JRNL AUTH 2

X.W.KOH,N.F.M.ASGAR, D.LI, D. YIM, R.A.JACKSON, J. YEW, J.QIAN,

JRNL AUTH A.IYU, Y. P. LIM, X.ZHOU, S.K.SZE, G.R.GUY, J.SIVARAMAN

JRNL TITL STRUCTURE OF A NOVEL PHOSPHOTYROSINE-BINDING

DOMAIN HAKAI

JRNL TITL THAT TARGETS E-CADHERIN

JRNL REF EMBO J. ^' 2012

JRNL REFN ESSN 1460

JRNL DOI 10. I038/EMBOJ.2011.496

REMARK

REMARK RESOLUTION. 1.90 ANGSTROMS.

REMARK

REMARK REFINEMENT. REMARK 3 PROGRAM PHENIX (PHENIX. REFINE: 1.6_289)

REMARK 3 AUTHORS PAUL ADAMS, PAVEL AFONINE, VICENT CHEN, IAN

REMARK 3 DAVIS, KRESHNA GOPAL, RALF GROSSE- ^'

REMARK 3 KUNSTLEVE, LI-WEI- HUNG, ROBERT IMMORMINO,

REMARK 3 TOM IOERGER, AIRLIE MCCOY, ERIK MCKEE, NIGEL

REMARK 3 MORIARTY, REETAL PAI , RANDY READ, JANE

REMARK 3 RICHARDSON, DAVID RICHARDSON, TOD ROMO, JIM

REMARK 3 SACCHETTINI, ICHOLAS SAUTER, JACOB SMITH,

REMARK 3 LAURENT STORONI,TOM TERWILLIGER, PETER

REMARK 3 ZWART

REMARK 3

REMARK 3 REFINEMENT TARGET .: ML

REMARK 3

REMARK 3 DATA USED IN REFINEMENT.

REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) 1.90

REMARK 3 RESOLUTION RANGE LOW _. (ANGSTROMS) 25.23

REMARK ^"3 MIN (FOBS/SIGMA_FOBS) 0.240

REMARK 3 COMPLETENESS FOR RANGE (%) 95.8

REMARK 3 NUMBER OF REFLECTIONS 21478

REMARK 3

REMARK 3 FIT TO DATA USED IN REFINEMENT.

REMARK 3 R VALUE (WORKING + TEST SET)- 0.222

REMARK 3 R VALUE (WORKING SET) 0.219

REMARK 3 FREE R VALUE 0.242

REMARK 3 FREE R VALUE TEST SET SIZE . (%) 10.020

REMARK 3 FREE R VALUE TEST SET COUNT 2153

REMARK 3

REMARK 3 FIT TO DATA USED IN .REFINEMENT (IN BINS) .

REMARK 3 BIN RESOLUTION RANGE COMPL . NWORK NFREE RWORK

RFREE

REMARK 3 1 25.2305 4.0821 0.97 1970 217 0.2219

0.2351

REMARK 3 2 4.0821 3.2422 0.99 1999 225 0.1990

0.2212

REMARK 3 3.2422 2.8330 0.98 1991 226 0.2246

0.2724

REMARK 3 2.8330 2.5743 0.-97 1954 213 0.2294

0.2498

REMARK 3 2.5743 2.3899 0.96 1934 212 0.217-3

0.2349

REMARK 3 2.3899 2.2491 0.96 1947 213 0.2058

0.2143

REMARK 3 7 2.2491 2.1365 0.96 1932 220 0.2156

0.2559

REMARK 3 8 2.1365 2.0436 0.95 1921 216 0.2249

0.2400

REMARK 3 9^' 2.0436 1.9649 0.93 1874 206 0.2372

0.2645

REMARK 3 10 1.9649 1.8971 0.89 1803 205 0.2445

0.2554

REMARK 3

REMARK 3 BULK SOLVENT MODELLING

REMARK 3 METHOD USED FLAT BULK SOLVENT MODEL

REMARK 3 SOLVENT RADIUS 1.11

REMARK 3 SHRINKAGE RADIUS 0.90

REMARK 3 K_SOL 0.42

REMARK 3 B SOL 60.07

REMARK 3 REMARK 3 ERROR ESTIMATES.

REMARK 3 COORDINATE ERROR (MAXIMUM-LIKELIHOOD BASED) 0.230 REMARK 3 PHASE ERROR (DEGREES, MAXIMUM-LIKELIHOOD BASED) 22.600 REMARK 3

REMARK 3 B VALUES.

REMARK 3 FROM WILSON PLOT (A**2) 31.95

REMARK 3 MEAN B VALUE (OVERALL, A**2) 38.94

REMARK 3 OVERALL ANISOTROPIC B VALUE.

REMARK 3 Bll (A**2) 3 .48460

REMARK 3 B22 (A**2)^~ 3 .48460

REMARK 3 B33 (A**2). - 6.96930

REMARK 3 B12 (A**2) 0 .00000

REMARK 3 B13 (A**2) - 0.00000

REMARK 3 B23 (A**2) 0 .00000

REMARK 3

REMARK 3 TWINNING INFORMATION.

REMARK 3 ■FRACTIO : NULL

REMARK 3 OPERATOR: NULL

REMARK 3

REMARK 3 DEVIATIONS FROM IDEAL VALUES

REMARK 3 RMSD COUNT

REMARK 3 BOND : 0.008 771

REMARK 3 ANGLE : 1.172 1031

REMARK 3 CHIRALITY : 0.081 108

REMARK 3 PLANARITY : 0.006 133

REMARK 3 DIHEDRAL : 13.796 297

REMARK 3

REMARK 3 TLS DETAILS

REMARK 3 NUMBER OF TLS GROUPS : NULL

REMARK 3

REMARK 3 NCS DETAILS

REMARK 3 NUMBER OF NCS GROUPS : NULL

REMARK 3

REMARK 3 OTHER REFINEMENT REMARKS: SF FILE CONTAINS FRIEDEL PAIRS. REMARK 4

REMARK 4 3VK6 COMPLIES WITH FORMAT V. 3.30, 13-JUL-ll

REMARK 100

REMARK 100 THIS ENTRY HAS BEEN PROCESSED BY PDBJ ON 22-NOV-ll. ^■ REMARK 100 THE RCSB ID CODE IS RCSB095146.

REMARK 200

REMARK 200 EXPERIMENTAL DETAILS

REMARK 200 EXPERIMENT TYPE X-RAY DIFFRACTION

REMARK 200 DATE OF DATA COLLECTION 20-JUL-10

REMARK 200 TEMPERATURE (KELVIN) 100

REMARK 200 PH

REMARK 200 NUMBER OF CRYSTALS USED

REMARK 200

REMARK 200 SYNCHROTRON (Y/N) Y

REMARK 200 RADIATION SOURCE NSLS

REMARK 200 BEAMLINE X4A

REMARK 200 X-RAY GENERATOR MODEL NULL

REMARK 200 MONOCHROMATIC OR LAUE (M/L) M

REMARK 200 WAVELENGTH OR RANGE (A) 0.979^'

REMARK 200 MONOCHROMATOR SAGITALLY FOCUSED SI (111) REMARK 200 OPTICS NULL

REMARK 200

REMARK 200 DETECTOR TYPE CCD

REMARK 200. DETECTOR MANUFACTURER ADSC QUANTUM 4 REMARK 200 INTENSITY-INTEGRATION SOFTWARE: HKL-2000

REMARK 200 DATA SCALING SOFTWARE HKL-2000

REMARK 200

REMARK 20^'0 NUMBER OF UNIQUE REFLECTIONS 21480

REMARK 200 RESOLUTION RANGE HIGH (A) 1.900

REMARK 200 RESOLUTION RANGE LOW (A) 50.000

REMARK 200 REJECTION CRITERIA (SIGMA(I)) 2.000

REMARK 200

REMARK 200 OVERALL .

REMARK 200 COMPLETENESS FOR RANGE (%) 99.7

REMARK 200 DATA REDUNDANCY ^' 22.800

REMARK 200 R MERGE (I) NULL

REMARK 200 R SYM (I) 0.05200

REMARK 200 <I/SIGMA(I)> FOR THE DATA SET 21.3000

REMARK 200

REMARK 200 IN THE HIGHEST RESOLUTION SHELL.

REMARK 200 HIGHEST RESOLUTION SHELL, RANGE HIGH (A) : 1.90

REMARK 200 HIGHEST RESOLUTION SHELL, RANGE LOW (A) : 1.97

REMARK 200 COMPLETENESS FOR SHELL (%): 99.7

REMARK 200 DATA REDUNDANCY IN SHELL : 22.80

REMARK 200 R MERGE FOR SHELL (I) : NULL

REMARK 200 R SYM FOR SHELL (I): 0.05200

REMARK 200 <I/SIGMA(I)> FOR SHELL : NULL

REMARK 200

REMARK 200 DIFFRACTION PROTOCOL: SINGLE WAVELENGTH

REMARK 200 METHOD USED TO DETERMINE THE STRUCTURE: SAD

REMARK 200 SOFTWARE USED: SOLVE

REMARK 200 STARTING MODEL: NULL

REMARK 200

REMARK 200 REMARK:^■ SF FILE CONTAINS FRIEDEL PAIRS.

REMARK 280

REMARK 280 CRYSTAL

REMARK 280 SOLVENT CONTENT, VS (%): 61.08

REMARK 280 MATTHEWS COEFFICIENT, VM (ANGSTROMS* *3/DA) : 3.16

REMARK 280

REMARK 280 CRYSTALLIZATION CONDITIONS: 200MM LI2S04, 100MM TRIS,

PEG 5000

REMARK 280 MME, PH 8, VAPOR DIFFUSION, HANGING DROP, TEMPERATURE

REMARK 290

REMARK 290 CRYSTALLOGRAPHIC SYMMETRY

REMARK 290 SYMMETRY OPERATORS FOR SPACE GROUP: P 62 2 2

REMARK 290

REMARK 290 SYMOP SYMMETRY

REMARK 290 NNNMMM OPERATOR

REMARK 290 1555 Χ,Υ,Ζ

REMARK 290 2555 -Y,X-Y,Z+2/3

REMARK 290 3555 -X+Y, -X, Z+l/3

REMARK 290 4555 -X,-Y,Z

REMARK 290 5555 Y, -X+Y, Z+2/3

REMARK 290 6555 X-Y, X, Z+l/3

REMARK 290 7555 Y,X, -Z+2/3

REMARK 290 8555 X-Y,-Y,-Z

REMARK 290 9555 -X, -X+Y, -Z+l/3

REMARK 290 10555 -Y,-X, -Z+2/3

REMARK 290 11555 -X+Y,Y,-Z

REMARK 290 12555 - X,X-Y, -Z+l/3

REMARK 290

REMARK 290 WHERE NNN -> OPERATOR NUMBER REMARK 290 MMM -> TRANSLATION VECTOR

REMARK 290

REMARK 290 CRYSTALLOGRAPHIC SYMMETRY TRANSFORMATIONS

REMARK 290 THE FOLLOWING TRANSFORMATIONS OPERATE ON THE ATOM/HETATM REMARK 290 RECORDS IN THIS ENTRY TO PRODUCE CRYSTALLOGRAPHICALLY REMARK 290 RELATED MOLECULES.

REMARK 290 SMTRY1 1 1 .000000 0. .000000_. 0 .000000 0. .00000 REMARK 290 SMTRY2 1 0 .000000 1. .000000 0 .000000 0. .00000 REMARK 290 SMTRY3 1 0 .000000 0. , 000000 1 .000000 0. .00000 REMARK 290 SMTRY1 2 -0 .500000 -0. .866025 0 .000000 0. .00000 REMARK 290 SMTRY2 2 0 .866025 -0. .500000 0 .000000 ο·. .00000 REMARK 290 SMTRY3 2 _.0 .000000 0. .000000 1 .000000 80. .69000 REMARK 290 SMTRY1 3 -0 .500000 0. ,866025 0 .000000 0. .00000 REMARK 290 SMTRY2 3 -0 .866025 -0. .500000 0 .000000 0'. .00000 REMARK 290 SMTRY3 3 0, .000000 0. .000000 1 .000000 40. .34500 REMARK 290 SMTRY1 4 -1, .000000 0. 000000 0 .000000 0. , 00000 REMARK 290 SMTRY2 4 0. .000000 -1. 000000 0. .000000 0. .00000 REMARK 290 SMTRY3 4 0. .000000 0. 000000 1. .000000 0. 00000 REMARK 290 SMTRY1 5 0. .500000 0. 866025 0, .000000 0. 00000 REMARK 290 SMTRY2 5 -0. .866025 ^' 0. 500000 0. .000000 0. 00000 REMARK 290 SMTRY3 5 0. .000000 0. 000000 1. .000000 80. 69000 REMARK 290 SMTRY1 6 0. .500000 -0. 866025 0 , .000000 0. 00000 REMARK 290 SMTRY2 6 0. .866025 0. 500000 0. .000000 0. 00000 REMARK 290 SMTRY3 6 0. .000000 0. 00000.0 1. .000000 40. 34500 REMARK 290 SMTRY1 7 -0. .500000 0. 866025 0. .000000 0. 00000 REMARK 290 SMTRY2 7 0. .866025 0. 500000 0. .000000 0. 00000 ^' REMARK 290 SMTRY3 7 0. .000000 0. 000000 -1. .000000 80. 69000 REMARK 290 SMTRY1 8 1. .000000 0. 000000 0. .000000 0. 00000 REMARK 290 SMTRY2 8 0. , 000000 -1. 000000 0. .000000 Ό. 00000 REMARK 290 SMTRY3. 8 0. ,000000 0. 000000 -1. ,000000 0. 00000 REMARK 290 SMTRY1 ■ 9 -0. .500000 -0. 866025 0. , 000000 0. 00000 REMARK 290 SMTRY2 9 -0. .866025 0. 500000 0. ,000000 0. 00000 REMARK 290 SMTRY3 9 0. 000000 0. 000000 -1. ,000000 40. 34500 REMARK 290 SMTRY1 10 0. 500000 -0. 866025 0. 000000 ^■ 0. 00000 REMARK 290 SMTRY2 10 -0. .866025 -0. 500000 0. , 000000 0. 00000 REMARK 290 SMTRY3 10 0. 000000 0. 000000 -1. ,000000 80. 69000 REMARK 290 SMTRY1 11 -1. 000000 0. 000000 0. 000000 0. 00000 REMARK 290 SMTRY2 11 0: 000000 1. 000000 0. 000000 0. 00000 REMARK 290 SMTRY3 11 0. 000000 0. 000000 -1. 000000 0. 00000 REMARK 290 SMTRY1 12 0. 500000 0. 866025 0. 000000 0. 00000 REMARK 290 SMTRY2 12 0. 866025 -0. 500000 0. 000000 0. 00000 REMARK 290 SMTRY3 12 0. 000000 0. 000000 -1. 000000 40. 34500 REMARK 290

REMARK 290 REMARK: NULL

REMARK 300

REMARK 300 BIOMOLECULE: 1

REMARK 300 SEE REMARK 350 FOR THE AUTHOR PROVIDED AND/OR PROGRAM REMARK 300 GENERATED ASSEMBLY INFORMATION FOR THE STRUCTURE IN REMARK 300 THIS ENTRY. THE REMARK MAY ALSO PROVIDE INFORMATION ON REMARK 300 BURIED SURFACE AREA.

REMARK 350

REMARK 350 COORDINATES FOR A COMPLETE MULTIMER REPRESENTING THE KNOWN REMARK 350 BIOLOGICALLY SIGNIFICANT^' OLIGOMERIZATION STATE OF THE REMARK 350 MOLECULE CAN BE GENERATED BY APPLYING BIOMT TRANSFORMATIONS REMARK 350 GIVEN BELOW. BOTH NON-CRYSTALLOGRAPHIC AND

REMARK 350 CRYSTALLOGRAPHIC OPERATIONS ARE GIVEN.

REMARK 350

REMARK 350 BIOMOLECULE: 1

REMARK 350 AUTHOR DETERMINED BIOLOGICAL UNIT: DIMERIC REMARK 350 SOFTWARE DETERMINED QUATERNARY STRUCTURE: DIMERIC

REMARK 350 SOFTWARE ^· USED ': PISA

REMARK 350 TOTAL BURIED SURFACE AREA: 3680 . ANGSTROM**2

REMARK 350 SURFACE AREA OF THE COMPLEX: 12910 ANGSTROM* *2

REMARK 350 CHANGE IN SOLVENT FREE ENERGY: - 116.0 KCAL/MOL

REMARK 350 APPLY THE FOLLOWING TO CHAINS: A

REMARK 350 BIOMT1 1 1.000000 0.000000 0.000000 0. .00000

REMARK 350 ^' BIOMT2 1 0.000000 1.000000 0.000000 0. .00000

REMARK 350 BIOMT3 1 0.000000 0.000000 1.000000 0. .00000

REMARK 350 BIOMT1 2 0.500000 0.866025 0.000000 0. .00000

REMARK 350 BIOMT2 2 0.866025 -0.500000 0.000000 0. .00000

REMARK 350 BIOMT3 2 0.000000 0.000000 -1.000000 40. .34500

REMARK 375

REMARK 375 SPECIAL POSITION

REMARK 375 THE FOLLOWING ATOMS ARE FOUND TO BE WITHIN 0.15 ANGSTROMS REMARK 375 OF A SYMMETRY RELATED ATOM AND ARE ASSUMED TO BE ON SPECIAL REMARK 375 POSITIONS.

REMARK 375

REMARK 375 ATOM RES CSSEQI

REMARK 375 HOH A 163^' LIES ON A SPECIAL POSITION.

REMARK 375 HOH A 138 LIES ON A SPECIAL POSITION.

REMARK 465

REMARK 465 MISSING RESIDUES

REMARK 465 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE

REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN REMARK 465 IDENTIFIER; SSSEQ=SEQUENCE NUMBER; I=INSERTION CODE.) REMARK 465

REMARK 465 M RES C SSSEQI

REMARK"465 LEU A 97

REMARK 465 GLU A 98

REMARK 465 ^■ ASN A 99

REMARK 465 VAL A 100 ^' .

REMARK 465 HIS A 101

REMARK 470

REMARK 470 MISSING ATOM

REMARK 470 THE FOLLOWING RESIDUES HAVE MISSING ATOMS (M=MODEL NUMBER; REMARK 470 RES=RESIDUE NAME; C=CHAIN IDENTIFIER; SSEQ=SEQUENCE NUMBER; REMARK 470 I=INSERTION CODE) :

REMARK 470 M RES CSSEQI ATOMS

REMARK 470 ALA A 95 CB

REMARK 500

REMARK 500 GEOMETRY AND STEREOCHEMISTRY

REMARK 500 SUBTOPIC: TORSION ANGLES

REMARK 500

REMARK 500 TORSION ANGLES OUTSIDE THE EXPECTED RAMACHANDRAN REGIONS: REMARK 500 (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN IDENTIFIER; REMARK 500 SSEQ=SEQUENCE NUMBER; I=INSERTION CODE) .

REMARK 500

REMARK 500 STANDARD TABLE:

REMARK 500 FORMAT : ( 10X, 13 , IX, A3, IX, Al , 1 , Al , 4X, F7.2 , 3X, F7.2 )

REMARK 500

REMARK 500 EXPECTED VALUES: GJ KLEYWEGT AND TA JONES (1996). PHI/PSI- REMARK 500 CHOLOGY: RAMACHANDRAN REVISITED. STRUCTURE 4, 1395 - 1400 REMARK 500

REMARK 500 M RES CSSEQI PSI PHI

REMARK 500 ALA A 95 53.06 -55.19

REMARK 500

REMARK 500 REMARK: NULL

REMARK 620 REMARK- 620 METAL COORDINATION- REMARK 620 (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN IDENTIFIER; REMARK 620_, SSEQ=SEQUENCE NUMBER; I=INSERTION CODE) :

REMARK 620

REMARK 620 COORDINATION ANGLES FOR: M RES CSSEQI METAL

REMARK 620 ^' ZN A 104 ZN

REMARK 620 N RES CSSEQI ATOM

REMARK 620 1 HIS A 22 ND1

REMARK 620 2 CYS A 40 SG 117. .5

REMARK- 620 3 CYS A 20 SG 103. .9 105.8

REMARK 620 4 CYS A 43 SG 107. .3 108.9

REMARK 620 N 1 2- 3

REMARK 620

REMARK 620 COORDINATION ANGLES FOR: M RES CSSEQI METAL

REMARK 620 ZN A 102 ZN

REMARK 620 N RES CSSEQI ATOM

REMARK 620 1 HIS A 80 NE2

REMARK 620 2 HIS A 85 NE^'2 101.8

REMARK 620 3 CYS A 67 SG 107.5 116.8

REMARK 620 N 1 2 ..

REMARK 620

REMARK 620 COORDINATION ANGLES FOR: M RES CSSEQI METAL

REMARK 620 ZN A 103 ZN

REMARK 620 N RES CSSEQI ATOM

^'REMARK 620 1 CYS A 7 SG

REMARK 620 2 CYS A ' 25 SG 112 .9

REMARK 620 3 CYS A 4 SG 104 .5 107. 2

REMARK 620 4 CYS A 28 SG 111 .0 110. 3

REMARK 620 N 1 2

REMARK 800

REMARK 800 SITE

REMARK 800 SITE IDENTIFIER: AC1

REMARK 8.00 EVIDENCE CODE: SOFTWARE

REMARK 800 SITE DESCRIPTION: BINDING SITE FOR RESIDUE ZN A

REMARK 800

REMARK 800 SITE IDENTIFIER: AC2

REMARK 800 EVIDENCE^' CODE: SOFTWARE

REMARK 800 SITE DESCRIPTION: . BINDING SITE FOR RESIDUE ZN A

REMARK 800

REMARK 800 SITE IDENTIFIER: AC3

REMARK 800 EVIDENCE CODE: SOFTWARE

REMARK 800 SITE DESCRIPTION: BINDING SITE FOR RESIDUE ZN A

DBREF 3VK6 A 1 101 UNP Q9JIY2 HAKAI MOUSE 106 206

SEQRES 1 A 101 VAL HIS PHE CYS ASP LYS CYS GLY LEU PRO ILE LYS VAL

SEQRES 2 A 101 TYR GLY ARG MET ILE PRO CYS LYS HIS VAL PHE CYS TYR

SEQRES 3 101 ASP CYS ALA ILE LEU HIS GLU LYS LYS GLY ASP LYS MET

SEQRES 4 A^" 101 CYS PRO GLY CYS SER ASP PRO VAL GLN ARG ILE GLU GLN

SEQRES 5 A 101 CYS THR ARG GLY SER LEU PHE MET CYS SER ILE VAL GLN

SEQRES 6 A 101 GLY CYS LYS ARG THR TYR LEU SER GLN ARG ASP LEU GLN

SEQRES 7 A 101 ALA HIS ILE AS HIS ARG HIS MET ARG ALA GLY LYS PRO

SEQRES 8 A 101 VAL THR ARG ALA SER LEU GLU^' ASN VAL HIS

HET ZN A 102 1

HET ZN ^"A 103 1

HET ZN A 104 1

HETNAM ZN ZINC ION

FORMUL 2 ZN 3 ( ZN 2+)

FORMUL 5 HOH *70(H2 O)

HELIX 1 1 TYR A 26 LYS A 35 1 10

HELIX 2 GLY A 56 LEU A 58

3

HELIX 3 SER A 73 HIS A 85

13

SHEET 1 A 3 VAL A 23 CYS A 25 0

SHEET 2 A 3 VAL A 13 ILE A 18 -1 N GLY A 15 PHE A 24

SHEET 3 A 3 ARG A 49 THR A 54 -1 O CYS A 53 TYR A 14

LINK ND1 HIS A 22 ZN ZN A 104 1555

1555 .98

LINK NE2 HIS A 80 ZN Z A 102 1555

1555 .08

LINK NE2 HIS A 85 ZN ZN A 102 • 1555

1555 .12

LINK SG CYS A 40 ZN ZN A 104 1555

1555 .29

LINK SG CYS A 20 ZN ZN A 104 1555

1555 .32

LINK SG CYS A 67 ZN ZN A 102 1555

1555 .33

LINK SG CYS A 7 ZN ZN A 103 1555

1555 .33

LINK SG CYS A 25 ZN ZN A 103 1555

1555 ,35

LINK SG CYS A 43 ZN ZN A 104 1555 ^■

1555 .36

LINK SG CYS A 4 ZN ZN A 103 1555

1555 .38

LINK SG CYS A 28 ZN ZN A 103 1555

1555 39

CISPEP 1 ILE A 18 PRO A 19 0 9.66

SITE 1 AC1 4 CYS A 61 CYS A 67 HIS A 80 HIS A 85

SITE^' 1. AC2 4 CYS A 4 CYS A 7 CYS A 25 CYS A 28

SITE 1 AC3 4. CYS A 20 HIS A 22 CYS A 40 CYS A 43

CRYST1 64.657 64.657 121.035 90.00 90.00 120.00 P 62 2 2 12

0RIGX1 1.000000 0.000000 0.000000 0.00000

ORIGX2 0.000000 1.000000 0.000000 0.00000

ORIGX3 0.000000 0.000000 1.000000 0.00000

SCALE1 0.015466 0.008929 0.000000 0.00000

SCALE2 0.000000 0.017859 0.000000 0.00000

SCALE3 0.000000 0.000000 0.008262 0.00000

ATOM N VAL A 1 14.217 -31.908 31.023 1.00 43.24

N

ATOM CA VAL A 1 -13.680 -32.592 29.844 1.00 38.28 C

ATOM C VAL A 1 -13.920 -31.761 28.590 1.00 39.47 C .

ATOM O VAL A 1 -15.044 -31.691 28.097 1.00 45.65 0

ATOM CB VAL A 1 -14.318 -34.009 29.682 1.00 42.95 C

ATOM CGI VAL A ■ 1 -13.873 -34.673 28.399 1.00 40.15 C

ATOM 7 CG2 VAL A 1 -13.966 -34.909 30.880 1.00 42.58 C

ATOM 8 N HIS A 2 -12.866 -31.123 28.088 1.00 34.05 N

ATOM 9 CA HIS A 2 -12.914 -30.352 26.861 0.98 37.02 C

ATOM 10 C HIS A 2 11.919 -30.960 25.866 1.00 34.85 C

ATOM 11 0 HIS A 2 10.717 -31.057 26.161 1.00 32.70 0

ATOM 12 CB HIS A 2 12.537 -28.897 27.149 1.00 36.14 C

ATOM 13 CG HIS A 2 12.569 -28.009 25.947 0.64 37.61 C

ATOM 14 ND1 HIS A 2 13.311 -28.301 24.823 0.20.37.81 N

ATOM 15 CD2 HIS^' A 2 11.962 -26.823 25.700 0.30 38.48 c -

ATOM 16 CE1 HIS A 2 13.151 -27.339 23.931 0.76 39.37 C

ATOM 17 NE2 HIS A 2 12.341 -26.428 24.440 0.38 39.67 N

ATOM 18 N PHE A 3 12.411 -31.379 2 .702 1.00 32.80 N

ATOM 19 CA PHE A 3 11.522 -31.929 23.678 1.00 32.28 C

ATOM 20 C PHE A 3 10.999 -30.855 22.739 1.00 35.87 C

ATOM 21 O PHE A 3 11.754 -29.978 22.295 1.00 34.57 0

ATOM 22 CB PHE A 3 12.233 -33.040 22.888 1.00 33.26 C

ATOM 23 CG ^' PHE A 3 12.316 -34.336 23.622 1.00 33.39 C

ATOM 24 CD1 PHE A 3 13.262 -34.525 24.623 1.00 35.97 C

ATOM 25 CD2 PHE A 3 11.451 -35.372 23.326 1.00 32.57 C

ATOM 26 CE1 PHE A 3 13.334 -35.723 25.305 1.00 35.87 C

ATOM 27 CE2 PHE A 3 11.524 -36.571 24.015 1.00 31.51

C^■

ATOM 28 CZ PHE A 3 12.467 -36.746 24.994 1.00 35.56 C

ATOM 29 N CYS A^' 4 -9.710 -30.929 22.426 1.00 32.09 N

ATOM 30 CA CYS A 4_. -9.109 -30.014 21.473 1.00 32.02 C

ATOM 31 C CYS A 4 -9.560 -30.347 20.055 1.00 35.66 C

ATOM 32 O CYS A 4 -9.502 -31.506 19.642 1.00 32.63 0

ATOM 33 CB CYS A 4 -7.591 -30.073 21.550 1.00 31.91 C

ATOM 34 SG CYS A 4 -6.800 -28.933 20.409 1.00 31.07 S

ATOM 35 N ASP A 5 10.005 -29.334 19.310 1.00 35.12 N

ATOM 36 CA ASP A 5 10.470 -29.558 17.937 1.00 35.99 C

ATOM 37 C ASP A 5 -9.344 -29.551 16.921 1.00 38.96 C ATOM 38 0 ASP A 5 -9.578 -29.737 15.724 1.00 37.64 0

ATOM 39 CB ASP A 5 -11.572 -28.568 17.529 1.00 41.25 C

ATOM 40 CG ASP A 5 -11.108 -27.117 17.538 1.00 43.64 C

ATOM 41^' GDI ASP A 5 -9.894 -26.855 17.668 1.00 41.44 Ό

ATOM 42 OD2 ASP A 5 -11.981 -26.227 17.403 1.00 50.17 O

ATOM 43 N LYS A 6 -8.123 -29.340 1.7.398 1.00 36.54 N

ATOM 44 CA LYS A 6 -6.946 -29. 20 16.542 1.00 37.90 C

ATOM 45 C LYS A 6 -6.322 -30.798 16.637 1.00 39.23 C

ATOM 46 0 LYS A 6 -6.194 -31.513 15.637 1.00 37.67 0

ATOM 47 CB LYS A 6 -5.901 -28.389 16.971 1.00 37.88 C

ATOM 48 CG LYS A 6 -5.696 -27.224 16.022 1.00 46.51 C

ATOM 49 CD LYS A 6 -6.967 -26.414 15.818 1.00 47.95 C

ATOM 50 CE LYS A 6 -7.667 -26.817 14.525 1.00 48.63 C

ATOM 51 NZ LYS A 6 -6.673 -27.052 13.439 1.00 49.54 N

ATOM 52 N CYS A 7 -5.921 -31.164 17.850 1.00 34.08 N

ATOM 53 CA CYS A 7 -5.224 -32.427 18.071 1.00 34.54 C

ATOM 54 C CYS A 7 -6.175 -33.574 18.410 1.00 35.78 C

ATOM 55 0 CYS A 7 -5.827 -34.734 18.246 1.00 37.45 0

ATOM 56 CB CYS A 7 -4.139 -32.280 19.151 1.00 33.57 C

ATOM 57 SG CYS A 7 -4.745 -32.011 20.856 1.00 31.74 S

ATOM 58 N GLY A 8 -7.367 -33.251 18.901 1.00 31.74 N

ATOM 59 CA GLY A 8 -8.353 -34.265 19.226 1.00 31.07 C

ATOM 60 C GLY A 8 -8.282 -34.867 20.616 1.00 31.77 C

ATOM 61 0 GLY A 8 -9.075 -35.742 20.951 1.00 33.42 0

ATOM 62 N LEU A 9 -7.351 -3 .397 21.441 l.^'OO 27.25 N

ATOM 63 CA LEU A 9 -7.193 -34.943 22.779 1.00 28.34 C

ATOM 64 C ^■ LEU A 9 -7.833 -34.036 23.842 1.00 31.76 C

ATOM 65■ 0 LEU A 9 -7.957 -32.820 23.643 1.00 27.98 0

ATOM 66 CB LEU A ^' 9 -5.723 -35.158 23.107 1.00 29.93 C ATOM 67 CG LEU A 9 4.938 -36.179 22.267 1.00 29.89 C

ATOM 68 CD1 LEU A 9 3.457 -36.036 22.586 1.00 31.72 C

ATOM 69 CD2 LEU A 9 5.408 -37.611 22.531 1.00 28.30 C

ATOM 70 N PRO A 10 8.250 -34.640 24.964 1.00 31.04 N

ATOM 71 CA PRO A 10 8.829 -33.883 26.078 1.00 32.07 C

ATOM 72 C PRO A 10 7.798 -32.927 26.647 1.00 34.78 C

ATOM 73 O PRO A 10 6.625 -33.283 26.803 1.00 33.68 0 .

ATOM 74 CB PRO A 10 9.221 -3 .965 27.092 1..00 35.67 C

ATOM 75 CG PRO A 10 8.466 -36.187 26.703 1.00 31.83 C

ATOM 76 CD PRO A 10 8.201 -36.089 25.225 1.00 32.04 C

ATOM 77 N ILE A 11 8.223 -31.699 26. 19 1.00 31.68 N

ATOM 78 CA ILE A 11 7.300 -30.668 27.362 1.00 31.74 C

ATOM 79 C ILE A 11 7.214 -30.674 28.888 1.00 34.92 C

ATOM 80 O ILE A 11 8.236 -30.642 29.584 1.00 34.62 0

ATOM 81 CB ILE A 11 7.750 -29.276 26, 884 1.00 32.66 C

ATOM 82 CGI ILE A 11 7.891 -29.260 25.355 1.00 30.36 C

ATOM 83 CG2 ILE A 11 6.774 -28.194 27.351 1.00 32.29 C

ATOM 84 CD1 ILE A 11 8.789 -28.127 24.849 1.00 34.02 C

ATOM 85 N LYS A 12 5.992 -30.728 29.397 1.00 34.77 N

ATOM 86 CA LYS A 12 5.780 -30.600 30.832 1.00 39.37 C

ATOM 87 C LYS A 12 5.033 -29.313 31.165 1.00 37.98 C

ATOM 88 O LYS A 12 5.190 -28.761 32.262 1.00 39.91 0

ATOM 89 CB LYS A 12 5.066 -31.832 31.402 1.00 45.11 C

ATOM 90 CG LYS A 12 3.700 -32.098 30.818 1.00 43.92 C

ATOM 91 CD LYS A 12 3.607 -33.512 30.238 1.00 50.37 C

ATOM 92 CE LYS A 12 2.198 -33.790 29.715 1.00 48.20 C

ATOM 93 NZ LYS A. 12 1.625 -32.564 29.075- 1.00 48.39 N

ATOM 94 N VAL A 13 4.236 -28.826 30.222 1.00 33.60 N

ATOM 95 CA VAL A 13 3.549 -27.553 30.399 1.00 35.83 C ATOM 96 C VAL A ^'13 3.859 -26.618 29.238 1.00 35.15 C

.ATOM 97 0 VAL A 13 3.737 -27.019 28.083 1.00 32.14 0

ATOM 98 CB VAL A 13 2.019 -27.736 30.490 1.00 37.74 C

ATOM 99 CGI VAL A 13 1.327 -26.386 30.600 1.00 36.88 C

ATOM 100 CG2 VAL A 13 1.657 -28.615 31.690 1.00 40.88 C

ATOM 101 N TYR A 14 4.239 -25.377 29.541 1.00 34.26 N

ATOM 102 CA TYR- A 14 4.574 -24.398 28.495 1.00.34.13 C

ATOM 103 C TYR A 14 3.478 -23.392 28.262 1.00 33.08 C

ATOM 104 O TYR A 14 2.719 -23.066 29.170 1.00 33.96 0

ATOM 105 CB TYR A 14 5.839 -23.608 28.849 1.00 31.93 C

ATOM 106 CG TYR A 14 7.105 -24.402 28.787 1.00 33.92 C

■ATOM 107 CD1 TYR A 14 7.898 -24.396 27.636 1.00 33.65 C

ATOM 108 CD2 TYR A 14 7.518 -25.148 29.866 1.00 36.49 C

ATOM 109 CE1 TYR A 14 9.063 -25.126 27.583 1.00 36.26 C

^•ATOM 110 CE2 TYR A 14 8.667 -25.874 29.822 1.00 40.39 C .

ATOM 111 CZ TYR A 14 9.435 -25.866 28.680 1.00 37.92 C

ATOM 112 OH TYR A 14 0.591 -26.602 28.660 1.00 45.77 O

ATOM 113 N GLY A 15 3.417 -22.880 27.035 1...00 34.07 N

ATOM 114 CA GLY A 15 2.627 -21.704 26.737 1.00 33.20 C

ATOM 115 C GLY A 15 3.575 -20.518 26.672 1.00 32.07 C

ATOM 116 O GLY A 15 4.593 -20.571 25.974 1.00 33.11 0

ATOM 117 N ARG A 16 3.271 -19.468 27.423 1,00 32.41 N

ATOM 118 CA ARG A 16 4.098 -18.265 27.432 1.00 29.19 C

ATOM 119 C ARG A 16 3.351 -17.200 26.646 1.0,0 33.70 C

ATOM 120 O ARG A 16 2.185 -16.905 26.933 1.00 33.66 0

ATOM 121 CB ARG A 16 4.380 -17.818 28.863 1.00 33.11 C

ATOM 122 CG ARG A 16 5.149 -16.514 29.013 1.00 29.75 C

ATOM 123 CD ARG A 16 5.644 -16.415 30.460 1.00 32.31 C

ATOM 124 NE ARG A 16 6.408 -15.202 30.765 1.00 31.60 N ATOM 125 . CZ ARG A 16 5.905 -14.131 31.373 1.00 33.56 C

ATOM 126 NH1 ARG A 16 4.625 -14.105 31.726 1.00 33.52 N

ATOM 127 NH2 ARG A 16 6.683 -13.080 31.627 1.00 30.00 N

ATOM 128 N MET A 17 3.993 -16.675 25.605 1.00 30.01 N

ATOM 129 CA MET A 17 3.333 -15.700 24.758 1.00 29.69 C

ATOM 130 ^' C MET A 17 3.315 -14.355 25.468 1.00 32.77 C

ATOM 131 O MET A 17 4.326 -13. 24 26.014 1.00 34.13 0

ATOM 132 CB MET A 17 4.073 -15.569 23.410 1.00 31.74 C

ATOM 133 CG MET A 17 4.284 -16.884 22.634 1.00 30.41 C

ATOM 134 SD MET A 17 2.796 -17.730 22.031 1.00 30.02 S

ATOM 135 CE MET A 17 2.728 -18.978 23.313 1.00 33.13 C

ATOM 136 N ILE A 18 2.174 -13.682 25.461 1.00 33.95 N

ATOM 137 CA ILE A 18 2.129 -12.317 25.961 1.00 36.17 C

ATOM 138 C ILE A 18 1.858 -11.383 24.774 1.00 37.58 C

ATOM 139 0 ILE A 18 0.904 -11.614 24.032 1.00 37.31- 0

ATOM 140 CB ILE A 18 1.041 -12.163 27.041 1.00 37.48 C

ATOM 141 CGI ILE A 18 1.304 -13.127 28.214 1.00 36.97 C

ATOM 142 CG2 ILE A 18 0.929 -10.717 27.512 1.00 40.45 C

ATOM 143 CD1 ILE A 18 2.556 -12.814 29.034 1.00 37.15 C

ATOM 144 N PRO A 19 2.657 -10.297 24.624 1.00 36.04 N

ATOM 145 CA PRO A 19 3.649 -9.786 25.581 1.00 34.39 C

ATOM 146 C PRO A 19 5.128 -10.060 25.277 I.00 36.76 C

ATOM 147 O PRO A 19 5.988 -9.586 26.035 1.00 37.23 0

ATOM 148 CB PRO A 19 3.401 -8.276 25.533 1.00 41.24 C

ATOM 149 CG, PRO A 19 2.984 -8.020 24.101 1.00 38.16 C

ATOM 150 CD PRO A 19 2.380 -9.311 23.561 1.00 39.06 C

ATOM 151 N CYS A 20 5.437 -10.817 24.230 1.00 33.89 N

ATOM 152 CA CYS A 20 6.840 -11.^"018 23.866 1.00 35.21 C

ATOM 153 C CYS A 20 7.598 -11.950 24.829 1.00 33.91 C ATOM 154 0 GYS A 20 -8.834 -11.931 24.875 1.00 31.07 O

ATOM 155 CB CYS A 20 -6.960 -11.494 22.416 1.00 33.97 C

ATOM 156 SG CYS A 20 -6.504 -13,233 22.179 1.00 33.10^' S

ATOM 157 N LYS A 21 -6.845 -12.756 25.584 1.00 29.78 N

ATOM 158 CA LYS A 21 -7.359 -13.601 26.673 1.00 30.24 C

ATOM 159 C . LYS A 21 -8.178 -14.830 26.280 1.00 28.76 C

ATOM 160 O LYS A 21 -8.805 -15.463 27.142 1.00 27.96 0

ATOM 161 CB LYS A 21 -8.105 -12.768 27.722 1.00 28.56 C

ATOM^' 162 CG LYS A ^' 21 -7.219 -11.732 28.417 1.00 32.15 C

ATOM 163 CD LYS A 21 -8.070 -10.813 29.302 1.00 33.23 C

ATOM 164 CE LYS A 21 -7.292 -9.571 29.723 1.00 37.90 C

ATOM 165 NZ LYS A 21 -6.807 -8.785 28.560 1.00 38.35 N

ATOM 166 N HIS A 22 -8.181 -15.164 24.996 1.00 27.92 N .

ATOM 167 CA HIS A 22 -8.771 -16.428 24.551 1.00 27.15 C

ATOM 168 C HIS A 22 -7.908 -17.626 24.979 1.00 30.80 C

ATOM 169 O HIS A 22 -6.697 -17.481 25.177 1.00 30.10 O

ATOM 170 CB HIS A 22 -8.999 -16.415 23.046 1.00 29.51 C

ATOM 171 CG HIS A 22 -9.983 -15.372 22.615 1.00 30.39 C

ATOM 172 ND1 HIS A 22 -9.667 -14.373 21.717 1.00 30.28 N

ATOM 173 CD2 HIS A 22 -11.267 -15.152 22.988 1.00 30.99 C

ATOM 174 CE1 HIS A 22 -10.719 -13.585 21.550 1.00 30.62 C

ATOM 175 NE2 HIS A 22 -11.-705 -1 .037 22.300 1.00 29.25 N

ATOM 176 N VAL A 23 -8.522 -18.795 25.139 1.00 29.80 N

ATOM 177 CA VAL A 23 -7.745 -19.968 25.549 1.00 30.93 C

ATOM 178 C VAL A 23 -7.891 -21.135 24.590 1.00 33.12 C ^"

ATOM 179 O VAL A 23 -8.924 -21.304 23.914 1.00 29.29 0

ATOM 180 CB VAL A 23 -8.066 -20.441 26.987 1.00 29.60 C

ATOM 181 CGI VAL A 23 -7.597 -19.396 28.018 1.00 31.89 C

ATOM 182 CG2 VAL A 23 -9.542 -20.760 27.128 1.00 ^' 31.43 C ATOM 183 N PHE A 24 6.837 -21.940 24.536 Γ.00 30.81 N

ATOM 184 CA PHE A 24 6.787 -23.063 23.609 1.00 32.98 C

ATOM 185 C PHE A 24 5.949 -2 .168 24.244 1.00 34.89

C ,

ATOM 186 0 PHE A 24 5.245 -23.931 25.215 1.00 32.00 0

ATOM 187 CB PHE A 24 6.151 -22.618 22.295 1.00 33.83 C

ATOM 188 · CG PHE A ^■ 24 6.855 -21.446 21.658 1.00 34.75 C

ATOM 189 CD1 PHE A 24 7.930 -21.652 20.808 1.00 34.89

ATOM 190 CD2 PHE A 24 6.467 -20.148 21.950 1.00 34.03 C

ATOM 191 CE1 PHE A 24 8.602 -20.580 20.243 1.00 36.57 C

ATOM 192 CE2 PHE A 24 7.137 -19.059 21.379 1.00 34.37 C

ATOM 193 CZ PHE A 24 8.205 -19.286 20.532 1.00 33.08 C

ATOM 194 N CYS A 25 6.041 -25.366 23.685 1.00 33.09 N

ATOM 195 CA CYS A 25 5.190 -26.478 24.075 1.00 31.38 C

ATOM 196 C CYS A 25 3.748 -25.974 24.184 1.00 32.44 C

ATOM 197 0 CYS A 25 3.233 -25.376 23.252 1.00 32.95 O

ATOM 198 CB CYS A 25 5.309 -27.549 22.998 1.00- 31.93 C

ATOM 199 SG CYS A 25 4.058 -28.857 23.054 1.00 32.02 S

ATOM 200 N TYR A 26 3.098 -26.187 25.326 1.00 33.66 N

ATOM 201 CA TYR A 26 1.759 -25.617 25.515 1.00 35.02 C

ATOM 202 C TYR A 26 0.730 -26.142 24.505 1.00 37.39 C

ATOM 203 O TYR A 26 0.067 -25.376 23.950 1.00 35.25 0

ATOM 204 CB TYR A 26 1.251 -25.843 26.943 1.00 37.34 C

ATOM 205 CG TYR A 26 0.099 -25.208 27.180 1.00 37.27 C

ATOM 206 CD1 TYR A 26 0.221 -23.835 27.302 1.00 37.64 C

ATOM 207 CD2 TYR A 26 1.252 -25.981 27.267 1.00 41.59 C

ATOM 208 CE1 TYR A 26 1.444 -23.242 27.511 1.00 41.92 C

ATOM 209 CE2 TYR A 26 2.484 -25.397 27.469 1.00 42.21 C■

ATOM 210 CZ TYR A 26 2.574 -24.023 27.588 1.00 43.26 C

ATOM 211 OH TYR A 26 3.792 -23.415 27.799 1.00 48.53 0 ATOM 212 N ASP A 27 -0.740 -27.453 24.280 1.00 35.01 N

ATOM 213 CA ASP A 27 0.244 -28.080 23.405 1, 00 38.59 C ^■

ATOM 214 C ASP A 27 0.126 -27.552 21.973 1.00 38.88 C

ATOM 215 0 ASP A 27 1.127 -27.240 21.320 1.00 40.15 O.

ATOM 216 CB ASP A 27 0.09^'5 -29.603 23. 60 1.00 38.84 C

ATOM 217 CG ASP A 27 0.478 -30.161 24.813 1.00 42.03 C

ATOM 218 OD1 ASP A 27 1.639 -29.978 25.203 1.00 45.14 0

ATOM 219 OD2 ASP A 27 -0.374 -30.757 25.503 1.00 45.01 O

ATOM 220 N CYS A 28 -1.104 -27.410 21.500 1.00 36.98 N

ATOM 221 CA CYS A 28 -1.320 -26.867 20.167 1.00 38.40 C

ATOM 222 C CYS A 28 -0.965 -25.384 20.063 1.00 41.43 C

ATOM 223 0 CYS A 28 -0.406 -24.945 19.056 1.00 45.87 0

ATOM 224 CB CYS A 28 -2.746 -27.135 19.695 1.00 34.77 C

ATOM 225 SG CYS A 28 -3.035 -28.883 19.302 1.00 35.70 S

ATOM 226 N ALA A 29 -1.289 -24.615 21.098 1.00 38.78 N

ATOM 227 CA ALA A 29 -0.875 -23.218 21.160 1.00 39.90 C

ATOM 228 C ALA A 29 0.650 -23.096 21.014 1.00 45.54 C

ATOM 229 0 ALA A 29 1.139 -22.325 20.188 1.00 47.21 0

ATOM 230 CB ALA A 29 -1.353 -22.575 22.460 1.00 37.71 C

ATOM 231 N ILE A 30 1.397 -23.860 21.809 1.00 45.46 N

ATOM 232 CA ILE A 30 2.859 -23.828 21.732 1.00 45.69 C

ATOM 233 C ILE A 30 3.340 -24.250 20.337 1.00 51.05 C

ATOM 234 O ILE A 30 4.131 -23.558 19.697 Γ.00 53.45 0

ATOM 235 CB ILE A 30 3.508 -24.733 22.804 1.00· 45.45 C

ATOM 236 CGI ILE A 30 3.182 -24.228 24.211 1.00 46.94 C

ATOM 237 CG2 ILE A 30 5.012 -24.816 22.605 1.00 51.64 C

ATOM 238 CD1 ILE A 30 3.496 -22.761 24.437 1.00 46.56 C

ATOM 239 N LEU A 31 2.847 -25.389 19.868 1.00 49.30 N

ATOM 240 CA LEU A 31 3.188 -25.887 18.546 1.00 51.97 C ATOM 241 C ^■ LEU A 31 3.024 -2 .789 17.501 1.00 55.25 C

ATOM 242- 0. LEU A 31 3.919 -24.543 16.689 1.00 58.11 0

ATOM 243 CB LEU A 31 2.300 -27.086 18.196 1.00 50.87 C

ATOM 244 CG LEU A 31 2.633 -27.802 16.891 1.00 53.86 C

ATOM 245 CD1 LEU A 31 3.997 -28.479 17.002 1.00 53.57 C

ATOM 246 CD2 LEU A 31 1.552 -28.812 16.541 1.00 53.40 C

ATOM 247 N HIS A 32 1.865 -24.139 17.535 1.00 52.60 N

ATOM 248 CA HIS A 32 1.534 -23.028 16.650 1.00 56.03 C

ATOM 249 C HIS A 32 2.584 -21.915 16.792 1.00 56.91 C

ATOM 250 O HIS A 32 3.056 -21.360 15.800 1.00 57.85 0

ATOM 251 CB HIS A 32 0.136 -22.516 17.016 1.00 53.15 C

ATOM 252 CG HIS A 32 -0.545 -21.731 15.938 1.00 59.33 C■

ATOM 253 ND1 HIS A 32 -1.025 -20.454 16.140 1.00 59.34 N

ATOM 254. CD2 HIS A 32 -0.857 -22.051 14.658 1.00 62.21 C

ATOM 255 CE1 HIS A 32 -1.590 -20.014 15.029 1.00 56.20 C

ATOM 256 NE2 HIS A 32 -1.501 -20.964 1 .114 1.00 63.21 N

ATOM 257 N GLU A 33 2.950 -21.606 18.035 1.00 54.43 N

ATOM 258 CA GLU A 33 3.949 -20.580 18.329 1.00 55.34 C

ATOM 259 C GLU A 33 5.322 -20.956 17.767 1.00 58.50 C

ATOM 260 0 GLU A 33 6.002 -20.128 17.158 1.00 57.87 0

ATOM 2-61 CB GLU A 33 4.038 -20.352 19.839 1.00 54.98 C

ATOM 262 CG GLU A 33 5.090 -19.351 20.282 1.00 52.81 C ^■

ATOM 263 CD GLU A 33 5.094 -19.153 21.786 1.00 53.52 C

ATOM 264 OE1 GLU A 33 5.323 -20.138 22.521 1.00 5 .05 0

ATOM 265 OE2 GLU A 33 4.869 -18.012 22.240 1.00 52.87 0

ATOM 266 N LYS A 34 5.712 -22.213 17.971 1.00 57.95 N

ATOM 267 CA LYS A 34 6.956 -22.746 17.423 1.00 59.98 C

ATOM 268 C LYS A 34 7.040 -22.564 15.908 1.00 61.96 C

ATOM 269 0 LYS A 34 8.125 -22.392 15.353 1.00 63.76 0 ATOM 270 CB LYS A- 34 7.107 -24.233 17.769 1.00 58.57 C

ATOM 271 CG . LYS A 34 8.312 -24.889 17.105 1.00 64.23 C - ATOM 272 CD LYS A 34 8.291 -26.412 17.221 1.00 66,49 C

ATOM 273 CE LYS A 34 9.352 -27.042 16.315 1.00 67.32 C

ATOM 274 NZ LYS A 34 9.274 -28.534 16.270 1.00 66.12 N

ATOM 275 N LYS A 35 5.891 -22.603 15.243 1.00 61.93 N

ATOM 276 CA LYS A 35 5.841 -22.520 13.790 1.00 60.26 C

ATOM 277 C LYS A 35 5.967 -21.084 13.286 1.00 61.88 C

ATOM 278 O LYS A 35 6.106 -20.851 12.086 1.00 63.28 0

ATOM 279 CB LYS A 35 4.539 -23.137 13.287 1.00 61.91 C

ATOM 280 CG LYS A 35 4.479 -23.386 11.795 1.00 65.10 C

ATOM 281 CD LYS A 35 3.206 -24.133 11.449 1.00 66.63 C

ATOM 282 CE LYS A 35 2.878 -25.173 12.524 1.00 66.79 C

ATOM- 283 NZ LYS A 35 3.998 -26.131 12.772 1.00 67.01 N

ATOM 284 N GLY A 36 5.908 -20.125 1 .204 1.00 61.73 N

ATOM 285 CA GLY A ' 36 6.056 -18.722 13.854 1.00 62.76 C

ATOM 286 C GLY A 36 4.758 -17.930 13.844 1.00 61.41 C

ATOM 287 O GLY A 36 4.731 -16.747 13.479 1.00 60.53 0

ATOM 288 N ASP A 37 3.672 -18.574 14.253 1.00 59.63 N

ATOM 289 CA ASP A 37 2.375 -17.916 14.231 1.00 56.51 C

ATOM 290 C ASP A 37 2.220 -16.999 15.437 1.00 54.20 C

ATOM 291 O ASP A 37 2.408 -17.419 16.576 1.00 55.12 0

ATOM 292 CB ASP A 37 1.252 -18.950 14.151 1.00 58.37 C

ATOM 293 CG ASP A 37 1.321 -19.774 12.878 1.00 62.78 C

ATOM 294 OD1 ASP A 37 2.199 -19.479 12.032 1.00 64.75 0

ATOM 295 OD2 ASP A 37 0.510 -20.712 12.718 1.00 63.46 0

ATOM 296 N LYS A 38 1.902 -15.736 15.178 1.00 49.95 N

ATOM 297 CA LYS A 38 1.803 -14.753 16.245 1.00 47.72

C .

ATOM 298 C LYS A 38 0.400 -14.173 16.347 1.00 41.91 C ATOM 299 O LYS A 38 0.186 -13.133 16.959 1.00 45.45 0

ATOM 300 CB LYS A _.38 2.842 -13.647 16.059 1.00 53.14 C

ATOM 301 CG LYS A 38 4.278 -14.163^' 16.040 1.00 55.05 C

ATOM 302 CD LYS A 38 5.264 -13.042 16.325 Ί.00 57.30 C

ATOM 303 CE LYS A 38 6.682 -13.571 16.445 1.00 59.98 C

ATOM 304 NZ LYS A 38 7.643 -12.484 16.795 1.00 56.91 N

ATOM 305 N ME A 39 -0.556 -14.864 15.747 1.00 41.93 N

ATOM 306 CA MET A 39 -1.946 --14.440 15.803 0.66 41.24 C

ATOM 307 C MET A 39 -2.768 -15.455 16.592 0.74 39.91 C ^'

ATOM 308 O MET A 39 -2.489 -16.656 16.556 1.00 39.24 0

ATOM 309 CB MET A 39 -2.506 -14.249 14.385 1.00 40.08 C

ATOM 310 CG MET A 39 -2.063 -12.946 13.710 1.00 43.42 C

ATOM 311 SD MET A. 39 -2.931 -12.665 12.149 0.53 44.40 S

ATOM 312 CE MET A 39 -2.908 -10.878 12.068 0.80 45.34 C

ATOM 313 N CYS A 40 -3.760 -14.967 17.332 1.00 36.62 N

ATOM 314 CA CYS A 40 -4.649 -15,-845 18.089 1.00 34.95 C

ATOM 315 C CYS A 40 -5.444 -16.695 17.109 1.00 35.02 C

ATOM 316 O CYS A 40 -6.008 -16.182 16.149 1.00 34.37 0

ATOM 317 CB CYS A 40 -5.589 -15.032 18.996 1.00 33.32 C

ATOM 318 SG CYS A 40 -7.025 -15.916 19.715 1.00 30.59 S

ATOM 319 N PRO A 41 -5.493 -18.008 17.345 1.00 37.22 N

ATOM 320 CA PRO A 41 -6.175 -18.878 16.376 1.00 36.05 C

ATOM 321 C PRO A 41 -7.679 -18.622 16.323 1.00 35.38 C

ATOM 322 O PRO A 41 -8.323 -19.057 15.381 1.00 35.44 0

ATOM 323 CB PRO A 41 -5.925 -20.291 16.916 1.00 38.79 C

ATOM 324 CG PRO A 41 -4.893 -20.158 17.999 1.00 40.0? C

ATOM 325 CD PRO A 41 -4.916 -18.745 18.484 1.00 35.53 C

ATOM 326 N GLY A 42 -8.233 -17.970 17.341 1.00 34.46 N

ATOM 327 CA GLY A 42 -9.671 -17.780 17.405 1.00 33.75 C ATOM 328 C GLY A 42 -10.153 -16.408 16.950 1.00 34.93 C

ATOM 329 O GLY A 42 -11.202 -16.299 16.333 1.00 33.81 0

ATOM 330 N ^■ CYS A 43 -9.407 -15.357 17.275 1.00 33.06 N

ATOM 331 CA CYS A 43 -9.853 -13.998 16.949 1.00 34.01 C

ATOM 332 C CYS A 43 -8.862 -13.258 16.029, 1.00 34.94 C

ATOM 333 O CYS A 43 -9.170 -12.186 15.494 1.00 35.97 O

ATOM 334 CB CYS A 43 -10.095 -13.206 18.245 1.00 33.44 C

ATOM 335 SG CYS A 43 -8.576 -12.552 18.929 1.00 30.93 S ·

ATOM 336 N SER A 44 -7.680 -13.846 15.841 1.00 30.80 N

ATOM 337 CA SER A 44 -6.623 -13.280 15.002 1.00 36.41 C ^'

ATOM 338 C SER A 44 -5.887 -12.045 15.554 1.00 37.05 C

ATOM 339 O SER A 44 -5.013 -11.510 14.883 1.00 37.40 0

ATOM 340 CB SER A 44 -7.127 -13.006 13.573 1.00 37.78 C

ATOM 341 OG SER A 44 -7.544 -14.209 12.934 1.00 39.96 0

ATOM 342 N ASP A 45 -6.203 -11.597 16.765 1.00 37.13 N

ATOM 343 CA ASP A 45 -5.427 -10.504 17.345 1.00 37.68 C

ATOM 344 C ASP A 45 -3.971 -10.937 17.469 1.00 41.55 C

ATOM 345 O ASP A 45 -3.686 -12.129 17.584 1.00 41.64 0

ATOM 346 CB ASP A 45 -5.961 -10.095 18.724 .1.00 37.82 C

ATOM 347 CG ASP A 45 -7.058 -9.054 18.644 1.00 42.00 C

ATOM 348 ■OD1 ASP A 45 -7.441 -8.675 17.518 1.00 42,27 0

ATOM 349 OD2 ASP A 45 -7.553 -8.620 19.706 1.00 44.29 0

ATOM 350 N PRO A 46 -3.037 -9.973 17.417 1.00 43.11 N

ATOM 351 CA PRO A 46 -1.637 -10.254 17.748 1.00 43.08 C

ATOM 352 C PRO A 46 -1.517 -10.858 19.140 1.00 3,8.17 C

ATOM 353 O PRO A 46 -2.247 -10.466 20.039 1.0.0 39.81 O

ATOM 354 CB PRO A 46 -0.995 -8.862 17.727 1.00 43.77 C

ATOM 355 CG- PRO A 6- -1.796 -8.119 16.696 1.00 44.17 C

ATOM 356 CD PRO A 46 -3.216 -8.610 16.883 1.00 43.74 C ATOM 357 N VAL A 47 -0.607 -11.^'809 19.308 1.00 41.56 N

ATOM 35-8 CA VAL A 47 -0.352 -12.400 20.614 1.00 40.19 C

ATOM 359 C VAL A 47 1.050 -12.033 21.091 1.00 40.92 C

ATOM 360 0 VAL A ^'47 2.033 -12.319 20.409 1.00 43.59 0

ATOM 361 CB VAL A 47 -0.512 -13.929 20.574 1.00 40.22 C

ATOM 362 CGI VAL A 47 -0.285 -14.528 21.958 1.00 39.30 C

ATOM 363 CG2 VAL A 47 -1.903 -14.292 20.070 1.00 39.64 C

ATOM 364 N GLN A 48 1.122 -11.378 22.247 1.00 37.96 N

ATOM 365 CA GLN A 48 2.381 -10.977 22.867 1.00 39.29 C

ATOM 366 C GLN A 48 3.055 -12.186 23.504 1.00 42.53 C

ATOM 367 0 GLN A 48 4.248 -12.396 23.333 1.00 44.80 0

ATOM 368 CB GLN A 48 2.147 -9.900 23.939 1.00 45.07 C

ATOM 369 CG GLN A 48 1.609 -8.561 23.419 1.00 48.20 C

ATOM 370 CD GLN A 48 1.103 -7.642 2 .534 1.00 50.16 C

ATOM 371 OE1 GLN A 48 1.450 -7.810 25.708 1.00 56.66 0

ATOM 372 NE2 GLN A 48 0.282 -6.660 24.165 1.00 52.21 N

ATOM 373 N ARG A ■ 49 2.286 -12.978 24.247 1.00 41.50 N

ATOM 374 CA ARG A 49 2.821 -14.182 24.879 1.00 43.00 C

ATOM 375 C ARG A 49 1.706 -15.163 25.231 1.00 39.26 C

ATOM 376 O ARG A 49 0.530 -14.810 25.227 1.00 36.89 0

ATOM 377 CB ARG A 49 3.637 -13.834 26.135 1.00 42.27 C

ATOM 378 CG ARG A 49 2.814 -13.280 27.287 1.00 42.54 C

ATOM 379 CD ARG A 49 3.687 -12.917 28.491 1.00 44.32 C

ATOM 380 - NE ARG A 4^'9 2.860 -12.525 29.636 1.00 46.91 N

ATOM 381 CZ ARG A 49 2.324 -11.319 29.779 1.00 48.55 C

ATOM 382 NH1 ARG A 49 2.531 -10.389 28.854 1.00 53.07 N

ATOM 383 NH2 ARG A 49 1.579 -11.041 30.836 1.00 50.50 N

ATOM 384 N ILE A 50 2.085 -16.399 25.526 1.00 40.07 N

ATOM 385 CA ILE A 50 1.111 -17.414 25.919 1.00 39.68 C ATOM 386 C ILE A 50 1.328 -17.833 27.367 1.00 40.24 C

ATOM 387 0 ILE A 50 2.439 -18.207 27.733 1.00 45.42 0

ATOM 388 CB ILE A 50 1.204 -18 ^~648 25.014 1.00 39.47 C

ATOM 389 ^' CGI ILE A 50 1.021 -18.247 23.548 1.00 40.35 C

ATOM 390 CG2 ILE A 50 0.159 -19.680 25.419 1.00 37.06 C

ATOM 391 CD1 ILE A 50 1.412 -19.333 22.574 1.00 40.23 C

ATOM 392 N GLU A 51 0.276 -17.753 28.182 1.00 35.65 N

ATOM 393 CA GLU A 51 0.320 -18.170 29.582 1.00 40.35 C

ATOM 394 C GLU A 51 -0.148 -19.604 29.745 1.00 40.32 C

ATOM 395 O GLU A 51 -1.108 -20.025 29.107 1.00 34.76 0

ATOM 396 CB GLU A 51 -0.607 -17.311 30.433 1.00 41.50 C

ATOM 397 CG GLU A 51 -0.241 -15.858 30.549 1.00 46.13 C

ATOM 398 CD GLU A 51 -1.098 -15.176 31.590 1.00 46.41 C

ATOM 399 OE1 GLU A 51 -2.132 -15.767 31.994 1.00 44.73 0

ATOM 400 OE2 GLU A 51 -0.754 -14.050 31.989 1.00 49.07 0

ATOM 401 N GLN A 52 0.514 -20.353 30.619 1.00 38.05 N

ATOM 402 CA GLN A 52 0.064 -21.703 30.910 1.00 36.62 C

ATOM 403 C GLN A 52 -0.926 -21.669 32.076 1.00.39.43 C

ATOM 404 O GLN A 52 -0.708 -20.979 33.087 1.00 40.34 0

ATOM 405 CB GLN A 52 1.263 -22.626 31.180 1.00 41.16 C

ATOM 406 CG GLN A 52 0.879 -24.071 31.374 1.00 42.97 C

ATOM 407 CD GLN A 52 2.073 -24.942 31.746 1.00 47.68 C

ATOM 408 OE1 GLN A 52 3.167 -24.783 31.201 1.00 46.85 0

ATOM 409 NE2 GLN A 52 1.864 -25.861 32.683 1.00 50.11 N

ATOM 410 N CYS A 53 -2.032 -22.390 31.926 1.00 34.22 N

ATOM 411 CA CYS A 53 -3.144 -22.295 32.859 1.00 35.35 C

ATOM 412 C CYS A 53 -3.805 -23.651 33.018 1.00 38.22 C

ATOM 413 O CYS A 53 -4.101 -24.328 32.027 1.00 36.28 0

ATOM 414 CB CYS A 53 -4.173 -21.294 32.325 1.00 38.17 C ATOM 415 SG CYS A 53 -3.497 -19.649 32.029 1.00 46.52 S

ATOM 416 N THR A 54 -4.022 -24.066 34.260 1.00 39.16 N

ATOM 417 CA THR A 54 -4.683 -25.338 34.504 1.00 41.46 C

ATOM 418 C THR A 54 -6.176 -25.129 34.378 1.00 43.34 C

ATOM 419 0 THR A 54 -6.672 -24.015 34.567 1.00 42.65 0

ATOM 420 CB THR A 54 -4.359 -25.908 35.887 1.00 41.22 C

ATOM 421 OG1 THR A 54 -4.948 -25.068 36.886 1.00 46.40 0

ATOM 422 CG2 THR A 54 -2.847 -25.961 36.092 1.00 40.13 C

ATOM 423 N. ARG A 55 -6.882 -26.211 3 .065 1.00 46.70 N

ATOM 424 CA ARG A 55 -8.299 -26.153 33.721 1.00 50.15 C

ATOM 425 C ARG A 55 -9.124 -25.313 34.690 1.00 48.88 C

ATOM 426 0 ARG A 55 -9. 14 -24.464 34.269 1.00 52.41 O

ATOM 427 CB ARG A 55 -8.881 -27.569 33.650 1.00 49.70 C

ATOM 428 CG ARG A 55 -7.838 -28.640 33.435 1.00 50.03 C

ATOM 429 CD ARG A 55 -7.991 -29.788 34.421 1.00 53.70 C

ATOM 430 NE ARG A 55 -9.257 -30.497 34.252 1.00 57.51 N

ATOM 431 CZ ARG A ^'55 -9.372 -31.720 33.747 1.00 55.27 C

ATOM 432 NH1 ARG A 55 -8.294 -32.386 33.356 1.00 53.16 N

ATOM 433' NH2 ARG A 55 -10.571 -32.282 33.641 1.00 56.78 N

ATOM 434 N GLY A 56 -8.945 -25.545 35.985 1.00 47.20 N

ATOM 435. CA GLY A 56 -9.822 -24.943 36.980 1.00 45.88 C

ATOM 436 C GLY A 56 -9.436 -23.565 37.486 1.00 -42.61 C

ATOM 437 O GLY A 56 -10.011 -23.067 38.450 1.00 46.54 0

ATOM 438 N SER A 57 -8.470 -22.932 36.842 1.00 39.66 N

ATOM 439 CA SER A 57 -8.030 -21.622 37.290 1.00 39.65 C

ATOM 440 C SER A 57 -8.649 -20.458 36.511 1.00 39.69 C

ATOM 441 O SER A 57 -8.324 -19.302 36.777 1.00 36.93 0

ATOM 442 CB SER A 57 -6.512 -21.521 37.201 1.00 40.98 C

ATOM 443 OG SER A 57 -6.091 -21.673 35.863 1.00 43.18 O ATOM 444 N LEU A 58 -9.537 -20.749 35.566 1.00 36.57 N

ATOM 445 CA LEU A 58 -10.024 -19.690 34.666 1.00 36.78 C

ATOM 446 C LEU A 58 -11.515 -19.368 34.813 1.00 36.43 C

ATOM 447 0 LEU A ' 58 -12.337 -20.241 35.136 1.00 33.28 0

ATOM 448 CB LEU A 58 -9.660 -20.024 33.213 1.00 34.54 C

ATOM 449 CG LEU A 58 -8.140 -19.981 32.965 1.00 38.83 C

ATOM 450 CD1 LEU A 58 -7.742 -20.499 31.615 1.00 36.89 C

ATOM 451 CD2 LEU A 58 -7.583 -18.580 33.178 1.00 38.56 C

ATOM 452 N PHE A 59 -11.851 -18.099 34.588 1.00 30.19 N

ATOM 453 CA PHE A 59 -13.230 -17.650 34.635 1.00 27.99 C

ATOM 454 C PHE A 59 -13.549 -17.087 33.240 1.00 33.11 C

ATOM 455 0 PHE A 59 -13.038 -16.041 32.883 l^'.OO 31.87 0

ATOM 456 CB PHE A 59 -13.396 -16.555 35.670 1.00 32.16 C

ATOM 457 CG PHE A 59 -13.142 -17.018 37.082 1.00 32.53 C

ATOM 458 CD1 PHE A 59 -14.185 -17.456 37.875 1.00 34.55 C

ATOM 459 CD2 PHE. A 59 -11.855 -17.018 3_.7.604 1.00 35.46 C

ATOM 460 CE1 PHE -A 59 -13.959 -17.881 39.208 1.00 38.65 C

ATOM 461- CE2 PHE A 59 -11.614 -17.440 38.930 1.00 33.93 C

ATOM 462 CZ PHE A 59. -12.667 -17.873 39.719 1.00 36.69 C-

ATOM 463 N MET A 60 -14.378 -17.792 32. 76 1.00 28.24 N

ATOM 464 CA MET A 60 -14.619 -17.445 31.066 1.00 28.77 C

ATOM 465 C MET A 60 -15.888 -16.594 30.926 1.00 29.09 C

ATOM 466 O MET A 60 -16.909 -16.850 31.569 1.00 27.92 0

ATOM 467 CB MET A 60 -14.725. -18.717 30.199 1.00 30.69 C

ATOM 468 CG MET A 60 -15.102 -18.458 28.681 1.00 27.68 C

ATOM 469 SD _. MET A 60 -15.153 -19.920 27.622 1.00 24.08 S

ATOM 4^'70 CE MET A 60 -13.406 7_.20.162 27.342 1.00 33.15 C

ATOM 471 ^' N CYS A 61 -15.816 -15.570 30.086 1.00 30.13 N

ATOM 472 CA CYS A 61 -16.999 -14.778 29.780 1.00 29-30 C ATOM 473 C CYS A 61 17.812 -15.524 28.717 1.00 26.48 C

•ATOM 474 0 CYS A 61 17.276 -15.876 27..6-87 1.00 27.76 0

ATOM 475 CB CYS A 61 16.584 -13.392 29.249 1.00 28.24 C

ATOM 476 SG CYS A 61 18.035 -12.420 28.853 1.00 28.26 S

ATOM 477 N SER A ^' 62 19.085 -15.791 28.975 1.00 26.86 N

ATOM 478 CA SER A 62 19.893 -16.494 27.982 1.00 31.16 C

ATOM 479 C SER A 62 20.993 -15.628 27.378 1.00 29.12 C

ATOM 480 O SER A 62 21.983 -16.141 26.888 1.00 30.07 O

ATOM 481 CB SER A 62 20.514 -17.756 28.575 1.00 31.22 C

ATOM 482 OG SER A 62 21.347 -17.439 29.678 1.00 36.37 O

ATOM 483 N ILE A 63 20.838 -14.320 27. 56 1.00 29.37 N

ATOM 484 CA ILE A 63 21.866 -13.420 26.949 1.00 27.31 C

ATOM 485 C ILE A 63 21.860 -13.533 25.425 1.00 25.97 C

ATOM 486 0 ILE A 63 20.803 -13.540 24.817 1.00 26.26 0

ATOM 487 CB ILE A 63 21.601 -11.990 27.394 1.00 29.81 C

ATOM 488 CGI^' ILE A 63 21.991 -11.831 28.879 1.00 29.87 C

ATOM 489 CG2 ILE A 63 22.374 -10.983. 26.516 1.00 28.87 C

ATOM 490 CD1 ILE A 63 21.453 -10.589 29.496 1.00 33.99 C

ATOM 491 N VAL A 64 23.053 -13.646 24.846 1.00 25.62 N

ATOM 492 CA VAL A 64 23.255 -13.,779 23.401 1.00 27.13 C

ATOM 493 C VAL A 64 23.928 -12.489 22.898 1.00 27.28 C

ATOM 494 O VAL A 64 25.052 -12.185 23.294 1.00 26.51 0

ATOM 495 CB VAL A 64 24.182 -14.963 23.088 1.00 25.78 C

ATOM 496 CGI VAL A 64 24.352 -15.150 21.576 1.00 25.68 C

ATOM 497 CG2 VAL A 64 23.647 -16.264 23.747 1.00 32.01 C

ATOM 498 N GLN A 65 23.214 -11.717 22.082 1.00 28.06 N

ATOM 499 CA GLN -A 65 23.769 -10.487 21.517 1.00 28.01 C

ATOM 500 C GLN A 65 23.390 -10.383 20.050 1.00 28.25 C

ATOM 501 O GLN A 65 22.483 -11.072 19.588 1.00 26.46 0 ATOM 502 CB GLN A 65 23.247 -9.266 22.265 1.00 31.43 C

ATOM 503 · CG GLN A 65 23.887 -9.035 23.613 1.00 34.36 C

ATOM 504 CD- GLN A 65 23.152 -8.008 24.430 1.00 33.33 C

ATOM ^' 505 OE1 GLN A 65 21.928 -7.885 24.337 1.00 32.76 0

ATOM 506 NE2 GLN A 65 23.896 -7.260 25.253 1.00 34.07 N

ATOM 507 N GLY A 66 24.059 -9.494 19.322 1.00 29.44 N

ATOM 508 CA GLY A 66 23.681 -9.230 17.945 1.00 26.86 C

ATOM 509 C GLY A 66 22.366 -8.463 17.836 1.00 25.43 C

ATOM 510 O GLY A 66 21.961 -7.740 18.750 1.00 28.16 0

ATOM 511 N CYS A 67 21.692 -8.625 16.702 1.00 27.03 N

ATOM 512 CA CYS A 67 20.429 -7.948 16.426 1.00 26.02 C

ATOM 513 C CYS A 67 20.612 -6.441 16.213 1.00 28.23 C

ATOM 514 O CYS A 67 20.018 -5.631 16.912 1.00 ^'25.82 0

ATOM 515 CB CYS A 67 19.794 -8.549 15.171 1.00 30.57 C

ATOM 516 SG CYS A 67 18.247 -7.773 14.685 1.00 29.97 S

' ATOM 517. N LYS A 68 21.413 -6.095 15.206 1.00 27.96. N

ATOM . 518 CA LYS A 68 21.794 -4.705 14.901 1.00 28.07 C

ATOM 519 C LYS A 68 20.683 -3.786 14.394 1.00 28.78 C

ATOM 520 0 LYS A 68 20.910 -2.584 14.293 1.00 30.23 0

ATOM 521 CB LYS A 68 22.509 -4.053 16.088 1.00 27.80 C

ATOM 522 CG LYS A 68 23.660 -4.893 16.623 1.00 31.00 C

ATOM 523 CD LYS A 68 24.513 -4.103 17: 586 1.00 32.54 C

ATOM 524 CE LYS A 68 25.516. -5.006 18.309 1.00 36.47 C ^■

ATOM 525 NZ LYS A 68 26.482 -4.203 19.117 1.00 37.71 N

ATOM 526 N ARG A 69 19.498 -4.322 14.096 1.00 27.32 N

ATOM 527 CA ARG A 69 18.454 -3.527 13.445 1.00 26.84 C

ATOM 528 C ARG A 69 18.908 -3.233 12.020 1.00 25.54 C .

ATOM 529 O ARG A 69 19.608 -4.041 11.432 1.00 24.67 0

ATOM 530 CB ARG A 69 17.118 -4.275 13.396 1.00 27.49 C ATOM 531 CG ARG A .69 . -16.516 4.546 14.794 1.00 27.95 C

ATOM 532 CD ARG A 69 -15.050 5.003 14.645 1.00 31.81 C

ATOM 533 NE ARG A 69 -14.586 5.745 .15.821 1.00 34.85 N

ATOM 534 CZ ARG A 69 -14.100 5.190 16.930 1.00 37.73 C

ATOM 535 NH1 ARG A 69 -14.013 3.873 .17.042 1.00 33.68 N

ATOM 536 NH2 ARG A 69 -13.699 5.959 17.941 1.00 37.33 N

ATOM 537 N THR A 70 · -18.493 2.082 11.488 1.00 25.54 N

ATOM 538 CA THR A 70 -18.948 1.615 10.169 1.00 24.97 C

ATOM 539 C THR A 70 -17.802 1.500 9.193 1.00. 27.68 C

ATOM 540 O THR A 70 -16.672 1.191 _. 9.587 1.00 27.19 0

ATOM 541 ^' CB THR A 70 -19.660 0.236 10.208 1.00 24.25 C

ATOM 542 OG1 THR A 70 -18.789 0.765 10.747 1.00 28.64 O

ATOM 543 CG2 THR A 70 -20.927 0.305 11.060 1.00 27.10 C

ATOM 544 N TYR A 11 -18.126 1.688 7.909 1.00 27.34 N

ATOM 545 CA TYR A 71 -17.134 1.679 6.855 1.00 28.22 C

ATOM 546 C TYR A 71 -17.659 0.806 5.717 1.00 31.88 C

ATOM 547 O TYR A 71 -18.871 0.592 5.623 1.00^' 28.57 0

ATOM 548 CB TYR A 71 -16.785 3.113 6.420^' 1.00 30.27 C

ATOM 549 CG TYR A 71 -16.130 3.779 7.607 1.00 29.96 C

ATOM 550 CD1 TYR A 71 -14.777 3.626 7.840 1.00 30.86 C

ATOM 551 CD2 TYR A 71 -16.897 4.427 . 8.558 1.00 31.39 C

ATOM 552 CE1 TYR A 71 -14.176 4.165 8.977 1.00 33.06 C

ATOM 553 CE2 TYR A 71 -16.305 4.980 9.719 1.00 31.59 C

ATOM 554 CZ TYR A 71 - -14.949 4.844 9.901 1.00 30.89 C

ATOM 555 OH TYR A 71 -14.343 5.342 11.032 1.00 33.17 0

ATOM 556 N LEU A 72 -16.744 0.294 4.897 1.00 32.47 N

ATOM 557 CA LEU A 72^' -17.106 0.651 3.828 1.00 34.44 C

ATOM 558 C LEU A- 72 -17.426 0.059 2.520 1.00 34.37 C

ATOM 559 O LEU A 72 -17.977 0.539 1.583 1.00 32.44 0 ATOM 560 CB LEU A 72 -16.008 1.707 3.648 1.00 33.04 C

ATOM 561 CG LEU A 72 -15.725 2.532 4.910 1.00 37.77 C

ATOM 562 CD1 LEU A 72 -14.622 3.565 ^■ 4.700 1.00 43.02 C

ATOM 563 CD2 LEU A 72 -16.996 3.190 5.425 1.00 37.13 C

ATOM 564 N SER A 73 -17.112 -1.351 2.460 1.00 31.48 N

ATOM 565 CA SER A 73 -17.438 -2.152 1.297 1.00 34.16 C

ATOM 566 C SER A 73 -17.814 -3.555 1.727 1.00 36.21 C

ATOM ^■ 567 O SER A 73 -17.384 -4.035 2.798 1.00 32.85 0

ATOM 568 CB SER A 73 -16.244 -2.220 0.340 1.00 35.70 C

ATOM 569 OG SER A 73 -15.226 -3.083 0.845 1.00 35.50 0

ATOM 570 N GLN A 74 -18.605 -4.217 0.889 1.00 32.46 N

ATOM 571 CA GLN A 74 -19.006 -5.579 1.170 1.00 37.49 C

ATOM 572 C GLN A 74 -17.761 -6. 52 1.187 1.00 39.18 C

ATOM 573 O GLN A 74 -17.623 -7.344 2.028 1.00 35.24 0

ATOM 574 CB GLN A 74 -20.018 -6.066 0.144 1.00 38.66 C

ATOM 575 CG GLN A 74 -20.398 -7.513 0.315 1.00 39.76 C

ATOM 576 ^'CD GLN A 74 -21.084 -7.770 1.628 1.00 44.01 C

ATOM 577 OE1 GLN A 74 -21.715 -6.873 2.202 1.00 44.47 0

ATOM 578 NE2 GLN A 74 -20.966 -9.002 _.2.123 1.00 43.00 N

ATOM 579 N ARG A 75 -16.840 -6.175 0.269 1.00 35.46 N

ATOM 580 CA ARG A 75 -15.565 -6.879 0.236 1.00 37.99 C

ATOM 5.81 C ARG A 75 -14.797 -6,793 1.567 1.00 36.30 C

ATOM 582 O ARG A 75 -14.220 -7.793 2.037 1.00 35.88 0

ATOM 583 CB ARG A 75 -14.702 -6.345 -0.914 1.00 42.31 C

ATOM 584 CG ARG A 75 -13.287 -6.886 -0.939 1.00 45.80 C

ATOM 585' CD ARG A 75 -12.512 -6.368 -2.155 1.00 47.25 C

ATOM 586 NE ARG A 75 -13.005 -6.943 -3.404 1.00 50.42 N

ATOM 587 CZ ARG A 75 ^•12.604 -6.555 -4.614 1.00 50.59 C

ATOM 588 NH1 ARG A 75 -11.711 -5.582 ^" -4.746 1.00 50.87 N ATOM 589 NH2 ARG A 75 13.102 -7.132 -5.68-9 1.00 49.38 N

ATOM 590 N ASP A 76 14.762 -5.605 ^'2.164 1.00 33.72 N

ATOM 591 CA ASP A 76 14.075 -5.439 3.444 1.00 33.53 C

ATOM 592 C ASP A 76 14.810 -6.177 4.566 1.00 33.79 C

ATOM 593 O ASP A 7_6 14.180 -6.750 5.464 1.00 33.84 O

ATOM 594 CB ASP A 76 13.907- -3.961 3.796 1.00 34.76 C

ATOM 595 CG ASP A 76 12.816 -3.281 2.968 1.00 37.59 C

ATOM 596 OD1 ASP A 76 12.127 -3.980 2.190 1.00 37.52 0

ATOM 597 OD2 ASP A 76 12.656 -2.050 3.092 1.00 34.86 0

ATOM 598 N LEU A 77 16.136 -6.173 4.516 1.00 32.61 N^'

ATOM 599 CA LEU A 77 16.914 -6.920 5.507.- 1.00 32.90 C

ATOM 600 C LEU A 77 16.631 -8.411 5.394 1.00 35.63 C

ATOM 601 · O LEU A 77 16.482 -9.100 6.411 1.00 29.68 0

ATOM 602 CB LEU A 77 18.406 -6.662 5.343 1.00 33.65 C

ATOM 603 CG LEU A 77 19.378 -7.549 6.123 1.00 33.62 C

ATOM 604 CD1 LEU A 77 19.113 -7,475 7.643 1.00 32.25 C

ATOM^' 605 CD2_. LEU A 77 20.803 -7.133 5.798 1.00 33.57 C

ATOM 606 N GLN A 78 16.554 -8.915 4.159 1.00 33.82 N

ATOM 607 CA GLN A 78 16.246 -10.319 3.962 1.00 34.23 C

ATOM 608 C GLN A 78 14.838 -10.655 4.464 1.00 36.22 C

ATOM 609 O GLN A 78 14.630 -11.688 5.114 1.00 34.64 0

ATOM 610 CB GLN A 78 16.435 -10.738 2.495 1.00 37.69 C

ATOM 611 CG' GLN A 78 16.346 -12.241 2.304 1.00-40.01 C

ATOM 612 CD GLN A 78 17.376 -12.995 3.124 1.00 42.39

C ^'

ATOM 613 OE1 GLN A 78 18.573 -12.694 3.073 1.00 44.55 O

ATOM 614 NE2 GLN A 78 16.917 -13.982 3.889 1.0D 43.06 N

ATOM 615 N ALA A 79 13.875 -9.779 4.193_^ 1.00 34.75 N

ATOM 616 CA ALA A 79 12.520 -10.000 4.692 1.00 36.58 C

ATOM 617 C ALA A 79 12.480 -10.019 6.228 1.00 35.62 C ATOM 618 0 ALA A 79 -11.756 -10.829 6.836 1.00 33.94 0

ATOM 619 CB ALA A 7.9 -11.565 -8·.964 4.142 1.00 37.21 C

ATOM 620 N HIS A 80 -13.262 -9.139 6.849 1.00 31.82 N

ATOM 621 CA HIS A 80 -13.408 -9.126 8.309 ^' 1.00 32.56 C

ATOM 622 C HIS A 80 -13.974 -10.441 8.859 1.00 32.70 C

ATOM 623 O HIS A 80 -13.490 -10.966 9.870 1.00 33.75 0

ATOM 624 CB HIS A 80 -14.286 -7.940 748 1.00 29.-70 C

ATOM 625 CG HIS A 80 -14.909 -8.112 10.100 1.00 29.10 C

ATOM 626 ND1 HIS A 80 -14.187 -7.980 11.269 1.00 30.37 N

ATOM 627 CD2 HIS A 80 -16.176 -8.414 10.-472 1.00 28.07 ^■C

ATOM 628 CE1 HIS A 80 -14.987 -8.172 12.302 1.00 29.59 C

ATOM 629 NE2 HIS A 80 -16.197 -8.442 11.850 1.00 31.18 N - ATOM 630 N ILE A 81 -15.012 -10.955 8.209 1.00 31.23 N

ATOM 631 CA ILE A 81 -15.630 -12.215 8.598 1.00 32.10 C

ATOM 632 C ILE A · 81 -14.624 -13.369 8.476 1.00 34.77 C

ATOM 633 0 ILE A 81 -14.532 -14.217 9.379 1.00 32.08 0

ATOM 634^' CB ILE A 81 -16.906 -12.513 7.756 1.00 35.04 C

ATOM 635 CGI ILE A 81 -17.989 -11.459 8.016 1.00 32.21 C

ATOM 636 CG2 ILE A 81 -17.446 -13.912 052 1.00 36.43 C

ATOM 637 CD1 ILE A 81 -19.226 -11.632 7.119 1.00 36.21 C

ATOM 638 N ASN A 82 -13.871 -13.404 7.372 1.00 34.-36 NT

ATOM 639 CA ASN A 82 -12.858 -14.441 7.176 1.00 36.28 C

ATOM 640 C ASN A 82 -11.737 -14.366 8.207 1.00 38.04 C

ATOM 641 0 ASN A 82 -11.144 -15.390 8.573 1.00 38.73 0

ATOM 642 CB ASN A 82 -12.255 -14.375 5.761 1.00 38.99 C

ATOM 643 CG ASN A 82 -13.242 -14.775 .684 1.00 41.72 C

ATOM 644 OD1 ASN A 82 -14.334 -15.265 4.976 1.00 42.85 0

ATOM 645 ND2 ASN A 82 -12.858 -14.575 3.420 1.00 45.66 N

ATOM 646 N HIS A 83 -11.460 -13.153 675 1.00 35.37 N ATOM 647 CA HIS .A. 83 -10.331 -12.875 9.554 1.00 34.29 C

ATOM 648 C HIS A 83 -10.701 -13.089 11.020 1.00 35.78 C

ATOM 649 0 HIS A 83 -9.883 -13.538 11.818 1.00 35.91 0

ATOM 650 CB HIS A 83 -9.873 -11.426 9.329 1.00 35.58 C

ATOM 651 CG HIS A 83 -8.599 -11.059 10.028 1.00 39.32 C

ATOM 652 ND1 HIS A 83 -7.400 -11.692 9.779 1.00 41.42 N

ATOM 653 CD2 HIS A 83 -8.333 -10.101 10.949 1.00 38.45 C

ATOM 654 CE1 HIS A 83 -6.454 -11.149 10.525 1.00 39.51 C

ATOM 655 NE2 HIS A 83 -6.994 -10.181 11.243 1.00 39.21 N

ATOM 656 N ARG A 84 -11.945 -12.778 11.364 1.00 32.69 N

ATOM 657. CA ARG A 84 -12.347 -12.706 12.753 1.00 31.89 C

ATOM 658 C ARG A 84 -13.289 -13.803 13.232 1.00 32.14 C

ATOM 659 O ARG A 84 -13.349 -14.071 14.455 1.00 32.10 0

ATOM 660 CB ARG A 84 -13.025 -11.353 13.033 1.00 34.34 C

ATOM 661 CG ARG A 84 -12.160 -10.131 12.762 1.00 31.51 C

ATOM 662 CD ARG A 84 -10.873 -10.141 13.567 1.00 36.82 C

ATOM 663 NE ARG A 84 -10.141 -8.888 13.373 1.00 40.30 N

ATOM 664 CZ ARG A 84 -9.047 -8.548 14.039 1.00 42.06 C

ATOM 665 NH1 ARG A 84 -8.549 -9.367 14.960 1.00 41.45 N

ATOM 666 NH2 ARG A 84 -8.454 ^■ -7.389 13.784 1.00 42.29 N

ATOM 667 N HIS A 85 -14.056 -14.411 12.323 1.00 29.73 N

ATOM 668 CA HIS A 85 -15.210 -15.197 12.768 1.00 31.97 C

ATOM 669 C 'HIS A 85 -15.194 -16.676 12.427 1.00 31.51 C

ATOM 670 O HIS A 85 -16.056 -17.417 12.874 1.00 31.76 O

ATOM 671 CB HIS A 85 -16.530 -14.605 12.261 1.00 28.59 C

ATOM 672 CG HIS A 85 -16.862 -13.274 12.853 1.00 29.77 C

ATOM 673 ND1 HIS A 85 ^■17.219 -13.115 14.176 1.00 29.35 N

ATOM 674 CD2 HIS A 85 -16.894 -12.036 12-300 1.00 29.31 C

ATOM 675 CE1 HIS A ^'85 -17.457 -11.836 14.412 1.00 31.55 C ATOM 676 NE2 HIS A 85 -17.269 -11.164 13.289 1.00 30.35 N

ATOM 677 N MET A 86 -14.230 -17.107 11.632 1.00 32.79 N

ATOM 678 CA MET A 86 -14.327 -18'.442 11.056 1.00 34.18 C

ATOM 679 C MET A 86 -13.955 -19.533 12.047 1.00 33.62 C

ATOM 680 0 MET A 86 -14.366 -20.680 11.907 1.00 35.47 0

ATOM 681 CB MET A 86 -13.499 -18.510. _; 9-770 1.00 36.54 C

ATOM 682 CG MET A 86 -13.969 -17.458 8.747 1.00 37.71 C

ATOM 683 SD MET A ■ 86 -15.698 -17.715 8.289 1.00 40.08 S

ATOM 684 CE MET A 86 -16.610 -16.883 9.591 1.00 45.05 C

ATOM 685 N ARG A 87 -13.195 -19.170 13.067 1.00 34.82 N

ATOM 686 CA ARG A 87 -12.837 -20.137 14.091 1.00 33.59 C

ATOM 687 C ARG A 87 -13.287 -19.665 15.461 1.00 36.52 C

ATOM 688 0 ARG A 87 -12.706 -20.050 16.477 1.00 34.66 O

ATOM 689 CB ARG A 87 -11.333 -20.368■ 14.096 1.00 34.57 C

ATOM 690 CG ARG A 87 -10.834 -21.136 12.882 1.00 35.24 C

ATOM 691 CD ARG A 87 -9.326 -21.355 12.967 1.00 40.27 C

ATOM 692 NE ^' ARG A 87 -8.580 -20.103 12.822 1.00 41.91 N

ATOM 693 CZ ARG A 87 -8.294 -19.523 11.656 1.00 44.35 C

ATOM 694 NH1 ARG A 87 -8.688 -20.070 10.504 1.00 44.24 N

ATOM 695 NH2 ARG A 87 -7.617 -18.385 11.640 1.00 42.72 N

ATOM 696 N ALA A 88 -14.330 -18.843 15.496 1.00 32.66 N

ATOM 697 CA ALA A 88 -14.786 -18.284 16.761 1.00 34.53 C

ATOM 698 C ALA A 88 -16..095 -18.899 17.235 1.00 36.39 C

ATOM 699 O ALA A 88 -16.788 -18.325 1.8.068 1.00 35.80 0

ATOM 700 CB ALA A 88 -14.915 -16.747 16.649 1.00 33.57 C

ATOM 701 N GLY A 89 -16.446 -20.058 16.691 1.00 35.10 N

ATOM 702 CA GLY A 89 -17.614 -20.780 17.152 1.00 36.38 C

ATOM 703 C GLY A 89 -18.794 -20.635 16.215 1.00 38.93 C

ATOM 704 O GLY A 89 -18.808 -19.746 15.370 1.00 37.16 0 ATOM 705 N LYS A 90 19.769 -21 528 16.361 1.00 36.16 N

ATOM 706 CA LYS A 90 20.985 -21 508 15.560 1.00 38.69 C

ATOM- 707 C LYS A 90 21.741 -20 192 15.756 1.00 37.65 C

ATOM 708 O LYS A 90 22.085 -19 820 16.894 1.00 36.61 O

ATOM 709 CB LYS A 90 21.863 -22 688 15.971. 1.00 38.06 C

ATOM 710 CG LYS A 90 23.152 -22 821 15.206 1.00 36.86 C

ATOM 711 CD LYS A 90 22.87_.4 -23 074 13.743 1.00 40.04 C

ATOM 712 CE LYS A 90 24.159 -23 133 12.967 1.00 44.28 C

ATOM . 713 NZ LYS A 90 24.880 -24 400 13.273 1.00 49.46 N

ATOM 714 N PRO A 91 21.967 -19 449 14.662 1.00 36.52 N

ATOM 715 CA PRO A 91 22.789 -18 244 14.795 1.00 34.92 C

ATOM 716 C PRO A 91 24.216 -18 595 15.217 1.00 34.80 C

ATOM 717 O PRO A 91 24.795 -19 550 14.697 1.00 35.15 O

ATOM 718 CB PRO A 91 22.779 -17 652 13.380 1.00 38.19 C

ATOM 719 CG PRO^' A 91 21.548 -18 207 12.742 1.00 38.19 C

ATOM 720 CD PRO A 91 21. 19 -19 598 13.297 1.00 38.17 C

ATOM 721 N VAL A 92 24.761 -17 838 16.166 1.00 35.09 N

ATOM 722 CA VAL A 92 26.127 -18 024 16.626 1.00 31.59 C

ATOM 723 C VAL A 92 27.108 -17 395 15.633 1.00 37.41 C

ATOM 724 O VAL A 92 27.016 -16 201 15.330 1.00 34.69 O

ATOM 725 CB VAL A 92 26.321 -17 373 18.000 1.00 31.23 C

ATOM 726 CGI VAL A 92 27.714 -17 667 18.552 1.00 31.95 C

ATOM 727 CG2 VAL A 92 25.235 -17 869 18.953 1.00 30.42 C

ATOM 728 N THR A 93 ■28.045 -18 193 15.132 1.00 36.04 N

ATOM 729 CA THR A 93 29.046 -17 681 14.191 1.00 37.77 C

ATOM 730 C THR A 93 30.426 -18 252 14.499 1.00 42.83 C

ATOM 731 O THR A 93 30.552 -19 304 15.134 1.00 40.93 O

ATOM 732 CB THR A 93 .28.690 -18 051 12.742 1.00 40.67 C

ATOM ^■ 733 OGl THR A 93 28.707 -19 478 12.599 1.00 40.63 ATOM 734 CG2 THR A 93 27.318 -17.523 12.375 1.00 36.17 C

ATOM 735 N ARG A 94 31.467 -17.565 14.-043 1.00 42.61 N

ATOM 736 CA ■ ARG A_. 94 32.813 -18.074 14.233 1.00 4'6.84 C

ATOM , 737 C ARG A ■ 94 32.887 -19.463 13.605 1.00 48.40 C

ATOM 738 O ARG A 94 33.514 -20.365 14.157 1.00 53.23 0

ATOM 739 CB ARG A 94 33.852 -17.140 13.604 1.00 48.48 C

ATOM 740 CG ARG A 94 35.198 -17.137 14.313 1.00 48.83 C

ATOM 741 CD ARG A 94 35.166 -16.283 15.589 1.00 48.63 C

ATOM 742 - NE ARG A 94 34.982 -14.858 15.291 1.00 50.73 N ^■

ATOM 743 CZ ARG A 94 35.102 -13.888 16.193 1.00 45.7.6 C

ATOM 744 NH1 ARG A 94 35.410 -14.199 17.451 1.00 46.43

N ^"

ATOM 745 NH2 ARG A 94 34.913 -12.609 15.843 1.00 46.44 N

ATOM 746 N ALA A 95 32.226 -19.628 12.460 1.00 46.13 N

ATOM 747 CA ALA A 95 32.143 -20.916 11.792 1.00 51.36 C

ATOM 748 C ALA A 95 31.590 -22.006 12.698 1.00 54.42 C

ATOM 749 0 ALA A 95 30.629 -22.701 12.355 1.00 56.30 0

ATOM 750 N SER A 96 32.205 -22.148 13.865 1.00 55.84 N

ATOM 751 CA SER A 96 31.807 -23.140 14.851 1..00 55.27 C

ATOM .752 C SER A 96 32.813 -23.162 15.994 1.00 52.69 C

ATOM 753 O SER A 96 32.822 -22.265 16.835 1.00 56.07 O

ATOM 754 CB SER A 96 30.403 -22.840 15.382 1.00 48.36 C

ATOM 755 OG SER A 96 29.753 -24.044 15.750 1.00 49.69 O

TER 756 SER A 96

HETATM 757 ZN ZN A 102 17.863 -9.174 12.859 1.00 29.08 ZN

HETATM 758 ZN ZN A 103 - .598 -29.683 20.918 1.00 32.14

ZN^'

HETATM 759 ZN ZN A.104 -8.028 -14.076 20.646 1.00 30.65 ZN

HETATM 760 O HOH A 105 19.233 2.894 1.594 1.00 35.38 O

HETATM 761 O HOH A 106 14.073 -0.850 5.272 1.00 32.56 O

HETATM 762 O HOH A 107 21.695 -0.549 15.633 1.00 34.73 O

HETATM 763 O HOH A 108 -6.561 -16.739 13.292 1.00 39.36 0

HETATM 764 HOH A 109 -11.651 -5.706 6.282 1.00 38.91 0

HETATM 765 HOH A 110 4.861 -17.203 25.097 1.00 43.19 0

HETATM .766 HOH A 111 -2.432 -29.387 25.840 1.00 40.53 0

HETATM - 767 HOH A 112 -3.818 -11.998 21.940 1.00 39.04 0

HETATM 768 HOH A 113 -6.220 -8.046 21.791 1.00 42.88 0

HETATM 769 HOH A 114 -13.950 -13.159 16.933 1.00 38.40 O

HETATM 770 HOH A 115 -11.466 -16.154 11.230 1.00 36.25 O

HETATM 771 HOH A 116 -22.491 -5.588 20.179 1.00 ^' 35.34■ 0

HETATM 772 O HOH A 117 -19.672 -7.568 22.670 1.00 40.74 0

HETATM 773 O HOH A 118 -24.545 -16.383 27.799 1.00 35.98 0

HETATM 774 O HOH A 119 -13.658 -3.558 19.920 1.00 40.14 0

HETATM 775 O HOH A 120 -20.047 -11.751 22.752 1.00 33.58 O

HETATM 776 O HOH A 121 -25.322 -2.448 20.859 1.00 41:05 0

HETATM 777 O HOH A 122 3.604 -28.435 21.581 1.00 44.70 0

HETATM 778 O HOH A 123 -17.357 -5.034 -2.201 1.00 38.85 0

HETATM 779 O HOH A 124 -30.326 -15.110 12.762 1.00 44.36 0 .

HETATM 780 O HOH A 125 -25.462 -7.548 20.543 1.00 33.55 O

HETATM ^'781 O HOH - A 126 -1.422 -11.998 32.919 1.00 49.51 O

HETATM 782 O HOH A 127 -13.187 -2.381 -0.702 1.00 47.92 O

HETATM 783 O HOH A 128 -17.813 -19.489 12.355 1.00 44.34 O

HETATM 784 O HOH A 129 -27.996 -21.031 15.769 1.00 43.87 O

HETATM 785 O HOH A 130 -3.768 -7.837 20.660 1.00 44.02 O

HETATM 786 O HOH A 131 -15.959 -22.030 14.409 1.00 43.41 O

HETATM- 787 O HOH A 132 -28.328. -5.658 20.252. 1.00 39.22 O

HETATM 788 O HOH A 133 -9.368 -11.836 5.463 1.00 43.91 O

HETATM 789 O HOH A 134 -3.490 -30.798 27.911 1.00 40.68 O

HETATM 790 O HOH. A 135 -13.174 -9.921 0.765 1.00 43.43 O

HETATM 791 O HOH A 136 0.594 -17.772 19.073 1.00 50.26 O

HETATM 792 O HOH A 137 -19.675 -8.839 20.259 1.00 39.57 o

HETATM 793 0 HOH A 138 -19.846 -11.458 20.172 0.50 36.25 0

HETATM 794 0 HOH A 139 -9.988 -12.893 3.009 1.00 44.74 O

HETATM 795 0 HOH A 140 -18.158 -10.035 22.941 1.00' 43.75 0

HETATM 796 0 HOH A 141 -10.707 -32.328 29.653 1.00 43.83 0

HETATM 797 0 HOH A 142 -30.476 -6.061 21.420 1.00 42.97 0

HETATM 798 0 HOH A 143 -0..652 -19.968 19.205 1.00 50.89 0

HETATM 799 0 HOH A 144 -24.044 -7.312 3.837 1.00 50.44 0

HETATM 800 0 HOH A 145 -15.706 -7.177 -6.055 1.00 48.03 O

HETATM 801 0 HOH A 146 -9.644 -5.974 4.279 1.00 48.97 0

^'HETATM 802 0 HOH A 147 -19.804 -18.159 31.771 1.00 46.13 0

HETATM 803 0 HOH A 148 -10.861 -26.936 20.920 1.00 39.93 0

HETATM 804 0 HOH A 149 -17.926 -6.975 -3.948 1.00 46.61 0

HETATM 805 0 HOH A 150 -31.814 -17.618 10.743 1.00 49.52 0

HETATM 806 0 HOH A _.151 -10.783 -8 ,505 0.322 1.00 49.93 0

HETATM 807 O HOH A 152 -20.303 -14.779 31.506 1.00 37.14 O

HETATM 808 O HOH A 153 0.567 -30.943 28.221 1.00 49.73 O

HETATM 809 O HOH A 154 -22.603 -16.784 9.031 1.00 55.70 O

HETATM 810 O HOH A 155 -21.112 -20.013 19.169 1.00 40.60 O

HETATM 811 O HOH A 156 -15.593 -22.142 9.967 1.00 48.31 O

HETATM 812 O HOH A 157 -24.211 -15.828 10.933 1.00 47.89 O

HETATM 813 O HOH A 158 -10.633 -2.972 0.221 1.00 48.48 O

HETATM 814 O HOH A 159 -15.655 -30.288 33.333 1.00 44.20 O

HETATM 815 O HOH A 160 -25.743 -9.294 2.182 1.00 52.85 O

HETATM 816 O HOH A 161 -23.286 0.569 17.469 1.00 44.29 O

HETATM 817 O HOH A 162 3.808 -20.595 28.087 1.00 47.57 O

HETATM 818 O HOH A 163 -16.164 -27.997 23.919 0.50 51.28 O

HETATM 819 O HOH A 164 -20.017 -17.238 8.965 1.00 52.69 O

HETATM 820. O HOH A 165 1.569, -13.803 31.336 1.00 53.12 - O

HETATM 821 O HOH A 166 -14.149 -8.523 15.634 1.00 37.46 HETATM 822 O HOH A 167 -27-.104 -25.440 13.010 1.00 48.06

0.

HETATM 823 O HOH A 168 5.717 -19.488 25.848 1.00 52.04

0

HETATM 824 O HOH A 169 0.236 -9.773 33.989 1.00 65.32

0

HETATM 825 O HOH A 170 -24.901 -14.986 13.780 1.00 28.09

0

HETATM 826 O HOH A 171 -11.685 -16.732 13.707 1.00 34.19

0

HETATM 827 O HOH A 172 -7.651 -26.089 21.388 1.00 34.03

0

HETATM 828 O HOH A 173 -28.432 -14.172 14.244 1.Ό0 32.48

0

HETATM 829 O HOH A 174 -14.063 -12.438 21.639 1.00 33.49

0

CONECT 34 758

CONECT 57 758

CONECT 156 759

CONECT 172 759

CONECT 199 758

CONECT 225 758

CONECT 318 759

CONECT 335 759

CONECT 516 757

CONECT 629 757

CONECT 676 757

CONECT 757 - 516 629 676

CONECT 758 34 57 199

CONECT 759 156 172 318

MASTER 326 0 3 828 1 14

END

Claims

CLAIMS . A drug screening method comprising:

TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLEN VH (a.a. 159-206) SEQ ID NO:1 or a sequence at least 31 % homologous thereto wherein the following amino acids are conserved C166, C172, H185, and H190;

2. The method according to claim 1 wherein the phosphotyrosine-binding domain is characterised by sequence structure:

TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENVH

(a.a. 159-206) SEQ ID NO:1 or a sequence at least 76% homologous thereto.

3. The method according to claim 1 wherein the polypeptide is characterised by sequence structure:

VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDPVQRIEQ CTRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENVH

(a.a. 106-206) SEQ ID NO: 2 or a sequence at least 23 % homologous thereto wherein the following amino acids are conserved C109, C112, C125, H127, C130, C133, C145 C148, C166, C172, H185, and H190.

4. The method according to claim 1 wherein the polypeptide is characterised by sequence structure:

(a.a. 106-206) SEQ ID NO: 2 or a sequence at least 71% homologous thereto.

5. The method according to claim 1 wherein said polypeptide comprises a phosphotyrosine-binding domain characterised by two of the following sequence structures:

TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENVH

(a.a. 159-206) SEQ ID NO:1 or a sequence at least 31% homologous thereto where the following amino acids are conserved C166, C172, H185, and H190, arranged as a dimer.

6. The method according claim 1 wherein said polypeptide comprises a phosphotyrosine-binding domain characterised by two of the following sequence structures:

TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENVH

(a.a. 159-206) SEQ ID NO:1 or a sequence at least 76% homologous thereto, arranged as a dimer.

7. The method according claim 1 wherein said polypeptide is characterised by two of the following sequence structures:

(a.a. 106-206) SEQ ID NO: 2 or a sequence at least 23% homologous thereto where the following amino acids are conserved C109, C1 2, C125, H127, C130, C133, C145 C148, C166, C172, H185, and H190, arranged as a dimer.

8. The method according claim 1 wherein said polypeptide is characterised by two of the following sequence structures: VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDPVQRIEQ CTRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLENVH

(a.a. 106-206) SEQ ID NO: 2 or a sequence at least 71 % homologous thereto, arranged as a dimer.

9. The method according to any one of claims 5-8 wherein said dimer is an anti-parallel dimer.

10. The method according to any preceding claim wherein said polypeptide is a ubiquitin 3 ligase or a fragment thereof.

11. The method according to any preceding claim wherein said polypeptide is selected from the group comprising Hakai, ZNF645, Ligand-of-Numb protein X1 and Ligand-of-Numb protein X2, or a fragment thereof.

12. The method according to any preceding claim wherein said polypeptide binds E-cadherin or DOK1 or cortacin.

13. The method according to any preceding claim wherein said polypeptide protein has the following conserved target binding residues H127 and H185.

14. The method according to claim 14 wherein the polypeptide further has the following conserved target binding residues R189 and/or Y176.

15. The method according to any preceding claim wherein, under part c), where said binding occurs concluding said compound may be useful in preventing cell migration or metastasis or invasion or cancer.

16. The method according to any one of claims 1 , 3, 5 or 7 wherein said sequence homology is 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44% or 45%.

17. The method according to any one of claims 2, 4, 6, or 8 wherein said sequence homology is 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

18. The method according to any preceding claim wherein said binding under part c) may be determined either in vitro, in vivo or in silico.

19. An isolated polypeptide selected from the group comprising:

i) TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLEN VH (a.a. 159-206) SEQ ID NO:1 or a sequence at least 31 % homologous thereto wherein the following amino acids are conserved C166, C172, H185, and H190;

ii) TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLE NVH (a.a. 159-206) SEQ ID NO:1 or a sequence at least 76% homologous thereto;

iii) VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDP VQRIEQCTRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPV TRASLENVH SEQ ID NO: 2 (a.a. 106-206) or a sequence at least 23 % homologous thereto wherein the following amino acids are conserved C109, C1 12, C125, H127, C130, C133, C145€148, C166, C172, H185, and H190;

iv) VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDP VQRIEQCTRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPV TRASLENVH SEQ ID NO: 2 (a.a. 106-206) or a sequence at least 71 % homologous thereto;

v) two of the following sequence structures:

TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLEN VH (a.a. 159-206) SEQ ID NO:1 or a sequence at least 31 % homologous thereto where the following amino acids are conserved C166, C172, H185, and H190, arranged as a dimer, ideally an anti- parallel dimer; vi) two of the following sequence structures:

TRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVTRASLEN VH (a.a. 159-206) SEQ ID NO:1 or a sequence at least 76% homologous thereto arranged as a dimer, ideally an anti-parallel dimer; vii) two of the following sequence structures:

VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDPV QRIEQCTRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVT RASLENVH (a.a. 106-206) SEQ ID NO:2 or a sequence at least 23% homologous thereto where the following amino acids are conserved C109, C112, C125, H127, C130, C133, C145 C148, C166, C172, H 85, and H 90, arranged as a dimer, ideally an anti-parallel dimer; viii) two of the following sequence structures:

VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGCSDPV QRIEQCTRGSLFMCSIVQGCKRTYLSQRDLQAHINHRHMRAGKPVT RASLENVH SEQ ID NO: 2 (a.a. 106-206) SEQ ID NO:2 or a sequence at least 71 % homologous thereto arranged as a dimer, ideally an anti- parallel dimer;

ix) an isolated polypeptide according to i), ii), v) and vi) in combination with a RING domain characterized by sequence structure:

VHFCDKCGLPIKIYGRMIPCKHVFCYDCAILHEKKGDKMCPGC (a.a. 106-148) SEQ ID NO:3;

x) IHFCDKCDLPIKIYGRIIPCKHAFCYHCANLYDKVGYKVCPRCRYPV LRIEAHKRGSVFMCSIVQQCKRTYLSQKSLQAHIKRRHKRARKQVTS ASLEKVR (a.a. 54-154 ZNF645) SEQ ID NO:4 or a sequence at least 71 % homologous thereto wherein, when aligned with Hakai a.a. 106- 206, the following amino acids are conserved C109, C112, C125, H127, C130, C133, C145 C148, C166, C172, H185, and H190;

xi) DLVCHICLLQPLLQPLDTPCGHTFCYKCLRNFLQEKDFCPLDRKRL HFKLCKKSSILVHKLLDKLLVLCPFSSVCKDVMQRCDLEAHLKNRCP GASHRRVALERRKTS (a.a. 47-153 LNX2) or a sequence at least 25% homologous thereto wherein, when aligned with Hakai a. a. 106- 206, the following amino acids are conserved C109, C112, C125, H127, C130, C133, C145 C148, C166, C172, H185, and H190; and xii)DLICHICLQALLDPLDTPCGHTYCTLCLTNFLVEKDFCPMDRKPLV LQHCKKSSILVNKLLNKLLVTCPFREHCT-

QVLQRCDLEHHFQTSCKGASHYGLTKDRKRRS (a.a. 38-144 LNX1) or a sequence at least 23% homologous thereto wherein, when aligned with Hakai a.a. 106-206, the following amino acids are conserved C109, C112, C125, H127, C130, C133, C145€148, C166, C172, H185, and H190.

20. Use of Methotraxate Hydrate, or a derivative or salt thereof, to treat a disease characterised by migration or metastasis or invasion or a lack of cell- cell adhesion.

21. Use of Methotraxate Hydrate, or a derivative or salt thereof, in the manufacture of a medicament to treat a disease characterised by migration or metastasis or invasion or a lack of cell-cell adhesion.

22. The Use according to claim 20 or 21 wherein said disease is cancer, dysplasia or hyperplasia.

23. A crystal form of the isolated polypeptide according to claim 19 wherein said crystal is characterised by the co-ordinates and structure factors described in Table 3.