The Crystal Structure and Mutational Binding Analysis of the Extracellular Domain of the Platelet-activating Receptor CLEC-2

Abstract

The human C-type lectin-like molecule CLEC-2 is expressed on the surface of platelets and signaling through CLEC-2 causes platelet activation and aggregation. CLEC-2 is a receptor for the platelet-aggregating snake venom protein rhodocytin. It is also a newly identified co-receptor for human immunodeficiency virus type 1 (HIV-1). An endogenous ligand has not yet been identified. We have solved the crystal structure of the extracellular domain of CLEC-2 to 1.6-Å resolution, and identified the key structural features involved in ligand binding. A semi-helical loop region and flanking residues dominate the surface that is available for ligand binding. The precise distribution of hydrophobic and electrostatic features in this loop will determine the nature of any endogenous ligand with which it can interact. Major ligand-induced conformational change in CLEC-2 is unlikely as its overall fold is compact and robust. However, ligand binding could induce a tilt of a 3–10 helical portion of the long loop region. Mutational analysis and surface plasmon resonance binding studies support these observations. This study provides a framework for understanding the effects of rhodocytin venom binding on CLEC-2 and for understanding the nature of likely endogenous ligands and will provide a basis for rational design of drugs to block ligand binding. The human C-type lectin-like molecule CLEC-2 is expressed on the surface of platelets and signaling through CLEC-2 causes platelet activation and aggregation. CLEC-2 is a receptor for the platelet-aggregating snake venom protein rhodocytin. It is also a newly identified co-receptor for human immunodeficiency virus type 1 (HIV-1). An endogenous ligand has not yet been identified. We have solved the crystal structure of the extracellular domain of CLEC-2 to 1.6-Å resolution, and identified the key structural features involved in ligand binding. A semi-helical loop region and flanking residues dominate the surface that is available for ligand binding. The precise distribution of hydrophobic and electrostatic features in this loop will determine the nature of any endogenous ligand with which it can interact. Major ligand-induced conformational change in CLEC-2 is unlikely as its overall fold is compact and robust. However, ligand binding could induce a tilt of a 3–10 helical portion of the long loop region. Mutational analysis and surface plasmon resonance binding studies support these observations. This study provides a framework for understanding the effects of rhodocytin venom binding on CLEC-2 and for understanding the nature of likely endogenous ligands and will provide a basis for rational design of drugs to block ligand binding. Platelet activation at sites of vascular injury is critical for primary hemostasis, but can also trigger arterial thrombosis in vascular disease. The C-type lectin-like protein CLEC-2 was recently shown to be expressed on platelets and signaling through CLEC-2 is sufficient to mediate platelet aggregation (1Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing G.D. Theakston R.D. Schweighoffer E. Zitzmann N. Morita T. Tybulewicz V.L. Ozaki Y. Watson S.P. Blood. 2006; 107: 542-549Crossref PubMed Scopus (382) Google Scholar). CLEC-2 was identified by sequence similarity to C-type lectin-like molecules with immune functions, such as the immunoreceptor NKG2D (2Colonna M. Samaridis J. Angman L. Eur. J. Immunol. 2000; 30: 697-704Crossref PubMed Scopus (188) Google Scholar). The gene encoding CLEC-2 is located in the human natural killer (NK) 2The abbreviations used are: NK, natural killer; HIV, human immunodeficiency virus; r.m.s.d., root mean square deviation; PEG, polyethylene glycol. complex on chromosome 12, along with the C-type lectin-like receptors NKG2D, LOX-1, and Dectin-1 (3Sobanov Y. Bernreiter A. Derdak S. Mechtcheriakova D. Schweighofer B. Duchler M. Kalthoff F. Hofer E. Eur. J. Immunol. 2001; 31: 3493-3503Crossref PubMed Scopus (97) Google Scholar). CLEC-2 is a 32-kDa type II transmembrane receptor, and transcripts have been identified in immune cells of myeloid origin, including monocytes, dendritic cells, and granulocytes, and in liver (2Colonna M. Samaridis J. Angman L. Eur. J. Immunol. 2000; 30: 697-704Crossref PubMed Scopus (188) Google Scholar). CLEC-2 has been shown to be a receptor on the surface of platelets for the snake venom toxin rhodocytin, which is produced by the Malayan pit viper Calloselasma rhodostoma (1Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing G.D. Theakston R.D. Schweighoffer E. Zitzmann N. Morita T. Tybulewicz V.L. Ozaki Y. Watson S.P. Blood. 2006; 107: 542-549Crossref PubMed Scopus (382) Google Scholar, 4Shin Y. Morita T. Biochem. Biophys. Res. Commun. 1998; 245: 741-745Crossref PubMed Scopus (75) Google Scholar). An endogenous CLEC-2 ligand has not yet been identified. Binding of rhodocytin to CLEC-2 leads to phosphorylation of a single tyrosine residue in a YXXL motif in the intracellular domain of CLEC-2. This phosphorylation promotes the binding of spleen tyrosine kinase (Syk), further downstream tyrosine phosphorylation events and the activation of PLCγ2 (1Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing G.D. Theakston R.D. Schweighoffer E. Zitzmann N. Morita T. Tybulewicz V.L. Ozaki Y. Watson S.P. Blood. 2006; 107: 542-549Crossref PubMed Scopus (382) Google Scholar). These signaling events result in platelet aggregation. The ability of rhodocytin or CLEC-2-specific antibodies to trigger platelet aggregation in the absence of other stimuli indicates the potency with which CLEC-2 can modulate platelet activity (1Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing G.D. Theakston R.D. Schweighoffer E. Zitzmann N. Morita T. Tybulewicz V.L. Ozaki Y. Watson S.P. Blood. 2006; 107: 542-549Crossref PubMed Scopus (382) Google Scholar). Therefore, CLEC-2 is a potential therapeutic target in thrombotic cardiovascular disease. In addition, snake bites remain a serious health issue in many countries. Envenomation from the viper family has a mortality rate of up to 15% and in Vietnam there are an estimated 30,000 snake bite victims per year, with the Malayan pit viper responsible for the majority (5Southeast Asian J. Trop. Med. Public Health. 1999; 30: 1-85Google Scholar). A molecular understanding of the effects of rhodocytin could lead to more effective therapy of snake envenomation. The mechanism of action of venoms is of great biological interest. CLEC-2 was also recently identified as a novel human immunodeficiency virus type 1 (HIV-1) attachment factor, which promotes virus capture by cells and platelets (6Chaipan C. Soilleux E.J. Simpson P. Hofmann H. Gramberg T. Marzi A. Geier M. Stewart E.A. Eisemann J. Steinkasserer A. SuzukiInoue K. Fuller G.L. Pearce A.C. Watson S.P. Hoxie J.A. Baribaud F. Pohlmann S. J. Virol. 2006; 80: 8951-8960Crossref PubMed Scopus (198) Google Scholar). To better understand the ligand binding properties of CLEC-2 and to provide a framework for the rational design of inhibitory drugs, we have crystallized the extracellular domain of CLEC-2 and used synchrotron x-ray diffraction to solve its atomic structure to 1.6-Å resolution. We used this structural information to design alanine mutations of surface residues and have studied the effects of these mutations on ligand binding using surface plasmon resonance. CLEC-2 Crystallization, Data Collection, and Structure Determination—The extracellular domain of CLEC-2 from residues 96 to 221 was cloned, expressed, purified, and crystallized as described previously (7O'Callaghan C.A. Cerwenka A. Willcox B.E. Lanier L.L. Bjorkman P.J. Immunity. 2001; 15: 201-211Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 8Watson A.A. O'Callaghan C.A. Acta Crystallogr. Sect F Struct. Biol. Cryst. Commun. 2005; 61: 1094-1096Crossref PubMed Scopus (21) Google Scholar). Preliminary diffraction data were collected to 2.0 Å as described (8Watson A.A. O'Callaghan C.A. Acta Crystallogr. Sect F Struct. Biol. Cryst. Commun. 2005; 61: 1094-1096Crossref PubMed Scopus (21) Google Scholar). Subsequent molecular replacement was unsuccessful with the programs Molrep, AMoRe, Resolve, and EPMR. Therefore, a composite search model was constructed for molecular replacement with CaspR from the following related proteins (PDB accession codes are in parentheses): LOX-1 (1YPQ), NKG2D (1KCG), DC-Sign (1K9I), Ly49C (1P1Z), Ly49A (1QO3), and DC-SignR (1XPH) (9Claude J.B. Suhre K. Notredame C. Claverie J.M. Abergel C. Nucleic Acids Res. 2004; 32: W606-W609Crossref PubMed Scopus (80) Google Scholar). The resulting solution was incomplete in two loops. After further crystal optimization, a 1.6-Å dataset was collected from a dissected crystal at the Synchrotron Radiation Source, Daresbury Laboratory, UK. This crystal was grown in 0.1 m bis-Tris pH6.5, 20% PEG monomethyl ether 5000, and cryoprotected in 20% glycerol. The synchrotron beam was collimated to 50 × 50 μm to focus it on the central high quality portion of the crystal. Data were collected using 1° oscillations and auto-indexed, integrated, and scaled using the HKL2000 programs (10Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38566) Google Scholar). The volume per unit weight (VM) of this crystal was 1.81 Å3 Da-1, which is typical of crystals displaying diffraction to this resolution (11Kantardjieff K.A. Rupp B. Protein Sci. 2003; 12: 1865-1871Crossref PubMed Scopus (602) Google Scholar). The structure was determined from this dataset using the incomplete CLEC-2 structure from the earlier 2.0-Å dataset as a new CaspR search model. Initial refinement was performed using rigid body refinement and energy minimization algorithms implemented by CNS solve and Refmac5 (12Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16965) Google Scholar, 13Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13865) Google Scholar). The ARP/wARP program was used to trace and build a model into the refined electron density map (14Perrakis A. Morris R. Lamzin Struct. Biol. 1999; PubMed Scopus Google Scholar). Subsequent and refinement used and Refmac5 P. K. Acta Crystallogr. D Biol. Crystallogr. 2004; PubMed Scopus Google Scholar). were to the and including the of refinement but we were to model the residues in the crystallized protein of of the electron density in this region. An of this is these of minimization E. K. Acta Crystallogr. D Biol. Crystallogr. PubMed Scopus Google Scholar, A.T. Full Text Full Text PDF PubMed Scopus Google Scholar). The and is the for determined at this resolution A.T. Full Text Full Text PDF PubMed Scopus Google Scholar). it was not to model the residues of the C-type lectin-like domain in the crystal of the related proteins NKG2D, LOX-1, and Ly49C S. B. M. P.D. Immunity. 2001; 15: Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. S. M. T. D. S. K. S. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J. R. K. N. P. Immunol. 2003; PubMed Scopus Google data and diffraction for the resolution are in of molecules in is the for the of an with and model and refinement of using data which were used for the in in in in mean square for the resolution are in is the for the of an with and using data which were used for the in a new The structure was in the with accession and J.M. J. Crystallogr. Google Scholar, J. PubMed Scopus Google Scholar). are to in the surface were using and were using and and with A. K.A. B. Struct. PubMed Scopus Google Scholar, W. A. K. J. PubMed Scopus Google Scholar, P.J. J. Crystallogr. Google Scholar, J. 1997; PubMed Scopus Google Scholar, E.A. Acta Crystallogr. D Biol. Crystallogr. PubMed Scopus Google Scholar). was used to and the likely of of CLEC-2 Acta Crystallogr. D Biol. Crystallogr. 2004; PubMed Scopus Google Scholar). The was to the crystal structure of CLEC-2 to an of protein which a of Acta Crystallogr. D Biol. Crystallogr. 2004; PubMed Scopus Google Scholar). from the were used to analysis on this Acta Crystallogr. D Biol. Crystallogr. 2004; PubMed Scopus Google Scholar). The of the were with W. A. K. J. PubMed Scopus Google Scholar, Acta Crystallogr. D Biol. Crystallogr. 2004; PubMed Scopus Google Scholar). and of the and of rhodocytin were constructed using the Protein program for more T. J. N. Nucleic Acids Res. 2003; 31: PubMed Scopus Google Scholar). The was by the and of rhodocytin a of the related C-type snake venom protein using This model of the was using energy minimization algorithms implemented by CNS solve (12Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16965) Google Scholar). of the complex were using a implemented in the program S. 2004; PubMed Scopus Google Scholar). The was used to and with surface and and electrostatic S. 2004; PubMed Scopus Google Scholar). were to minimization using to potential for more of the S. 2004; PubMed Scopus Google Scholar). To these a of biological was to the model. were on the basis of surface electrostatic and the of the CLEC-2 this model of the and from of the binding properties of C-type lectin-like CLEC-2 residues were for studies to determine in ligand binding 2003; PubMed Scopus (97) Google Scholar). were with R. W. J. 1998; Scopus Google Scholar). CLEC-2 and Binding was used to single alanine mutations in CLEC-2 at residues and These proteins were expressed, and in the as CLEC-2 and and (8Watson A.A. O'Callaghan C.A. Acta Crystallogr. Sect F Struct. Biol. Cryst. Commun. 2005; 61: 1094-1096Crossref PubMed Scopus (21) Google Scholar). plasmon resonance were performed using a and were as described previously for NKG2D and its ligands (7O'Callaghan C.A. Cerwenka A. Willcox B.E. Lanier L.L. Bjorkman P.J. Immunity. 2001; 15: 201-211Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). was from the venom of C. rhodostoma as described previously W. D. Eble J.A. R. H. C. R. B. J. Biol. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. B. A. J. Biol. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). rhodocytin was to using The nature of rhodocytin the of data with CLEC-2 on the were at in 1 1 of CLEC-2 and proteins were were from to the for a at was using the as implemented in the program In the from cells with rhodocytin was with the from cells with an resolution x-ray diffraction data were from crystals of protein quality electron density was for the protein from residue of the structure The of CLEC-2 key features of the C-type lectin-like with two by two and The structure was with related which protein ligands and for which ligand binding data is the density receptor LOX-1 and the immune receptors NKG2D and which complex molecules S. B. M. P.D. Immunity. 2001; 15: Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. S. M. T. D. S. K. S. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J. R. K. N. P. Immunol. 2003; PubMed Scopus Google Scholar). These the overall as CLEC-2 are Å Å and Å However, these related proteins are CLEC-2 as CLEC-2 was in solution as a and there is of in the crystal structure and with of CLEC-2 a (1Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing G.D. Theakston R.D. Schweighoffer E. Zitzmann N. Morita T. Tybulewicz V.L. Ozaki Y. Watson S.P. Blood. 2006; 107: 542-549Crossref PubMed Scopus (382) Google Scholar). human and CLEC-2 rhodocytin as not its overall similarity to other C-type lectin-like features of CLEC-2 are likely to for its is an 3–10 in the long loop region 2The semi-helical long loop region. semi-helical long loop region the surface of CLEC-2. The loop region is shown in with the to the the central is the and the the CLEC-2 is the C-type lectin-like molecule with a in the long loop region. The long loop of LOX-1, and NKG2D are and The the for long loop region is a which is the key of in of related molecules solved to 2003; PubMed Scopus (97) Google Scholar). The surface of CLEC-2 is and the semi-helical long loop region of the surface on the of the molecule to that of the CLEC-2 is the C-type lectin-like structure this loop a 2003; PubMed Scopus (97) Google Scholar). The long loop region of the immunoreceptor Ly49C a residue with 3–10 helical features and a key of the binding with its ligand J. R. K. N. P. Immunol. 2003; PubMed Scopus Google Scholar). In LOX-1, the long loop region a which with in T. T. S. M. T. D. S. K. S. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The loop in NKG2D key binding with its protein ligands and is by residues which the S. B. M. P.D. Immunity. 2001; 15: Full Text Full Text PDF PubMed Scopus Google Scholar). The residues in the loop and flanking of NKG2D and CLEC-2 are that are likely to a in rhodocytin binding by CLEC-2. CLEC-2 not on rhodocytin, as the venom protein is not This was by with and The absence of was by and by and CLEC-2 of the residues which are in the long loop region in related binding molecules at In the binding these residues a and the with the R. R. K. PubMed Scopus Google Scholar). are in the CLEC-2 crystal on CLEC-2 is unlikely to a in ligand as of the two potential sites and in the structure is to the binding the protein is expressed in and is but can to rhodocytin. in the is in the of conformational change in the binding of related such as NKG2D, which structural ligand binding S. B. M. P.D. Immunity. 2001; 15: Full Text Full Text PDF PubMed Scopus Google Scholar, C.A. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, T. D. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Major ligand-induced conformational in CLEC-2 are unlikely as energy be to the and hydrophobic which its and provide hydrophobic compact and through the of The hydrophobic region two tyrosine and by a tyrosine a in protein M. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The hydrophobic the two of CLEC-2 and two The hydrophobic region the long loop region and a motif in which the with a at The of these hydrophobic is by a further hydrophobic the motif in to the 2003; PubMed Scopus (97) Google Scholar). binding is unlikely to these hydrophobic but could change the long loop region. The of the hydrophobic described are with The of the the long loop region the helical portion is unlikely to of the loop residues on of it more binding to CLEC-2 could the of these more the to tilt on its long The absence of any or the is with this This of the of CLEC-2 is with of the crystal A for CLEC-2 the likely of the long loop region of CLEC-2 and that its are from the of the molecule This also that the 3–10 is likely to as an unit are by in the In the central and in the of is by a and the and of the This a on of with a of the surface available for ligand binding on the the of the long loop of CLEC-2 could an of the 3–10 In an of the more related protein a 3–10 in an a in ligand binding and is also in an P. S. M. E. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Mutational and Binding the Binding of the ligand binding properties of were constructed of rhodocytin, and of the of CLEC-2 and rhodocytin A and A binding the long loop region of CLEC-2 a surface of rhodocytin with a surface of are molecules at this surface in the CLEC-2 crystal structure and potential a to the energy of binding and to the of the The binding surface on CLEC-2 is by residues in the long loop region and its flanking region. residues and the long loop region two which hydrophobic with the ligand A and To the of these residues ligand binding and to the binding surface of we produced CLEC-2 proteins with alanine mutations of surface residues and The molecular of were by as described previously (8Watson A.A. O'Callaghan C.A. Acta Crystallogr. Sect F Struct. Biol. Cryst. Commun. 2005; 61: 1094-1096Crossref PubMed Scopus (21) Google Scholar). We used surface plasmon resonance to binding for rhodocytin with that of CLEC-2. We that CLEC-2 not to in a CLEC-2 rhodocytin with an of This is the of for the binding of other C-type lectin-like molecules (7O'Callaghan C.A. Cerwenka A. Willcox B.E. Lanier L.L. Bjorkman P.J. Immunity. 2001; 15: 201-211Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, Immunity. 2003; Full Text Full Text PDF PubMed Scopus (75) Google Scholar, S. M. Z. M. P.D. J. Immunol. PubMed Scopus Google Scholar). with the loop residue mutations and a in binding for rhodocytin as by the flanking residue to an alanine the of CLEC-2 with rhodocytin, with a in to surface plasmon resonance is from the is the are by the the in the and the type are from the type analysis of by from the type analysis of by from the type analysis of by from the type analysis of by from the type analysis of by from the type analysis of by from the type analysis of by from the type analysis of by in a new These are with model of the The long loop region with effects from the of the loop from the helical and from flanking on the of CLEC-2 or are for binding by rhodocytin in model of the complex not the The the energy determined for to the for the are with the of a with rhodocytin, as the of this from that of the by This study the crystal structure of human CLEC-2 and a analysis of the with rhodocytin. It provides a basis for studies of the of CLEC-2. The potential binding surface is by the long loop region and this loop a 3–10 Mutational analysis the of this loop region in ligand binding. A key is binding of a ligand to the extracellular domain of CLEC-2 intracellular phosphorylation events and platelet aggregation. surface receptors such as the receptor by a conformational change ligand binding M. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). binding to CLEC-2 is unlikely to a conformational change in the overall CLEC-2 but could in the semi-helical long loop region. of the long loop region this as with that are from of the of the These are in with in the binding to rhodocytin, resulting from mutations in these The to have the to resulting in a or tilt of the 3–10 the The flanking are also of which will the binding ligand be with of the surface available for binding and a tilt in the of the 3–10 The of and hydrophobic residues in the human CLEC-2 long loop and flanking region will binding properties to CLEC-2 and is in and CLEC-2. residues and the loop to alanine the of human CLEC-2 for rhodocytin by up to The energy of the binding of CLEC-2 as of CLEC-2 are with the of and with rhodocytin. This a for the loop and flanking region in ligand binding. The semi-helical loop region provide of and attachment to its with binding by flanking However, as this region is from the any change in the loop is unlikely to be through the compact extracellular domain of CLEC-2 to the binding the signaling of CLEC-2 molecules into is in signaling receptors such as the receptor tyrosine kinase and rhodocytin of CLEC-2 F. N. Res. 2003; PubMed Scopus Google Scholar). The related NKG2D, and Ly49C have hydrophobic that a S. B. M. P.D. Immunity. 2001; 15: Full Text Full Text PDF PubMed Scopus Google Scholar, J. R. K. N. P. Immunol. 2003; PubMed Scopus Google Scholar, K. 2000; PubMed Scopus Google Scholar). However, in CLEC-2 there is an alanine and a residue in this which not in solution and be unlikely to at the It is that this a in ligand-induced rhodocytin is and will an binding surface for ligand binding of CLEC-2 on the platelet surface K. Ozaki Y. M. Y. Y. Y. T. T. K. Morita T. J. Biol. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). surface of CLEC-2 receptors on platelets signaling for phosphorylation and by rhodocytin is likely to CLEC-2 on the platelet this the that an endogenous ligand also CLEC-2. This study the structure of CLEC-2 and the binding the of the long loop region with its 3–10 to have with the to with the semi-helical loop and flanking region of which are The structure and binding analysis of CLEC-2 indicates that an endogenous ligand is likely to be a protein with a binding surface and hydrophobic The ligand is likely to be by the CLEC-2 long loop with from flanking C-type lectin-like the fold of the of C-type but are not binding CLEC-2 rhodocytin and is unlikely to on its natural ligand it of the features with or binding. CLEC-2 also the binding motif in the related receptor, and is unlikely to the binding Y. T. Y. A. H. J. S. N. 2004; PubMed Scopus Google Scholar). In to into the binding of and endogenous ligands by CLEC-2 on the structure and binding analysis of CLEC-2 provides a for the rational design of inhibitory drugs of great potential in cardiovascular and snake and which target the long loop region be likely to activation of We at the and and Watson for and of the and to the two for a of rhodocytin. with