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• W2146604114 abstract "Most mammalian cells and some pathogenic bacteria are capable of adhering to collagenous substrates in processes mediated by specific cell surface adherence molecules. Crystal structures of collagen-binding regions of the human integrin α2β1 and a Staphylococcus aureus adhesin reveal a “trench” on the surface of both of these proteins. This trench can accommodate a collagen triple-helical structure and presumably represents the ligand-binding site (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512–28517; Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., Schneider, A., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 4, 833–838). We report here the crystal structure of the α subunit I domain from the α1β1 integrin. This collagen-binding protein also contains a trench on one face in which the collagen triple helix may be docked. Furthermore, we compare the collagen-binding mechanisms of the human α1 integrin I domain and the A domain from the S. aureus collagen adhesin, Cna. Although the S. aureus and human proteins have unrelated amino acid sequences, secondary structure composition, and cation requirements for effective ligand binding, both proteins bind at multiple sites within one collagen molecule, with the sites in collagen varying in their affinity for the adherence molecule. We propose that (i) these evolutionarily dissimilar adherence proteins recognize collagen via similar mechanisms, (ii) the multisite, multiclass protein/ligand interactions observed in these two systems result from a binding-site trench, and (iii) this unusual binding mechanism may be thematic for proteins binding extended, rigid ligands that contain repeating structural motifs. Most mammalian cells and some pathogenic bacteria are capable of adhering to collagenous substrates in processes mediated by specific cell surface adherence molecules. Crystal structures of collagen-binding regions of the human integrin α2β1 and a Staphylococcus aureus adhesin reveal a “trench” on the surface of both of these proteins. This trench can accommodate a collagen triple-helical structure and presumably represents the ligand-binding site (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512–28517; Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., Schneider, A., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 4, 833–838). We report here the crystal structure of the α subunit I domain from the α1β1 integrin. This collagen-binding protein also contains a trench on one face in which the collagen triple helix may be docked. Furthermore, we compare the collagen-binding mechanisms of the human α1 integrin I domain and the A domain from the S. aureus collagen adhesin, Cna. Although the S. aureus and human proteins have unrelated amino acid sequences, secondary structure composition, and cation requirements for effective ligand binding, both proteins bind at multiple sites within one collagen molecule, with the sites in collagen varying in their affinity for the adherence molecule. We propose that (i) these evolutionarily dissimilar adherence proteins recognize collagen via similar mechanisms, (ii) the multisite, multiclass protein/ligand interactions observed in these two systems result from a binding-site trench, and (iii) this unusual binding mechanism may be thematic for proteins binding extended, rigid ligands that contain repeating structural motifs. Trench-shaped binding sites promote multiple classes of interactions between collagen and the adherence receptors, α1β1 integrin andStaphylococcus aureusCna MSCRAMM.Journal of Biological ChemistryVol. 274Issue 40PreviewOne of the contributing authors, Dr. Jindrich Symersky, was inadvertently omitted from the author list. The correct list is shown above. Full-Text PDF Open Access Staphylococcus aureus collagen adhesin microorganism surface component recognizing adhesive matrix molecules metal ion-dependent adhesion site polyacrylamide gel electrophoresis surface plasmon resonance spectroscopy Collagen polypeptides are largely composed of repeats of the GP X tripeptide and associate to form triple-helical monomers. These monomers combine into macroscopic fibers. Prokaryotic and eukaryotic cells bind collagen via receptors on their cell surfaces (1Switalski L.M. Patti J.M. Butcher W. Gristina A.G. Spieziale P. Höök M. Mol. Microbiol. 1993; 7: 99-107Crossref PubMed Scopus (159) Google Scholar, 2Emödy L. Heesemann J. Wolf-Watz H. Skurnik K. Kapperud P. Wadström T. J. Bacteriol. 1989; 171: 6674-6679Crossref PubMed Scopus (79) Google Scholar, 3Westerlund B. Kuusela P. Ristelli J. Risteli L. Vartio T. Rauvala H. Virkola R. Korhonen T.K. Mol. Microbiol. 1989; 3: 329-337Crossref PubMed Scopus (109) Google Scholar, 4Vercelloti G.M. McCarthy J.B. Lindholm P. Peterson P.K. Jacob H.S. Furcht L.T. Am. J. Pathol. 1985; 120: 13-21PubMed Google Scholar, 5Santoro S.A. Cowen J.F. Collagen Relat. Res. 1982; 2: 31-43Crossref PubMed Scopus (29) Google Scholar, 6Wayner E.A. Cater W.G. J. Cell Biol. 1987; 105: 1873-1884Crossref PubMed Scopus (540) Google Scholar). We now hypothesize that to accommodate such an unusually shaped ligand, the collagen-binding surface proteins of these cells must adopt an atypical binding-site structure. Bacterial pathogens utilize this interaction as a means of adherence to collagenous host tissues. Some Staphylococcus aureus strains express an adhesin, Cna,1 of the MSCRAMM class that binds collagen (1Switalski L.M. Patti J.M. Butcher W. Gristina A.G. Spieziale P. Höök M. Mol. Microbiol. 1993; 7: 99-107Crossref PubMed Scopus (159) Google Scholar, 7Symersky J. Patti J.M. Carson M. House-Pompeo K. Teale M. Moore D. Jin L. Schneider A. DeLucas L.J. Höök M. Narayana S.V.L. Nat. Struct. Biol. 1997; 4: 833-838Crossref PubMed Scopus (123) Google Scholar, 8Patti J.M. House-Pompeo K. Boles J.O. Garza N. Gurusiddappa S. Höök M. J. Biol. Chem. 1995; 270: 12005-12011Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 9Patti J.M. Bremell T. Krajewska-Pietrasik D. Abdelnour A. Tarkowski A. Ryden C. Höök M. Infect. Immun. 1994; 62: 152-161Crossref PubMed Google Scholar, 10House-Pompeo K. Boles J.O. Höök M. Methods (Orlando). 1994; 6: 134-142Crossref Scopus (22) Google Scholar, 11Patti J.M. Boles J.O. Höök M. Biochemistry. 1993; 32: 11428-11435Crossref PubMed Scopus (118) Google Scholar, 12Patti J.M. Jonsson H. Guss B. Switalski L.M. Wiberg K. Lindberg M. Höök M. J. Biol. Chem. 1992; 267: 4766-4772Abstract Full Text PDF PubMed Google Scholar, 13Rich R.L. Demeler B. Ashby K. Deivanayagam C.C.S. Petrich J.W. Patti J.M. Narayana S.V.L. Höök M. Biochemistry. 1998; 37: 15423-15433Crossref PubMed Scopus (48) Google Scholar). Cna from S. aureus FDA 574 is depicted in Fig.1 a: it contains two major domains, A and B, in addition to features characteristic of cell-surface proteins on Gram-positive bacteria (11Patti J.M. Boles J.O. Höök M. Biochemistry. 1993; 32: 11428-11435Crossref PubMed Scopus (118) Google Scholar). The collagen-binding site has been localized within the Cna A domain (12Patti J.M. Jonsson H. Guss B. Switalski L.M. Wiberg K. Lindberg M. Höök M. J. Biol. Chem. 1992; 267: 4766-4772Abstract Full Text PDF PubMed Google Scholar). Binding analyses demonstrate that (i) a synthetic peptide mimicking a short sequence of the A domain can inhibit collagen binding to S. aureus (8Patti J.M. House-Pompeo K. Boles J.O. Garza N. Gurusiddappa S. Höök M. J. Biol. Chem. 1995; 270: 12005-12011Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar); (ii) the A domain/collagen interaction involves more than one affinity class and multiple sites of contact within a single collagen molecule (8Patti J.M. House-Pompeo K. Boles J.O. Garza N. Gurusiddappa S. Höök M. J. Biol. Chem. 1995; 270: 12005-12011Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10House-Pompeo K. Boles J.O. Höök M. Methods (Orlando). 1994; 6: 134-142Crossref Scopus (22) Google Scholar); and (iii) the B domain does not alter the collagen binding ability of the A domain (13Rich R.L. Demeler B. Ashby K. Deivanayagam C.C.S. Petrich J.W. Patti J.M. Narayana S.V.L. Höök M. Biochemistry. 1998; 37: 15423-15433Crossref PubMed Scopus (48) Google Scholar). The crystal structure of a truncated form of the Cna A domain reveals a binding-site “trench” on one face of the protein. In molecular modeling studies, this trench was found to accommodate a triple-helical peptide that mimics the collagen structure. Symersky et al. (7Symersky J. Patti J.M. Carson M. House-Pompeo K. Teale M. Moore D. Jin L. Schneider A. DeLucas L.J. Höök M. Narayana S.V.L. Nat. Struct. Biol. 1997; 4: 833-838Crossref PubMed Scopus (123) Google Scholar) noted that this trench complemented well the structure of a collagen triple helix and binding studies of site-specific mutants of the S. aureus Cna truncate revealed that (i) no single residue or area within the trench was responsible for collagen binding, but rather, a number of contacts contributed to the protein/collagen interaction and (ii) this binding-domain truncate bound to multiple sites along a collagen molecule. The affinity of Cna for an individual site within collagen may be the consequence of the number of “good” and “bad” contacts within the binding trench. Binding of eukaryotic cells to collagen serves not only as a mechanism of tissue adherence, but also may induce a complex signaling cascade in the cell. Attachment of eukaryotic cells to the extracellular matrix is primarily mediated by integrins. The integrins are transmembrane αβ heterodimeric proteins that direct cell-cell and cell-matrix interactions and are found on most mammalian cells. To date, several integrins, including α1β1, α2β1, α3β1, α9β1, α10β1, and αMβ2, have been reported to mediate cellular adherence to collagen (14Calderwood D.A. Tuckwell D.S. Humphries M.J. Biochem. Soc. Trans. 1995; 23: 504SCrossref PubMed Google Scholar, 15Forsberg E. Ek B. Engström Å. Johansson S. Exp. Cell Res. 1994; 213: 183-190Crossref PubMed Scopus (30) Google Scholar, 16Wayner E.A. Carter W.G. J. Cell Biol. 1987; 105: 1873-1884Crossref PubMed Google Scholar, 17Camper L. Hellman U. Lundgren-Åkerland E. J. Biol. Chem. 1998; 273: 20383-20389Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 18Walzog B. Schuppan D. Heimpel C. Hafezi-Moghadam A. Gaehtgens P. Ley K. Exp. Cell Res. 1995; 218: 28-38Crossref PubMed Scopus (71) Google Scholar). Of these, α1β1 and α2β1are apparently the primary collagen-binding integrins. The α1, α2, and α10 subunits each contain an “inserted” (I) domain near the N terminus (Fig.1 c). The I domains have been shown to contain a ligand-binding site and a MIDAS motif, which needs to be occupied by an appropriate cation for effective ligand binding by the integrin. Recombinant proteins duplicating these small (approximately 200 amino acids) I domain polypeptide segments effectively bind collagen, presumably it is these regions that are responsible for the integrins' binding to collagens. A binding site-trench in the I domain of the human α2β1 integrin was also suggested by the crystal structure of the α2 integrin I domain. Molecular modeling of this protein complexed with a collagen triple-helical peptide (19Emsley 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) demonstrated favorable ligand docking encompassing about 10 residues of the collagen sequence within a trench spanning the MIDAS motif. From this work Liddington and co-workers (19Emsley 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) suggested that the divalent cation is involved in direct ligand binding via coordination of an amino acid residue (most probably, glutamate) within the collagen molecule. Our previous work has shown that the full-length A domain of the S. aureus Cna protein binds collagen more efficiently than the binding-domain truncate does (7Symersky J. Patti J.M. Carson M. House-Pompeo K. Teale M. Moore D. Jin L. Schneider A. DeLucas L.J. Höök M. Narayana S.V.L. Nat. Struct. Biol. 1997; 4: 833-838Crossref PubMed Scopus (123) Google Scholar, 8Patti J.M. House-Pompeo K. Boles J.O. Garza N. Gurusiddappa S. Höök M. J. Biol. Chem. 1995; 270: 12005-12011Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10House-Pompeo K. Boles J.O. Höök M. Methods (Orlando). 1994; 6: 134-142Crossref Scopus (22) Google Scholar, 12Patti J.M. Jonsson H. Guss B. Switalski L.M. Wiberg K. Lindberg M. Höök M. J. Biol. Chem. 1992; 267: 4766-4772Abstract Full Text PDF PubMed Google Scholar). The causes of this behavior have not been investigated to date. In addition, detailed analysis of collagen-binding activity of the human α1integrin I domain, which binds Type I collagen more efficiently than the α2 integrin I domain does (14Calderwood D.A. Tuckwell D.S. Humphries M.J. Biochem. Soc. Trans. 1995; 23: 504SCrossref PubMed Google Scholar), has not been performed. The questions we seek to answer here include: (a) can the gross structure of I domains and the detailed topology of their MIDAS-centered binding site be determined from modeling experiments based on known structures? (b) Is a trench similar to that found on Cna the binding-site motif employed by the α1β1 integrin? (c) Does the α1 integrin I domain bind to a single or multiple class(es) of sites within a collagen macromolecule? To address these questions, we compare the structures and collagen-binding characteristics of the S. aureus Cna A and the human α1 integrin I domains. The expression plasmid pQE-α1I was constructed based on the vector pQE-30 (Qiagen Inc., Chatsworth, CA) using standard molecular biology protocols (20Sambrook J. Fritsch E.F. Maniatas T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar, 21Ausubel F.A. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing and Wiley-Interscience, New York1991Google Scholar). cDNA encoding the I domain of the human α1 integrin was obtained by polymerase chain reaction using a human hepatoma cDNA library as a template and the oligonucleotide primers, 5′-CGGATCCCCCACATTTCAAGTCGTGAAT-3′ and 5′-GCTGCAGTCATATTCTTTCTCCCAGAGTTTT-3′. The amplified gene fragment was digested with the Bam HI and the Pst I restriction endonucleases, purified by agarose gel electrophoresis (Geneclean kit, ISC BioExpress), and ligated into the vector pQE-30 (previously linearized by digestion with the same endonucleases). Ligation mixtures were subsequently transformed into Escherichia coli strain JM101. Plasmids from isolated transformants were analyzed by restriction digestions and automated DNA sequencing analysis (Molecular Genetics Core Facility, University of Texas Medical School, Houston, TX) to confirm the expected open reading frame. The α1 integrin I domain sequence examined here corresponds to the sequence published by Briesewitz et al. (22Briesewitz R. Epstein M.R. Marcantonio E.E. J. Biol. Chem. 1993; 268: 2989-2996Abstract Full Text PDF PubMed Google Scholar) except for nucleotide substitutions resulting in Lys174 → Glu and Thr230 → Ile. The α1 integrin I domain cDNA was also cloned into the glutathione S-transferase expression vector pGEX-KG (23Guan K.L. Dixon J.E. Anal. Biochemistry. 1991; 192: 262-267Crossref PubMed Scopus (1641) Google Scholar). Recombinant GST-α1I fusion proteins were purified by chromatography over glutathione-agarose and cleaved by digestion with thrombin as described in Ref. 23Guan K.L. Dixon J.E. Anal. Biochemistry. 1991; 192: 262-267Crossref PubMed Scopus (1641) Google Scholar. The construction of a plasmid for the expression of the Cna A domain has been previously described and consists of the gene segment encoding the S. aureus collagen MSCRAMM amino acids Ala30-Glu531 cloned between the Bam HI and Sal I restriction sites of the pQE-30 expression vector (13Rich R.L. Demeler B. Ashby K. Deivanayagam C.C.S. Petrich J.W. Patti J.M. Narayana S.V.L. Höök M. Biochemistry. 1998; 37: 15423-15433Crossref PubMed Scopus (48) Google Scholar). Large-scale preparations of recombinant protein were prepared and purified as follows. Overnight cultures (40 ml) of stationary-phase bacteria were used to inoculate 1 liter of Luria broth and the cells were allowed to grow for 2.5 h at 37 °C (OD600 nm ∼ 0.6). Protein expression was induced by addition of isopropyl-β-d-thiogalactopyranoside to a final concentration of 0.2 mm and the culture was incubated for an additional 3 h at 37 °C. Bacteria were then collected by centrifugation and resuspended in a minimal volume of 4 mmTris, 100 mm NaCl, pH 7.9, before being frozen at −80 °C. Induced bacteria were thawed and passed through a French press (11,000 p.s.i) twice to lyse the cells. Insoluble debris was removed by centrifugation at 14,000 rpm for 20 min and the supernatant was filtered through a 0.45 μm membrane. Imidazole was added to a final concentration of 6.67 mm and the lysates were applied to a 10 × 100-mm column of Ni2+-charged iminodiacetic acid/Sepharose. The column was washed with 50 ml of 4 mm Tris, 100 mm NaCl, 5 mmimidazole, pH 7.9, and bound protein eluted with a 200-ml linear gradient of 0–200 mm imidazole in 4 mm Tris, 100 mm NaCl, pH 7.9. Fractions containing the desired protein, as determined by SDS-PAGE, were pooled and concentrated using an Amicon ultrafiltration system. The isolated proteins were essentially pure and appeared as single bands on an overloaded SDS-PAGE gel. The isolated recombinant proteins were dialyzed against 3 × 1-liter changes of 1 mm EDTA, 50 mm HEPES, 150 mm NaCl, pH 7.4, to remove all cations, and then dialyzed against 3 × 1-liter changes of 50 mm HEPES, 150 mm NaCl, pH 7.4, to remove EDTA. All buffers for the α1 integrin I domain protein also contained 5 mm β-mercaptoethanol; the justification for adding a reducing agent to this sample solution is discussed below. During our initial analyses of the recombinant His6 tag α1 integrin I domain protein, we observed gradual precipitation of the protein within several days post-purification when the solution was kept at 4 °C. The presence of dimeric and higher-order multimers of the recombinant α1 integrin I domain protein in the solution was apparent by SDS-PAGE (data not shown). Addition of 5 mm β-mercaptoethanol delayed the protein precipitation for several weeks. All studies discussed here are the analyses of recombinant α1 integrin I domain within 2 weeks of expression and purification and in buffer containing 5 mm β-mercaptoethanol, unless noted otherwise. The far-UV CD spectra of freshly purified α1 integrin I domain in the presence and absence of 5 mm β-mercaptoethanol were identical (data not shown). Also, the SPR sensorgrams of freshly purified α1 integrin I domain flowed over immobilized collagen in the presence and absence of 5 mmβ-mercaptoethanol are identical (data not shown). The sensorgrams measured over a period of days for the α1 integrin I domain protein flowed over collagen in buffer containing the reducing agent remain unchanged; repeating this experiment in the absence of the reducing agent, however, revealed the gradual increase in association and decrease in dissociation of the protein-collagen complex over time. After approximately 2 weeks, the sensorgrams for the α1integrin I domain in the absence of β-mercaptoethanol duplicated those published previously (24Calderwood D.A. Tuckwell D.S. Eble J. Kühn K. Humphries M.J. J. Biol. Chem. 1997; 272: 12311-12317Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The increase in apparent affinity of the α1 integrin I domain protein after storage for collagen may be due to the contribution of multiple I domain elements in the protein aggregate binding at one location within the collagen macromolecule. The addition of the reducing agent is therefore necessary to preserve the monomeric state of the recombinant protein and does not alter its structure or function. Analyses were performed using the BIAcore system as described in Ref. 13Rich R.L. Demeler B. Ashby K. Deivanayagam C.C.S. Petrich J.W. Patti J.M. Narayana S.V.L. Höök M. Biochemistry. 1998; 37: 15423-15433Crossref PubMed Scopus (48) Google Scholar, with 5 mm β-mercaptoethanol and 0.25% octyl-β-d-glucopyranoside included in the buffer for α1 integrin I domain analyses. No mass transport effects were observed in these measurements. The data for the construction of the Scatchard plots was obtained from the equilibrium portion of the SPR sensorgrams and analyzed as described. 2Rich, R. L., Kreikemeyer, B., Owens, R. T., LaBrenz, S., Narayana, S. V. L., Murray, B., Weinstock, G. M., and Höök, M. (1999) J. Biol. Chem., in press. Assays were performed as described in Ref. 13Rich R.L. Demeler B. Ashby K. Deivanayagam C.C.S. Petrich J.W. Patti J.M. Narayana S.V.L. Höök M. Biochemistry. 1998; 37: 15423-15433Crossref PubMed Scopus (48) Google Scholar. For wells in which the buffer included MgCl2, all washes and incubations were performed in the presence of 1 mm MgCl2. The equilibrium dialysis experiments were carried out in a double acrylic microdialysis module (Hoffer, San Francisco, CA) as described by Yang et al. (26Yang V.W.-C. LaBrenz S.R. Rosenberg L.C. McQuillan D. Höök M. J. Biol. Chem. 1999; 274: 12454-12460Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Aliquots of 150 μl of thrombin-cleaved α1 integrin I domain protein in 10 mm Tris-HCl, 150 mm NaCl, pH 7.0, were added to the inner compartments. The same volume of 0–5 mmultrapure MgCl2 (Sigma) in 10 mm Tris-HCl, 150 mm NaCl, pH 7.0, was added to the outer compartments. After incubation, the concentration of Mg2+ in the outer compartments was determined using a Mg2+ detection kit (Sigma). An aliquot of 10 μl from each outer compartment and 100 μl of each kit component were mixed. The reaction was immediate and sample absorbance was measured at 525 nm using a Molecular Devices plate-reading visible spectrophotometer. Calculation of the Mg2+-complexed α1 integrin I domain fraction was performed as described in Ref. 27Rich R. Photophysics and Activity of Biological Systems Containing the Optical Probes 7-Azatryptophan and Its Analogs.Doctoral dissertation. Iowa State University, Ames, IA1995Google Scholar. Recombinant His6 tag α1 integrin I domain protein in 10 mm HEPES, 200 mm NaCl, 5 mm β-mercaptoethanol, pH 7.0, was further purified using a 300 × 7.5 Bio-sil-TSK125 gel-filtration column. The protein solution was then concentrated to 20 mg/ml using an Amicon ultrafiltration system and dialyzed against 10 mm HEPES, 200 mm NaCl, 5 mmMgCl2, 5 mm β-mercaptoethanol, pH 7.0. Crystallization trials were set up using the hanging-drop vapor-diffusion method. High quality crystals were obtained from a droplet made by mixing 2 μl of protein solution and 2 μl of 31% PEG2000, 50 mm HEPES, 200 mm NaCl, 5 mm MgCl2, 5 mm β-mercaptoethanol, pH 7.5 (solution A), and equilibrating it against 1 ml of solution A. Crystals were prism-shaped, with the largest having dimensions of 0.3 × 0.2 × 0.1 mm. Crystals were soaked in a synthetic mother liquor containing 15% glycerol as cryoprotectant and subsequently cryo-cooled using the Oxford cryosystem (Oxford Cryosystems, Oxford, United Kingdom). X-ray diffraction data were collected to 2.0-Å resolution using a RAXIS IV imaging plate system mounted on a RIGAKU RU-HBR rotating-anode generator (50 kV, 100 mA). A complete native data set was collected over 99 frames (oscillation of 2°, exposure time of 20 min, and crystal-to-image plate distance of 150 mm). The frames were indexed and scaled using DENZO and SCALEPACK (28Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The scaled data had 99% completeness, where 71.5% of the data in the last shell was above 3ς level with an Rsym value of 6.1%. The calculated Matthews coefficient, Vm, was 1.9 Å3 Da−1, suggesting two molecules exist in the asymmetric unit with an estimated solvent content of 35%. Data collection details are presented in TableI.Table ISummary of crystallographic data parameters and refinement statisticsCell parametersa = 37.44, b = 96.35, c = 53.27 Åα = 90.0, β = 104.28, γ = 90.0°Space groupP21Estimated molecular mass24.5 kDaMatthews coefficient (Vm)1.9 Å3 Da−1Vsolv35.3%Completeness99.2%Rsymm1-aRsymm = ΣhΣi‖Ihi− 〈Ihi〉‖/‖ΣhΣiΣIhi‖, where h specifies unique reflections and i indicates symmetry-equivalent observations of h.6.1%1/ς124.1% Reflections above 3ς77.7% (in the last resolution shell)R-factor20.6%Rfree24.3%Total number of protein atoms (asym unit)3008Total number of solvent molecules (asym unit)229Average B-factor23.65 Å2Average B-factor (MC)1-bMC, main chain atoms.21.84 Å2Average B-factor (SC)1-cSC, side chain atoms.24.61 Å2Average B-factor (solvent)29.36 Å2Root mean square deviations bonds0.006 ÅRoot mean square deviations angles1.20°Root mean square deviations (molec1 and molec2)0.231 Å1-a Rsymm = ΣhΣi‖Ihi− 〈Ihi〉‖/‖ΣhΣiΣIhi‖, where h specifies unique reflections and i indicates symmetry-equivalent observations of h.1-b MC, main chain atoms.1-c SC, side chain atoms. Open table in a new tab The crystal structure of the recombinant His6 tag α1 integrin I domain was determined by the molecular replacement method using the CCP4 integrated version of AmoRe (29Navaza J. Acta Crystallogr. Sec. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar, 30Collaborative Computer Project No. 4 Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). We used the molecular model of the human complement factor B middle domain as the initial molecular replacement unit (31Tuckwell D.S. Xu Y. Newham P. Humphries M.J. Volanakis J.E. Biochemistry. 1997; 36: 6605-6613Crossref PubMed Scopus (40) Google Scholar). The C-terminal helix and all the connecting loops were removed from the starting molecular replacement search model, which had 109 residues. Repeated rounds of rigid-body refinement and checking for acceptable crystal packing helped us to identify two solutions having high correlation factors and the lowest R-factors (0.62 and 41%, respectively, for 8.0–4.5-Å resolution data). The two molecules in the asymmetric unit were not related by an exact 2-fold non-crystallographic axis. Next, the side chains of the correctly positioned model were replaced with the corresponding homologous side chains of the α1 integrin I domain. Rigid body refinement to 3.0-Å resolution, where the individual secondary structural elements were treated as independent units in XPLOR (32Brunger A.T. X-PLOR manual, Version 3.1. Yale University Press, New Haven, CT1992Google Scholar), resulted in an R- factor of 39% and Rfree (calculated on 10% of the reflections) of 44%. Several rounds of manual refitting to 2 Fo − Fc maps using the graphics program “O” (33Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) and positional refinement in XPLOR were done while extending the resolution to 2.5 Å in small increments. At this stage, the R-factor was 31%, Rfree value was 41%, and a 2 Fo − Fc map calculated had visible density for the missing C-terminal helix and for most of the deleted loop regions. At this juncture, the resolution was extended to the final 2.0 Å and two rounds of simulated annealing and model rebuilding led to the tracing of the complete C-terminal end. After one refinement cycle of individual B-factors (r= 26% and Rfree = 30%), water molecules were added to the model by picking the peaks above 3ς level in a calculated (Fo − Fc) difference map. Two of these water molecules were identified as metal ions based on their bonding geometry. The OOPS program (34Kleywegt G.J. Jones T.A. Methods Enzymol. 1997; 276: 208-230Crossref Scopus (309) Google Scholar) was used throughout the cycles of rebuilding for quality checks. The cross-validated maximum likelihood refinements were performed with CNS-0.4 (35Adams P.D. Pannu N.S. Read R.J. Baugen A.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5018-5023Crossref PubMed Scopus (383) Google Scholar). Bulk solvent corrections were applied in the last few cycles of refinement. The final refinement yielded 229 water molecules, two Mg2+ions, 3008 non-hydrogen atoms, and four cis-prolines. For 24,537 reflections (out of 24,807 reflections) between 100.0 and 2.0-Å resolution, the final R-factor was 20.6% and Rfree was 24.3%. The final structure was checked using PROCHECK (36Morris A.C. MacArthur M.W. Hutchinson E.G. Thronton J. Proteins Struct. Funct. Genet. 1992; 12: 345-364Crossref PubMed Scopus (1420) Google Scholar, 37Leskowski R.A. MacArthur M.W. Moss D.S. Thronton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and WHAT_CHECK (38Hooft R.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1818) Google Scholar). The complete refinement statistics are presented in Table I. The α1integrin I domain structure was aligned with other integrin I domains and von Willebrand factor A3 domain crystal structures taken from the protein data bank (1AO3 for von Willebrand factor, 1AOX for the α2 integrin, 1JLM for the αM integrin, and1ZON for the αL integrin) using the program MODELER (39Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10636) Google Scholar). Non-crystallographic c" @default.
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