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• W2019503485 abstract "Integrin α2β1 is a major receptor required for activation and adhesion of platelets, through the specific recognition of collagen by the α2-I domain (α2-I), which binds fibrillar collagen via Mg2+-bridged interactions. The crystal structure of a truncated form of the α2-I domain, bound to a triple helical collagen peptide, revealed conformational changes suggestive of a mechanism where the ligand-bound I domain can initiate and propagate conformational change to the full integrin complex. Collagen binding by α2-I and fibrinogen-dependent platelet activity can be inhibited by snake venom polypeptides. Here we describe the inhibitory effect of a short cyclic peptide derived from the snake toxin metalloprotease jararhagin, with specific amino acid sequence RKKH, on the ability of α2-I to bind triple helical collagen. Isothermal titration calorimetry measurements showed that the interactions of α2-I with collagen or RKKH peptide have similar affinities, and NMR chemical shift mapping experiments with 15N-labeled α2-I, and unlabeled RKKH peptide, indicate that the peptide competes for the collagen-binding site of α2-I but does not induce a large scale conformational rearrangement of the I domain. Integrin α2β1 is a major receptor required for activation and adhesion of platelets, through the specific recognition of collagen by the α2-I domain (α2-I), which binds fibrillar collagen via Mg2+-bridged interactions. The crystal structure of a truncated form of the α2-I domain, bound to a triple helical collagen peptide, revealed conformational changes suggestive of a mechanism where the ligand-bound I domain can initiate and propagate conformational change to the full integrin complex. Collagen binding by α2-I and fibrinogen-dependent platelet activity can be inhibited by snake venom polypeptides. Here we describe the inhibitory effect of a short cyclic peptide derived from the snake toxin metalloprotease jararhagin, with specific amino acid sequence RKKH, on the ability of α2-I to bind triple helical collagen. Isothermal titration calorimetry measurements showed that the interactions of α2-I with collagen or RKKH peptide have similar affinities, and NMR chemical shift mapping experiments with 15N-labeled α2-I, and unlabeled RKKH peptide, indicate that the peptide competes for the collagen-binding site of α2-I but does not induce a large scale conformational rearrangement of the I domain. The integrins constitute a functionally versatile family of integral membrane receptors that mediate cell-cell and cell-extracellular matrix interactions through their regulation of cell adhesion, differentiation, migration, and the immune response (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9007) Google Scholar, 2Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6868) Google Scholar, 3Shimaoka M. Takagi J. Springer T.A. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 485-516Crossref PubMed Scopus (445) Google Scholar, 4Arnaout M.A. Mahalingam B. Xiong J.P. Annu. Rev. Cell Dev. Biol. 2005; 21: 381-410Crossref PubMed Scopus (419) Google Scholar, 5Luo B.H. Carman C.V. Springer T.A. Annu. Rev. Immunol. 2007; 25: 619-647Crossref PubMed Scopus (1245) Google Scholar). Signal transduction is bi-directional through both outside-in and inside-out mechanisms. All integrins are heterodimers composed of subunits α and β. Different combinations of subunits are expressed on different cell types with the interplay of 19 α and 8 β subunits, generating a family of 25 different heterodimers (3Shimaoka M. Takagi J. Springer T.A. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 485-516Crossref PubMed Scopus (445) Google Scholar, 5Luo B.H. Carman C.V. Springer T.A. Annu. Rev. Immunol. 2007; 25: 619-647Crossref PubMed Scopus (1245) Google Scholar). The integrin receptors share common structural features. The extracellular portions of the α and β subunits combine to form a globular “head” domain that is attached to a pair of membrane-spanning helical “stalks.” Signal transduction is believed to involve an allosteric rearrangement characterized by the separation and reorientation of the stalk segments. The bidirectional nature of signal transduction is complex. Extracellular ligands induce outside-in signals by binding to fixed motifs in the head domain, whereas inside-out signaling ensues from intracellular interactions between relatively short structurally plastic control elements and a large repertoire of cellular proteins. In nine of the human α subunits, ligand recognition is carried out by a 200-residue structurally conserved inserted (I) domain or a von Willebrand factor A domain (3Shimaoka M. Takagi J. Springer T.A. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 485-516Crossref PubMed Scopus (445) Google Scholar, 5Luo B.H. Carman C.V. Springer T.A. Annu. Rev. Immunol. 2007; 25: 619-647Crossref PubMed Scopus (1245) Google Scholar). The I and A domains adopt a Rossmann dinucleotide-binding fold, with a 6-stranded β-sheet surrounded by seven α-helices, and ligand recognition requires the binding of a single Mg2+ ion to a metal ion-dependent adhesion (MIDAS) 2The abbreviations used are: MIDAS, metal ion-dependent adhesion; HSQC, heteronuclear single quantum spectroscopy; ITC, isothermal titration calorimetry; HPLC, high pressure liquid chromatography. 2The abbreviations used are: MIDAS, metal ion-dependent adhesion; HSQC, heteronuclear single quantum spectroscopy; ITC, isothermal titration calorimetry; HPLC, high pressure liquid chromatography. motif (6Lee J.O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (804) Google Scholar, 7Emsley J. King S.L. Bergelson J.M. Liddington R.C. J. Biol. Chem. 1997; 272: 28512-28517Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). The importance of the α I domain for understanding conformational regulation and ligand binding for all integrins has been reviewed recently (5Luo B.H. Carman C.V. Springer T.A. Annu. Rev. Immunol. 2007; 25: 619-647Crossref PubMed Scopus (1245) Google Scholar). Integrin α2β1 is a member of the collagen/laminin receptor family and is a major receptor required for activation and adhesion of platelets, through the specific recognition of collagen by the α2-I domain (α2-I) (8Tuckwell D. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 108: 1629-1637Crossref PubMed Google Scholar), which binds fibrillar collagen via Mg2+-bridged interactions, supported by the MIDAS motif residues Asp-151, Ser-153, Thr-221, and Asp-254. The crystal structure of the α2-I domain, bound to a triple helical collagen peptide, revealed conformational changes from an unbound “closed” form to a bound “open” form, suggestive of a mechanism where the ligand-bound I domain can initiate and propagate conformational change to the full integrin complex (9Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar). Collagen binding and fibrinogen-dependent platelet activity can be inhibited by snake venom polypeptide toxins, enhancing the effects of hemorrhagic venom metalloproteases. These so-called disintegrins are functional homologues of the Arg-Gly-Asp (RGD) motif found in extracellular matrix proteins. Integrin α2β1 associates with Jararhagin, a 52-kDa metalloprotease isolated from the venom of the Brazilian pit viper Bothrops jararaca, that targets multiple components in hemostasis, including von Willebrand factor, fibrinogen, and platelet aggregation. Anti-platelet activity is thought to stem from its specificity for the α2β1 integrin (10Paine M.J. Desmond H.P. Theakston R.D. Crampton J.M. J. Biol. Chem. 1992; 267: 22869-22876Abstract Full Text PDF PubMed Google Scholar, 11De Luca M. Ward C.M. Ohmori K. Andrews R.K. Berndt M.C. Biochem. Biophys. Res. Commun. 1995; 206: 570-576Crossref PubMed Scopus (90) Google Scholar, 12Kamiguti A.S. Hay C.R. Theakston R.D. Zuzel M. Toxicon. 1996; 34: 627-642Crossref PubMed Scopus (186) Google Scholar, 13Kamiguti A.S. Hay C.R. Zuzel M. Biochem. J. 1996; 320: 635-641Crossref PubMed Scopus (151) Google Scholar, 14Laing G.D. Moura-da-Silva A.M. Toxicon. 2005; 45: 987-996Crossref PubMed Scopus (69) Google Scholar). Notably, a short cyclic peptide derived from the jararhagin metalloprotease domain, containing the specific amino acid sequence RKKH, is sufficient to prevent binding of type I collagen to α2-I in a competitive manner and is capable of disrupting cell adhesion to type I collagen (15Ivaska J. Kapyla J. Pentikainen O. Hoffren A.M. Hermonen J. Huttunen P. Johnson M.S. Heino J. J. Biol. Chem. 1999; 274: 3513-3521Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The cyclic RKKH peptide-binding site coincides with the collagen-binding site, near the I domain MIDAS motif (16Pentikainen O. Hoffren A.M. Ivaska J. Kapyla J. Nyronen T. Heino J. Johnson M.S. J. Biol. Chem. 1999; 274: 31493-31505Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The inhibitory effects of the RKKH peptides on the homologous α1β1 integrin have been suggested to reflect the ability of the peptide to mimic the natural type I collagen ligand by inducing or stabilizing a conformational transition from the closed to the open form of the I domain (17Nymalm Y. Puranen J.S. Nyholm T.K. Kapyla J. Kidron H. Pentikainen O.T. Airenne T.T. Heino J. Slotte J.P. Johnson M.S. Salminen T.A. J. Biol. Chem. 2004; 279: 7962-7970Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In this study we characterize the conformational dynamics of the α2-I domain and its interactions with collagen and RKKH peptides. Our studies show that the RKKH peptide binds the α2-I domain and inhibits its association with type I collagen, without inducing a conformational change in the α2-I domain. Materials—The pET-28b expression plasmid was from Invitrogen. (15NH4)2SO4, [13C]glucose, and D2O were purchased from Cambridge Isotopes Laboratories. Nickel-nitrilotriacetic acid-agarose was from Qiagen. The reverse phase Delta Pak C18 HPLC column (300 × 7.8 mm; 300 Å) was obtained from Waters. The 24-residue type I collagen peptide with the GFOGER recognition sequence (O = hydroxyproline, Hyp) was purchased in HPLC-purified powder form from Biomer Technology (Hayward, CA). The partially purified RKKH peptide was purchased from Genscript (Piscataway, NJ). The peptide sequences are shown in Fig. 1. Expression and Purification of Integrin α2-I Domain—The recombinant α2-I domain used in these studies corresponds to residues 144–334 of the human sequence (NCBI accession number NP_002194). The DNA coding for the sequence was inserted into the NdeI/XhoI site of the pET-28b expression plasmid (Invitrogen). For eventual chemical derivatization, Ser-334, at the C terminus, was mutated to Cys. Site-directed mutagenesis was used to convert Cys-150 to Leu. The resulting plasmid, pNHis-α2-I(144–334)C150L, has a His6 tag at the N terminus for purification. The sequence of the expressed protein is shown in Fig. 1. Recombinant α2-I domain was expressed in Escherichia coli strain BL21(DE3). To prepare 15N- and 13C-labeled protein for NMR experiments, cells bearing pNHis-α2-I(144–334)C150L were grown in minimal M9 media containing (15NH4)2SO4 and/or [13C]glucose. Induction at A600 = 1 with 1 mm isopropyl 1-thio-β-d-galactopyranoside for 2 h at 37 °C gave high level expression of the protein in the soluble fraction. Cells were lysed using a French press in buffer D (50 mm Tris, pH 8.0, 1 m NaCl, 30 mm imidazole), and the α2-I domain was purified by nickel-affinity chromatography on nickel-nitrilotriacetic acid-agarose, in buffer C (50 mm Tris, pH 7, 1 m NaCl, 300 mm imidazole). The purified protein solution was dialyzed against two changes of buffer A (50 mm Tris, pH 8.0, 150 mm NaCl) with 1 mm EDTA, followed by two changes of buffer B (50 mm PO4, pH 7.0, 150 mm NaCl, 5 mm MgSO4) with 1 mm dithiothreitol, using dialysis membranes with a molecular weight cutoff of 10,000. The final yield of purified protein was 20 mg/liter of cell culture. For NMR experiments, the protein solution was concentrated by ultrafiltration to 0.7 mm (ϵ = 17330 cm-1m-1). For experiments with the RKKH peptide, the surface-exposed C-terminal Cys residue of the α2-I domain was alkylated with iodoacetamide as follows. Purified protein was dialyzed against four changes of buffer A. The protein was removed from the dialysis bag, reacted with 10 mm iodoacetamide for 10 h at 4 °C, dialyzed against two changes of buffer B, and concentrated by ultrafiltration. Formation of Collagen Triple Helix—To obtain a collagen triple helix, the 24-residue collagen peptide powder was suspended in buffer B at 1 mm concentration, heated to 45 °C, and then allowed to equilibrate at 4 °C for at least 12 h, as described previously (18Feng Y. Melacini G. Taulane J.P. Goodman M. J. Am. Chem. Soc. 1996; 118: 10351-10358Crossref Scopus (191) Google Scholar). Formation of Cyclic RKKH Peptide—Formation of cyclic peptide was obtained by forming a disulfide link between the terminal Cys residues as described (15Ivaska J. Kapyla J. Pentikainen O. Hoffren A.M. Hermonen J. Huttunen P. Johnson M.S. Heino J. J. Biol. Chem. 1999; 274: 3513-3521Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The peptide was dissolved at 1 mg/ml concentration in 0.1 m NH4CO3 and incubated at 4 °C for 24 h. The reaction was flash-frozen and the water removed by lyophilization. The cyclized peptide was resuspended in HPLC grade water and purified by reverse phase HPLC. Peak fractions were combined, frozen, and lyophilized to powder. A colorimetric assay using 5,5′-dithiobis(2-nitrobenzoic acid), to test for the presence of free thiol, showed complete conversion to the cyclized product within the limits of detection. The cyclized peptide was suspended in buffer B immediately before experiments. NMR Spectroscopy—NMR experiments were performed on Bruker AVANCE 600- and 800-MHz spectrometers. The standard 1H/15N fast HSQC pulse sequence was used for experiments with peptides (19Mori S. Abeygunawardana C. Johnson M.O. Vanzijl P.C.M. J. Magn. Reson. 1995; 108: 94-98Crossref Scopus (569) Google Scholar). Backbone resonance assignments were made using a standard CBCA(CO)NH experiment (20Grzesiek S. Bax A. J. Am. Chem. Soc. 1992; 114: 6291-6293Crossref Scopus (927) Google Scholar) and by comparison with the assignments reported previously by Elshorst et al. (21Elshorst B. Jacobs D.M. Schwalbe H. Langer T. J. Biomol. NMR. 2003; 27: 191-192Crossref PubMed Scopus (1) Google Scholar) for the same polypeptide. The chemical shifts are referenced to the 1H2O resonance, set to its expected position of 4.87 ppm at 20 °C (22Cavanagh J. Fairbrother W.J. Palmer A.G. Skelton N.J. Protein NMR Spectroscopy: Principles and Practice. Academic Press, San Diego1996Google Scholar). The NMR data were processed using NMRPipe (23Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11548) Google Scholar), and the spectra were assigned and analyzed using Sparky (24Goddard T.D. Kneller D.G. SPARKY 3. University of California, San Francisco2004Google Scholar). All experiments were performed at millimolar concentrations of collagen or the RKKH to obtain saturation of the α2-I domain, for a single site binding model of interaction and the measured affinity of the peptides for α2-I (see below). Isothermal Titration Calorimetry (ITC)—The α2-I domain, the triple helix collagen peptide, and the cyclized RKKH peptide were all dissolved in buffer B. The pH of each solution was measured to ensure that no changes were produced by the polypeptide components. For the collagen binding experiments the concentration of α2-I domain in the cell was 100 μm and that of the collagen peptide solution 1 mm. For the RKKH binding experiments, the α2-I domain was Cys-alkylated with iodoacetamide, and its concentration in the sample cell was 100 μm. The concentration of cyclized RKKH was 1 mm. ITC experiments were performed with a Microcal VP-ITC calorimeter. Measurements were made by titration of collagen or RKKH peptide into the α2-I domain at a temperature of 10 °C. For titration experiments, the α2-I domain was degassed and placed in the 1.4-ml reaction cell. The collagen or RKKH peptides were loaded in the 250-μl injection syringe, and a series of 8-μl injections over 16 s were made, with a spacing of 500 s between injections over 300 min. The reference power was set to 20 μcal/s, and the stirring speed was 300 rpm. Parallel control experiments, to correct for the heat of mixing, were performed by adding the peptide to a sample cell containing only buffer without α2-I domain. The thermodynamic data were processed with the ORIGIN program (Microcal) to extract the enthalpic, entropic, and equilibrium constants. Nonlinear least squares fitting was done using a single site binding model. Size Exclusion Chromatography—Size exclusion chromatography was performed at 4 or 22 °C, using an Acta Prime flow system with a Superdex 75 10/300 column (GE Healthcare), running in buffer B plus 0.25 mm dithiothreitol. Samples of the α2-I domain (0.3 mm in buffer B) were combined with 0.15 mm of equilibrated collagen peptide at 4 °C in buffer B. Injections of the collagen and α2-I domain alone were done using the same buffer. The flow rate was 0.4 ml/min for the 22 °C experiments and 0.55 ml/min for the 4 °C experiments. The protein-collagen complexes were detected at UV wavelengths of 254 or 215 nm. For size comparison we utilized bovine serum albumin (66.3 kDa) and lysozyme (14.4 kDa). Association of α2-I with Type I Collagen Peptide and RKKH Disintegrin Peptide—In activated platelets α2β1 integrin has a high affinity for soluble collagen with an associated Kd of 35–90 nm (25Jung S.M. Moroi M. J. Biol. Chem. 1998; 273: 14827-14837Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), whereas the affinity of the α2-I domain for type I collagen measured by surface plasmon resonance is weaker, with a Kd in the low micromolar range (26Calderwood D.A. Tuckwell D.S. Eble J. Kuhn K. Humphries M.J. J. Biol. Chem. 1997; 272: 12311-12317Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 27Cruz M.A. Chen J. Whitelock J.L. Morales L.D. Lopez J.A. Blood. 2005; 105: 1986-1991Crossref PubMed Scopus (56) Google Scholar). Recognition by α2β1 resides totally within the collagen sequence GFOGER, where Glu cannot be replaced by Asp, and sequence recognition is entirely dependent upon the presence of a triple helical conformation (28Knight C.G. Morton L.F. Peachey A.R. Tuckwell D.S. Farndale R.W. Barnes M.J. J. Biol. Chem. 2000; 275: 35-40Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar). To determine the affinity of α2-I for the triple helical type I collagen peptide, we performed ITC experiments where the 26-residue collagen peptide, containing the GFOGER recognition sequence (Fig. 1B), was titrated into the α2-I domain. This peptide sequence is similar to that which was co-crystallized with α2-I (9Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar). To maintain the collagen peptide in its active triple helical form, the ITC cell temperature was kept at 10 °C, below the predicted melting temperature of about 20 °C (9Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar). The results are shown in Fig. 2A, and the free energy parameters are reported in Table 1. Fitting the ITC data to a single-site binding curve (Fig. 2C) yields a Kd of 7.8 μm.TABLE 1Thermodynamic binding constants for the α2-I domain measured by ITCPeptideKdΔGΔHTΔSμmkcal/molkcal/molkcal/mol(GPO)3GFOGER(GPO)3-NH27.8 ± 0.24–6.6–1.1715.44CTRKKHDNAQC-NH28.0 ± 0.33–6.6–0.2546.35 Open table in a new tab ITC was also performed to determine the affinity of α2-I for a cyclic RKKH peptide whose sequence had been previously identified to have the most potent inhibitory effect on α2-I (Fig. 1C) (15Ivaska J. Kapyla J. Pentikainen O. Hoffren A.M. Hermonen J. Huttunen P. Johnson M.S. Heino J. J. Biol. Chem. 1999; 274: 3513-3521Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). To enable direct comparison with the affinity determined for collagen, this study was also performed at 10 °C. Fig. 2B shows the titration profile, and the thermodynamic parameters are reported in Table 1. The data fit to a single-site binding model (Fig. 2D) with a Kd of 8.0 μm, consistent with the IC50 of 1.2 μm, estimated in competition assays for the inhibition of collagen binding (15Ivaska J. Kapyla J. Pentikainen O. Hoffren A.M. Hermonen J. Huttunen P. Johnson M.S. Heino J. J. Biol. Chem. 1999; 274: 3513-3521Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Characterization of the α2-I-Collagen Complex—To further characterize the formation of the α2-I-collagen complex, we performed size exclusion chromatography at temperatures below or above the melting transition of the collagen triple helix (Fig. 3, A and B). Isolated α2-I elutes with an apparent molecular mass near 30 kDa at both temperatures (peak a), whereas the collagen peptide elutes near 20 kDa (peak b). We attribute the differences between these observed values and those expected from the calculated molecular weights of the proteins (23 kDa for α2-I; 6.8 kDa for triple helical collagen peptide; 2.3 kDa for monomeric collagen peptide) to the hydrodynamic radii of the molecules, which govern the elution profiles. In particular, the elution of collagen is likely to be dominated by the rod-like shape of the triple helix. However, it is interesting to note that the peptide elutes at a slightly higher apparent molecular weight at 4 than 22 °C, reflecting triple helix formation below the melting temperature. This is further corroborated by the elution profiles of premixed α2-I and collagen. At 22 °C, the elution profile is identical to that of the individual components, with two resolved peaks corresponding to either α2-I (Fig. 3A, peak a) or collagen (Fig. 3A, peak b). However, when the molecules are combined and eluted at 4 °C, a new peak appears at a higher apparent molecular mass of about 45 kDa (Fig. 3B, peak c), indicating complex formation with triple helical collagen. These results are consistent with specific recognition by α2-I of the GFOGER sequence properly displayed in triple helical collagen (28Knight C.G. Morton L.F. Peachey A.R. Tuckwell D.S. Farndale R.W. Barnes M.J. J. Biol. Chem. 2000; 275: 35-40Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar). The size exclusion results in Fig. 3 further show that the α2-I domain and collagen form a stable long lived complex and help explain the NMR results, where the extreme broadening observed in the presence of collagen reflects the formation of a large slowly tumbling biomolecular species (see below). Effects of Mg2+and Collagen on α2-I—The α2-I domain possesses high affinity for type I collagen in the presence of the divalent metal cation Mg2+. The metal-binding site consists of MIDAS motif residues Asp-151, Ser-153, Ser-155, Thr-221, Asp-254, and Glu-256 which form an octahedral coordination sphere composed of direct and water-bridged interactions. The structural role of Mg2+ has been examined using both x-ray and NMR methods in the CD11a/LFA-1 I domain (29Qu A. Leahy D.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10277-10281Crossref PubMed Scopus (290) Google Scholar, 30Qu A. Leahy D.J. Structure (Lond.). 1996; 4: 931-942Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 31Legge G.B. Kriwacki R.W. Chung J. Hommel U. Ramage P. Case D.A. Dyson H.J. Wright P.E. J. Mol. Biol. 2000; 295: 1251-1264Crossref PubMed Scopus (61) Google Scholar, 32Shimaoka M. Xiao T. Liu J.H. Yang Y. Dong Y. Jun C.D. McCormack A. Zhang R. Joachimiak A. Takagi J. Wang J.H. Springer T.A. Cell. 2003; 112: 99-111Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar), where Mg2+ was found to play a role in ligand binding, and its removal did not cause large scale structural change in the CD11a/LFA-1 I domain. The crystal structure of α2-I bound to triple helical collagen suggests that collagen binding is accompanied by a large conformational rearrangement of the C-terminal helix, coupled with changes in the coordination the Mg2+ metal (9Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar). Collagen binding causes three concerted changes in the I domain; the loops of the MIDAS motif are perturbed because of a rearrangement upon insertion of a collagen Glu side chain into the metal coordination sphere; helices h6 and h7 rearrange to open up the top surface; and helix h7 moves downward to the opposite pole of the MIDAS motif. The rearrangement of helix h7 is thought to produce the large scale conformational changes experienced by the integrin heterodimer during signaling. To see if the protein dynamics and conformation associated with Mg2+ and collagen binding could be characterized in solution, we examined the 1H/15N HSQC NMR spectrum of α2-I in the presence or absence of metal and collagen. The 1H and 15N chemical shifts from protein backbone amide groups are very sensitive to changes in protein conformation or chemical environment and can be used to monitor the equilibrium exchange between states arising from free and ligand-bound protein (22Cavanagh J. Fairbrother W.J. Palmer A.G. Skelton N.J. Protein NMR Spectroscopy: Principles and Practice. Academic Press, San Diego1996Google Scholar). If the exchange rate is faster than the difference between the chemical shifts measured for the two states, then the system is in fast exchange, and one peak is observed at the population-weighted average chemical shift of the two states. NMR can be used to detect weak binding or minor conformational rearrangements, and chemical shift changes as small as 0.02 ppm have been reported for minor local effects on protein structure resulting from binding of small molecules or modifications, whereas much larger changes (>1 ppm) can reflect major conformational rearrangements (33Rajagopal P. Waygood E.B. Klevit R.E. Biochemistry. 1994; 33: 15271-15282Crossref PubMed Scopus (48) Google Scholar, 34Shuker S.B. Hajduk P.J. Meadows R.P. Fesik S.W. Science. 1996; 274: 1531-1534Crossref PubMed Scopus (1819) Google Scholar, 35Grzesiek S. Bax A. Clore G.M. Gronenborn A.M. Hu J.S. Kaufman J. Palmer I. Stahl S.J. Wingfield P.T. Nat. Struct. Biol. 1996; 3: 340-345Crossref PubMed Scopus (306) Google Scholar, 36Hoffman R.M. Li M.X. Sykes B.D. Biochemistry. 2005; 44: 15750-15759Crossref PubMed Scopus (22) Google Scholar). A sample of metal-free α2-I domain (isotopically enriched with 15N) was prepared by exhaustive dialysis against EDTA. The 1H-15N HSQC spectra of the metal-free and Mg2+-bound forms of the α2-I domain are shown in Fig. 4A. Several peaks undergo measurable frequency changes reflecting metal binding. A plot of the total change in 1H and 15N chemical shifts against amino acid number shows that the peaks with the largest frequency changes are localized to residues involved in direct coordination of Mg2+ in the MIDAS motif (Fig. 5).FIGURE 5Total change in α2-I domain backbone amide chemical shifts induced by the addition of Mg2+ from 0 to 1.7 mm (96% saturation). The total combined change in chemical shift (Δ) for each residue was calculated by adding the changes in 1H(ΔH) and 15N(ΔN) chemical shifts, according to the equation Δ= ((ΔH)2 + (ΔN/5)2)1/2, where the 15N chemical shift is scaled by 1/5 to account for the 5-fold difference between the chemical shift dispersions of 15N and 1H (35Grzesiek S. Bax A. Clore G.M. Gronenborn A.M. Hu J.S. Kaufman J. Palmer I. Stahl S.J. Wingfield P.T. Nat. Struct. Biol. 1996; 3: 340-345Crossref PubMed Scopus (306) Google Scholar, 36Hoffman R.M. Li M.X. Sykes B.D. Biochemistry. 2005; 44: 15750-15759Crossref PubMed Scopus (22) Google Scholar). Residue numbers are indicated for peaks with Δ ≥0.1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) This is further highlighted by mapping the frequency changes on the previously determined crystal structure of the α2-I domain (Fig. 6C). However, most of the peaks from other residues throughout the protein structure do not change, indicating that Mg2+ binding does not induce a large scale conformational change of the α2-I domain in solution. This is similar to the results reported for LFA-1 (31Legge G.B. Kriwacki R.W. Chung J. Hommel U. Ramage P. Case D.A. Dyson H.J. Wright P.E. J. Mol. Biol. 2000; 295: 1251-1264Crossref PubMed Scopus (61) Google Scholar). A potential indicator of close association between the metal-binding site and conformational change in helix h7 is the chemical shift change observed for Glu-318 in the spectra from metal-bound and unbound α2-I. Glu-318 is located in the loop preceding the C-terminal helix and is characterized by a distinctive downfield chemical shift (1H ∼ 11.5 ppm). In the comparison of the structures of the collagen-bound and unbound forms of the α2-I domain, it was noted that Glu-318 in the unbound form of α2-I domain is engaged in a salt bridge interaction with Arg-288, which is not present in the collagen-bound form (9Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abs" @default.
• W2019503485 created "2016-06-24" @default.
• W2019503485 creator A5000398535 @default.
• W2019503485 creator A5039256159 @default.
• W2019503485 creator A5050815439 @default.
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• W2019503485 date "2008-06-01" @default.
• W2019503485 modified "2023-09-28" @default.
• W2019503485 title "Competitive Interactions of Collagen and a Jararhagin-derived Disintegrin Peptide with the Integrin α2-I Domain" @default.
• W2019503485 cites W1532085934 @default.
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