Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

• 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.
• SH2 Src homology 2
• PTB phosphotyrosine-binding domains
• SH2 Src homology 2
• PTB phosphotyrosine-binding 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.
• 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).
• PKCd the human M2 pyruvate kinase
• 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).
• 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.
• 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).
• 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.
• a drug screening method comprising:
• 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
• sequence homology may be 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44% or 45%.
• a drug screening method comprising:
• H (a.a. 159-206) SEQ ID NO:1 or a sequence at least 76% homologous thereto;
• 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%.
• 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.
• 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%.
• H (a.a. 106-206) SEQ ID NO: 2 or a sequence at least 71% homologous thereto.
• 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%.
• said polypeptide comprises or consists of a phosphotyrosine-binding domain characterised by two of the following sequence structures:
• SEQ ID NO:1 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.
• said polypeptide comprises or consists of a phosphotyrosine-binding domain characterised by two of the following sequence structures:
• SEQ ID NO:1 (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.
• 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.
• 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.
• said method is undertaken using a ubiquitin 3 ligase protein or a polypeptide fragment thereof which comprises a phosphotyrosine-binding domain characterised by
• SEQ ID NO:1 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.
• 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 76% homologous thereto; or
• H (a.a. 106-206) SEQ ID NO: 2 or a sequence at least 71% homologous thereto.
• said protein is selected from the group comprising Hakai, ZNF645, Ligand-of-Numb protein X1 and Ligand-of-Numb protein X2.
• polypeptide or protein has the following conserved target binding residues H127 and H185.
• polypeptide or protein also has the following conserved target binding residues R189 and/or Y176.
• said polypeptide or protein has a 1 :1 binding relationship with its target molecule.
• the target molecule is E-cadherin, DOK1 or cortacin.
• 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.
• the HYB domain is therefore composed of four zinc-finger domains cooperating to bind pTyr residues, ideally, surrounded by acidic amino acids.
• 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.
• 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.
• 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.
• MG132 the proteosomal inhibitor
• cells lysates are collected using appropriate buffer.
• Target proteins are then precipitated using specific antibodies against those proteins.
• 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.
• GST- fusion proteins of the targets E-Cadherine, DOK1 and cortactin
• 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.
• 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.
• 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.
• 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.
• 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:
• 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:
• 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.
• 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.
• 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.
• PDB Protein Data Bank
• 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.
• 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.
• 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.
• 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.
• any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.
• 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 15 N-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.
• 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.
• 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.
• 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- 15 N-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.
• FIG. 1 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.
• Figure 9 shows the amino acid sequence structure of Hakai with the HYB domain highlighted.
• 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.
• Hakai constructs were cloned into pGEX6P-1 (GE Healthcare, UK). Point mutants and truncates were generated using the proofreading Pfu DNA polymerase.
• 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).
• 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.
• HEK293 cells were harvested 24 h post-transfection with a lysis buffer containing protease inhibitors (Roche) and 1 mM Na 3 V0 4 . Immunoprecipitations
• 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.
• ABB ammonium bicarbonate
• ACN acetonitrile
• the samples were processed and analysed as described (Zhang et al, 2010) using a LTQ-FT ultra mass spectrometer.
• 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.
• 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).
• N/ 3 C-labelled Hakai (aa 106-206) was obtained from cultures grown in M9 media supplemented with 15 N-labelled ammonium chloride and R elabelled 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, Düsseldorf, DE).
• the backbone assignment was obtained using standard 15 N-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).
• the 2D H- 15 N-HSQC spectra for the 15 N-labelled Hakai 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.
• the datasets were processed and scaled using HKL2000 (Otwinowski and Minor, 1997).
• 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.
• 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 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.
• 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://evicdna.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).
• H185 and H190 histidine residues
• C172 cysteine residue
• C166 cysteine residue from the adjacent monomer
• Figure 1 F Supplementary Figure S2
• ZnF classical C2H2 zinc finger
• 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 ⁇ -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 1650A 2 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
• 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.
• 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.
• DOK1 competed with endogenous cortactin for Hakai, implying that DOK1 and cortactin bind Hakai on the same site ( Figure 4A-C).
• 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
• Bond angles ( ) 1.202 Values in parentheses are for highest-resolution shell.
• a/3 ⁇ 4ym ⁇
• Macaca mulatta (Rhesus HVFCYDCAILHEKKGDKMCPGCSDPVQRIEQCTRGS.LFMCSIVQGCKRT monkey) YLSQRDLQ AHINHRH
• Rattus norvegicus (Norway HVFCYDCAILHEKKGD MCPGCSDPVQRIEQCTRGSLFMCSIVQGCKRT rat) YLSQRDLQ AHINHRH
• Mus musculus (House HVFCYDCAILHEKKGDKMCPGCSDPVQRIEQCTRGSLFMCSIVQGCKRT mouse) YLSQRDLQ AHINHRH
• Equus caballus (Horse) YLSQRDLQ AHINHRH
• Salmo salar (Atlantic HVFCYDCALLHEKKMEKMCPGLTLYSCTDPVQRIEQCLRGLLYMCSIVP salmon) GCKRTYLS QRDLQAHVNHRH
• COMPND FRAGMENT PHOSPHOTYROSINE BINDING DOMAIN, UNP RESIDUES 106-
• COMPND 6 PROTEIN 1 COMPND 6 PROTEIN 1, .
• J.SIVARAMAN JRNL AUTH A.IYU, Y. P. LIM, X.ZHOU, S.K.SZE, G.R.GUY, J.SIVARAMAN
• REMARK 200 REMARK: ⁇ SF FILE CONTAINS FRIEDEL PAIRS.
• ATOM 215 0 ASP A 27 1.127 -27.240 21.320 1.00 40.15 O.

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