Crystal Structure of the I Domain from Integrin α2β1
1997; Elsevier BV; Volume: 272; Issue: 45 Linguagem: Inglês
10.1074/jbc.272.45.28512
ISSN1083-351X
AutoresJonas Emsley, Sandra L. King, Jeffrey M. Bergelson, Robert Liddington,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoWe have determined the high resolution crystal structure of the I domain from the α-subunit of the integrin α2β1, a cell surface adhesion receptor for collagen and the human pathogen echovirus-1. The domain, as expected, adopts the dinucleotide-binding fold, and contains a metal ion-dependent adhesion site motif with bound Mg2+ at the top of the β-sheet. Comparison with the crystal structures of the leukocyte integrin I domains reveals a new helix (the C-helix) protruding from the metal ion-dependent adhesion site face of the domain which creates a groove centered on the magnesium ion. Modeling of a collagen triple helix into the groove suggests that a glutamic acid side chain from collagen can coordinate the metal ion, and that the C-helix insert is a major determinant of binding specificity. The binding site for echovirus-1 maps to a distinct surface of the α2-I domain (one edge of the β-sheet), consistent with data showing that virus and collagen binding occur by different mechanisms. Comparison with the homologous von Willebrand factor A3 domain, which also binds collagen, suggests that the two domains bind collagen in different ways. We have determined the high resolution crystal structure of the I domain from the α-subunit of the integrin α2β1, a cell surface adhesion receptor for collagen and the human pathogen echovirus-1. The domain, as expected, adopts the dinucleotide-binding fold, and contains a metal ion-dependent adhesion site motif with bound Mg2+ at the top of the β-sheet. Comparison with the crystal structures of the leukocyte integrin I domains reveals a new helix (the C-helix) protruding from the metal ion-dependent adhesion site face of the domain which creates a groove centered on the magnesium ion. Modeling of a collagen triple helix into the groove suggests that a glutamic acid side chain from collagen can coordinate the metal ion, and that the C-helix insert is a major determinant of binding specificity. The binding site for echovirus-1 maps to a distinct surface of the α2-I domain (one edge of the β-sheet), consistent with data showing that virus and collagen binding occur by different mechanisms. Comparison with the homologous von Willebrand factor A3 domain, which also binds collagen, suggests that the two domains bind collagen in different ways. The integrins are a family of plasma membrane proteins that transduce bidirectional signals between the cytoplasm and the extracellular matrix or other cells (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9139) Google Scholar). The integrin α2β1 is expressed on a variety of cell types, serving as the collagen receptor on platelets and fibroblasts, and as both a collagen and laminin receptor on endothelial and epithelial cells (2Elices M.J. Hemler M.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9906-9910Crossref PubMed Scopus (326) Google Scholar, 3Languino L.R. Gehlsen K.R. Wayner E. Carter W.G. Engvall E. Ruoslahti E. J. Cell Biol. 1989; 109: 2455-2462Crossref PubMed Scopus (289) Google Scholar). It also acts as the receptor for the human pathogen echovirus-1 (4Bergelson J.M. Shepley M.P. Chan B.M.C. Hemler M.E. Finberg R.W. Science. 1992; 255: 1718-1720Crossref PubMed Scopus (250) Google Scholar). In common with six other integrin α-chains (α1, αD, αE, αL, αM, and αX) the α2 chain contains a 200-amino acid inserted domain, the I domain, that is homologous to the von Willebrand factor A domains (5Colombatti A. Bonaldo P. Doliana R. Matrix. 1993; 13: 297-306Crossref PubMed Scopus (156) Google Scholar). Recombinant α2-I domain recapitulates many of the ligand binding properties of the parent integrin (6Bahou W.F. Potter C.L. Mirza H. Blood. 1994; 84: 3734-3741Crossref PubMed Google Scholar, 7Tuckwell D.S. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 108: 1629-1637Crossref PubMed Google Scholar, 8Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar). It exhibits specific binding to various fibrillar collagens, and two groups have shown that, like collagen binding to the complete receptor (9Santoro S.A. Cell. 1986; 46: 913-920Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 10Staatz W.D. Rajpara S.M. Wayner E.A. Carter W.G. Santoro S.A. J. Cell Biol. 1989; 108: 1917-1924Crossref PubMed Scopus (322) Google Scholar), binding to the I domain is cation-dependent, being supported by magnesium or manganese but not by calcium (7Tuckwell D.S. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 108: 1629-1637Crossref PubMed Google Scholar, 11Dickeson S.K. Walsh J.J. Santoro S.A. J. Biol. Chem. 1997; 272: 7661-7668Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The triple-helical structure of collagen is required for recognition by α2β1, but specific collagen sequences have not been identified (for review, see Ref. 12Tuckwell D. Humphries M. Cell Dev. Biol. 1996; 7: 649-657Crossref Scopus (38) Google Scholar). The first crystal structure of an integrin I domain, from αMβ2, showed that it adopts the dinucleotide-binding fold, with a central parallel β-sheet surrounded on both sides by α-helices (13Lee J.-O. Rieu P. Arnaout M.A. Liddington R.C. Cell. 1995; 80: 631-635Abstract Full Text PDF PubMed Scopus (812) Google Scholar). In this class of fold, a functional surface of the domain always lies at the C-terminal end of the β-sheet (14Branden C. Tooze J. Introduction to Protein Structure. Garland Publishing, Inc., New York1991Google Scholar). In the I domain, a novel cation coordination sphere is located there, and in the αM-I domain crystal structure with bound Mg2+, a glutamate side chain from a neighboring I domain in the crystal lattice completes the octahedral coordination sphere of the metal. This led to the suggestion that the glutamate behaves as a ligand mimetic, as most integrin ligands possess a critical aspartate residue (or glutamate) as a key feature of their integrin-binding motifs, and mutation of any of the metal-coordinating side chains of the I domain (8Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar, 15Michishita M. Videm V. Arnaout M.A. Cell. 1993; 72: 857-867Abstract Full Text PDF PubMed Scopus (322) Google Scholar, 16Edwards C.P. Champe M. Gonzales T. Wessinger M.E. Spencer S.A. Presta L.G. Berman P.W. Bodary S.C. J. Biol. Chem. 1995; 270: 12635-12640Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 17Kern A. Briesewitz R. Bank I. Marcantonio E.E. J. Biol. Chem. 1994; 269: 22811-22816Abstract Full Text PDF PubMed Google Scholar) abolishes binding in a dominant negative fashion. This motif was therefore dubbed the metal ion-dependent adhesion site (MIDAS) 1The abbreviations used are: MIDAS, metal ion-dependent adhesion site; vWF-A3, von Willebrand Factor A3 domain; αLMn-I, αL-I domain with bound Mn2+; αMMn-I, αM-I domain with bound Mn2+; αMMg-I, αM-I domain with bound Mg2+; RMS, root mean square. (13Lee J.-O. Rieu P. Arnaout M.A. Liddington R.C. Cell. 1995; 80: 631-635Abstract Full Text PDF PubMed Scopus (812) Google Scholar). Apart from the highly conserved residues that directly coordinate the metal, the upper surface of the domain surrounding the MIDAS motif is highly variable, suggesting that the metal-Glu/Asp bond contributes some but not all of the binding energy, with the rest of the energy, and the specificity, arising from further interactions (ionic/polar/hydrophobic) between complementary surfaces of the integrin and ligand. In support of this notion, Huang and Springer (18Huang C. Springer T.A. J. Biol. Chem. 1995; 270: 19008-19016Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) utilized mouse-human chimeras and site-specific mutagenesis to demonstrate that residues essential for the interaction of αLβ2 with intercellular adhesion molecule-1 are located on the MIDAS face surrounding the site of metal coordination. In addition, two of the epitopes for function-blocking antibodies map to the same face (19Champe M. McIntyre B.W. Berman P.W. J. Biol. Chem. 1995; 270: 1388-1394Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Similarly, Rieu et al. (20Rieu P. Sugimori T. Griffith D.L. Arnaout M.A. J. Biol. Chem. 1996; 271: 15858-15861Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) showed that residues essential for the binding of the hookworm pathogen, neutrophil inhibitory factor, a protein that blocks the binding of natural ligands to αMβ2, cluster around the MIDAS face of αM-I. Crystal structures have previously been reported for the αL-I and αM-I domains with bound Mg2+ and Mn2+ (13Lee J.-O. Rieu P. Arnaout M.A. Liddington R.C. Cell. 1995; 80: 631-635Abstract Full Text PDF PubMed Scopus (812) Google Scholar,21Lee J.-O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 22Qu A. Leahy D.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10277-10281Crossref PubMed Scopus (291) Google Scholar, 23Qu A. Leahy D.J. Structure. 1996; 4: 931-942Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The crystal structure of an A-domain from von Willebrand Factor (vWF-A3) has also been solved recently (24Bienkowska J. Cruz M. Atiemo A. Handin R. Liddington R. J. Biol. Chem. 1997; 272: 25162-25167Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). We now report the crystal structure of the α2-I domain, which we determined as a first step in understanding the atomic level determinants of ligand binding, and compare the structure with other A/I domains. vWF-A3 also binds collagen, suggesting that the two domains might have similar binding motifs. Our crystal structure suggests that they do not. Human α2-I domain (residues 140–337) was expressed as a glutathioneS-transferase fusion protein in Escherichia coli, cleaved, and purified as described previously (25King S.L. Cunningham J.A. Finberg R.W. Bergelson J.M. J. Virol. 1995; 69: 3237-3239Crossref PubMed Google Scholar). The protein was next loaded onto an affinity iminodiacetic acid-Sepharose column (Pharmacia) charged with Ni2+ and eluted with a linear gradient of 0–200 mm imidazole. Fractions were pooled, diluted 3-fold with distilled water and concentrated to 10 mg/ml. Crystals were grown by hanging drop vapor diffusion, by mixing 3 μl of protein and reservoir solution (16–20% polyethylene glycol 8 K, 50 mm HEPES pH 7.5, 10 mm MgCl2, 20% glycerol) at room temperature. Crystals grow to a typical size of 0.5 mm × 0.5 mm × 0.5 mm in 2–3 days and belong to the space group P1 with cell dimensions: a = 40.5 Å,b = 43.3 Å, c = 68.0 Å, α = 88.3°, β = 76.6°, γ = 66.7°. Isomorphous crystals grow in the presence of manganese. A 2.5-Å data set (see Table I) was collected from a single crystal mounted and frozen in a stream of boiled-off nitrogen at 100 K using a Rigaku RU-200 x-ray generator with focusing mirrors and an RAXIS II image plate. Data were reduced with DENZO and scaled with SCALEPACK (26Otwinowski Z. Sawyer L. Isaacs N. Bailey S. Data Collection and Processing. SERC, Daresbury, United Kingdom1993: 59-62Google Scholar) with an R merge = 4.2%, and 95% completeness to 2.5-Å resolution. This data set was used to perform the molecular replacement calculations and the early stages of refinement. A room temperature data set was subsequently collected from an imperfect twin. As the twins of the crystal were randomly oriented and diffracted with approximately equal intensity, both triclinic lattices could be indexed and data merged, with anR merge = 11.2% (31% in the outer shell) and completeness of 96.5% to 1.9-Å resolution. A 7ς peak on the κ = 180° section of a self-rotation function calculated using GLRF (27Tong L. Rossmann M.G. Acta Cryst. Sect. A. 1990; 46: 738-792Crossref Scopus (257) Google Scholar) indicated the presence of two molecules in the asymmetric unit, consistent with a solvent content of 55%. Molecular replacement was performed with AMoRe (28Collaborative Computational Project, No. 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19877) Google Scholar), using the superposed structures of αM-I (Mn2+-bound conformation (21Lee J.-O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar)) and αL-I domains (22Qu A. Leahy D.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10277-10281Crossref PubMed Scopus (291) Google Scholar) and the vWF-A3 domain (24Bienkowska J. Cruz M. Atiemo A. Handin R. Liddington R. J. Biol. Chem. 1997; 272: 25162-25167Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) as a search model. The top two peaks in a cross-rotation function gave the correct orientations of the two monomers, and their relative displacement was readily determined with a translation function. Applying this solution to all protein atoms in the αM-I structure resulted in a model with good crystal packing, and an R factor and correlation coefficient of 52% and .24, respectively (20-6 Å), improving to 50% and .48 on rigid body refinement. The αM-I model was next stripped of side chains and loop regions; the remaining 114 alanine and glycine residues constituted 5 α-helices (α7 omitted) and 6 β-strands, which were refined as individual rigid bodies in XPLOR (29Brünger A.T. X-PLOR Manual, version 3.1. Yale University, New Haven, CT1992Google Scholar) using 10–2.8 Å data. TheR factor at this stage was 41.6% andR free (calculated on 10% of reflections) was 47.9%. A 2F o − F c electron density map revealed a number of new features including several side chains and the C-terminal helix α7. At this point, the refinement was extended to the resolution limit of the room temperature data set (1.9 Å). Several cycles of simulated annealing XPLOR refinement, followed by inspection of 2F o − F c maps and manual model building, led to a complete polypeptide trace. Water molecules were then added at the positions of F o −F c peaks greater than 3ς, where reasonable hydrogen bonding partners existed. After applying a bulk solvent correction, the current R factor is 19.6% for all data between 15 and 1.9 Å (R free = 24.8%); the model includes residues 139–339 (including 2 residues at the C terminus from the expression construct) for both molecules in the asymmetric unit, and 267 water molecules. The only residue in an unfavored region of the Ramachandran plot is Ala188. Electron density for this residue is persuasive and stabilizing hydrogen bonds exist. There are two cis proline residues, Pro158 in the βA-α1 turn and Pro307 in the α6-βF turn.Table ISummary of crystallographic analysisResolutionNo. of observations (completeness/redundancy)R sym1-aR sym = Σ‖I−〈I〉‖/ΣI,where I = observed intensity, 〈I〉 = average intensity from multiple observations of symmetry related reflections. RMSD, root mean square deviation from ideal values.I/ςIoverall/outer shellData collection Native 115–1.9Å34,120 (94/3.5)11.0%20.7 /2.6 Native 215–2.5Å13,121 (95/4.3)4.3%24.2 /10.4Refinement statistics Resolution: 15–1.9 Å;R working = 0.196; R free = 0.248; No. of waters = 267; RMSD bond lengths, 0.018 Å; RMSD angle, 2.11°Space group P1, a = 40.5, Å, b = 43.3 Å, c = 68.0 Å, α = 88.3°, β = 76.6°, γ = 66.7°; 2 molecules/asymmetric unit; solvent content 55%.1-a R sym = Σ‖I−〈I〉‖/ΣI,where I = observed intensity, 〈I〉 = average intensity from multiple observations of symmetry related reflections. RMSD, root mean square deviation from ideal values. Open table in a new tab Space group P1, a = 40.5, Å, b = 43.3 Å, c = 68.0 Å, α = 88.3°, β = 76.6°, γ = 66.7°; 2 molecules/asymmetric unit; solvent content 55%. We built a collagen triple helix based on the published crystal structure (30Bella J. Eaton M. Brodsky B. Berman H.M. Science. 1994; 266: 75-81Crossref PubMed Scopus (903) Google Scholar) (PDB entry 1cag) of a collagen-like peptide (Pro-Hyp-Gly)4-Pro-Hyp-Ala-(Pro-Hyp-Gly)5. This peptide contains a single Gly to Ala substitution at the center of a 30-residue sequence which disrupts the (X-Y-Gly) n motif, creating a local untwisting of the helix. We used repeats 2 to 4 (residues 4–12) of each polypeptide chain and extended these along the helical axis to generate an ideal collagen triple helix 80 Å long. At the center of this triple helix, a proline (at the Xposition) or a hydroxyproline (at Y) was replaced with a glutamate in its preferred gauche(+),trans side chain conformation. The collagen model and I domain were then manually docked as rigid bodies by constraining the distance between the glutamate carboxylate and the metal ion to 2 Å (the observed bond distance in the αMβ2 I domain) and by minimizing intermolecular steric clashes (monitored using the CCP4 program CONTACT (28Collaborative Computational Project, No. 4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19877) Google Scholar)). With Glu at either theX or Y position, a convincing fit could be found with only minor steric clashes. These clashes could be relieved either by trimming 3 or 4 of the collagen Pro/Hyp side chains to Ala or Gly, or by allowing a small number of side chains on the I domain to adopt alternate rotamer conformations. When the Glu was replaced by an Asp, severe steric clashes occurred between backbone atoms of the two docking partners. The structure, as expected, adopts the classic dinucleotide-binding fold, with seven helices surrounding a core of five parallel β-strands and one short antiparallel β-strand (Figs. 1 and2). Compared with the αM and αL-I domains, the most striking difference is a new turn-and-a-half of α-helix, residues 284–288, which we call the C-helix, extending from the top of strand βE and protruding from the MIDAS face (see below). There is a buried glutamic acid in place of the usual glycine in the position following the MIDAS aspartic acid (DESNS), but this is accommodated without distortion of the MIDAS motif. Space is created by a 1-residue insertion in the α3-α4 loop that wraps over the top of βA and βB, and the charge is neutralized by a salt bridge to Arg192 from βC. A buried water molecule adjacent to the MIDAS motif is closely conserved in αM-I and αL-I; it makes hydrogen bonds to the main chain carbon oxygen of Gly255, to the side chain hydroxyls of Thr223and Thr253 and to the carboxylate of Glu299.Figure 2Sequence alignments of the human integrin I domains (α2-I, αM-I, and αL-I) and the vWF-A3, with α-helices and β-sheets indicated for the α2-I domain. Sequences are aligned based on structural superpositions (38May A.C.W. Johnson M.S. Protein Eng. 1994; 7: 475-485Crossref PubMed Scopus (55) Google Scholar). Lowercase letters denote a lack of structural similarity. Sequence identities with the α2-I domain are 26.7% (αM), 24.0% (αL), and 20.3% (vWF-A3). The αM and αL-I domains form a subfamily with 33.9% sequence identity. The α1-I domain sequence alignment is also shown; its structure has not been determined but is likely to be very similar to the α2-I domain.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The buried Phe in αM-I and αL-I (Ile in vWF-A3) at the top of helix α7 (which becomes exposed in the "active" conformer of αM-I (21Lee J.-O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar)) is replaced by a glutamic acid in the α2-I domain (Glu318). The side chain turns upwards to avoid complete burial, creating a cavity which is filled by a water molecule that is not found in αL-I, αM-I, or vWF-A3. The water molecule hydrogen bonds to the carboxylate of the MIDAS aspartate (Asp254), the main chain nitrogens of Gly284 and Tyr285, as well as the carboxylate of Glu318. A salt bridge is provided by the side chain of Arg288 from the C-helix. The orientation of the α7 helix is nevertheless very similar to that in the αM-IMn structure. Sequences of α2β1 have been reported from human, cow, mouse, and pig. Within the I domains, 43 positions are not invariant. These all lie on the surface of the molecule, except for two conservative changes in the hydrophobic core (Val182-Met and Leu328-Ile in bovine). None of the changes are found on the MIDAS face, including the new C-helix. Although the two molecules in the crystal unit cell were refined independently the overall structures are almost identical (RMSD = 0.25 Å for main chain atoms) except for the chain termini. Both chain termini are ordered to a greater extent than in other I domain structures. The N terminus extends 5 residues before strand βA, and the C terminus 3 residues beyond helix α7. In the crystal, a disulfide bridge is formed between the N-terminal cysteine residues (Cys140) of the two molecules in the asymmetric unit, and this results in different conformations of the termini. It is unlikely that such a disulfide bond forms in vivo, and Springer (31Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 65-72Crossref PubMed Scopus (393) Google Scholar) has predicted that Cys140 makes a disulfide bridge with a cysteine residue within the 4-1 loop of his propeller model. In one molecule, the C-terminal residues (335–337) pack into a crevice formed by the N-terminal loop (residues 138–142) and the α4-βD loop (residues 244–246). The side chain of Ile335 packs into this crevice, the main chain makes a number of β-sheet hydrogen bonds to both loops, and the N and C termini are brought into very close proximity. In the second molecule in the crystal, it appears that disulfide bond formation induces a new conformation in the N-terminal loop which squeezes the C-terminal residues out of the crevice, and we suggest that the first molecule better reflects the native conformation of the domain termini in the intact integrin. The MIDAS motif (Fig.3) binds a magnesium ion in the α2-I domain, as expected given the strict sequence conservation of the motif throughout the integrin I domains (the MIDAS motif in the vWF-A3 domain is not strictly conserved, and the vestigial motif does not bind a metal ion (24Bienkowska J. Cruz M. Atiemo A. Handin R. Liddington R. J. Biol. Chem. 1997; 272: 25162-25167Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar)). The metal is directly coordinated by three side chains (from residues Ser153, Ser155, and Asp254) and three water molecules, making strong bonds (2.0 ± 0.1 Å) in an octahedral arrangement. Asp151makes hydrogen bonds to Ser153 and a water molecule (2.7–2.9 Å) but no direct bond. Thr221 does not coordinate the metal directly (Mg-OH(Thr) = 4.1 Å), but makes a hydrogen bond (2.9 Å) to one of the water molecules. This coordination is very similar to that found in the Mn2+-bound structures of αL-I and αM-I (αLMn-I and αMMn-I, defined as the "inactive" form by Lee et al. (21Lee J.-O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar)), but different from the coordination observed in the αM-I structure with bound Mg2+ (αMMg-I, the active form). It is the first high resolution crystal structure of the inactive conformer with bound Mg2+, and confirms the coordination predicted from the lower resolution (2.8 Å) structure of αL-I with bound Mg2+ (23Qu A. Leahy D.J. Structure. 1996; 4: 931-942Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The role of the MIDAS threonine is intriguing; it is the only MIDAS residue that is absolutely critical for collagen binding to the recombinant α2-I domain (32Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar), and it is also critical for ligand binding in the αM-I and αL-I domains (33Kamata T. Wright R. Takada Y. J. Biol. Chem. 1995; 270: 12531-12535Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Only in the active αMMg-I domain does the threonine coordinate the metal directly, suggesting that the threonine is required for stability of the active conformer and supporting the theory of tertiary structure change within the I domain (21Lee J.-O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). It appears that using modified protein, the requirement for cation can be circumvented (see below), but it is puzzling that the threonine remains essential under those conditions also (32Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar). This issue will only be properly resolved by structure determination of an authentic I domain-ligand complex. The central core of five parallel β-strands and one short antiparallel β-strand is highly conserved among the integrin I domains and vWF-A3, with RMS deviations of 0.6–0.7 Å (Figs. 1 and 2). The βB-βC hairpin and a β-bulge at the end of βC are almost identical in all four structures. By contrast, the helices are more variable, with only helices α1 and α4 showing a general agreement of length and orientation. Helix α2 is replaced by a short turn and helix α3 extended by a turn in α2-I and vWF-A3. The C-terminal helix, α7, has a similar conformation in αM-IMn, α2-I, and vWF-A3, but is different in αL-I, where the helix splays out from the side of the domain, exposing a large hydrophobic crevice that is filled by a hydrophobic C-terminal sequence from another molecule in the crystal lattice (22Qu A. Leahy D.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10277-10281Crossref PubMed Scopus (291) Google Scholar). The conformation of helix α7 is also very different in the active conformer of αM-IMg, where a functional role in propagating structural changes from the MIDAS face to the rest of the integrin has been proposed (21Lee J.-O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). The loops surrounding the MIDAS motif, which comprise the MIDAS face, are βA-α1, α3-α4, βD-α5, and βE-α6 (Fig. 4). These loops, except for βE-α6, have been implicated in ligand binding to the αL-I and αM-I domains by mutagenesis experiments (18Huang C. Springer T.A. J. Biol. Chem. 1995; 270: 19008-19016Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 19Champe M. McIntyre B.W. Berman P.W. J. Biol. Chem. 1995; 270: 1388-1394Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 20Rieu P. Sugimori T. Griffith D.L. Arnaout M.A. J. Biol. Chem. 1996; 271: 15858-15861Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The loops have highly variable surface-exposed residues in all of the A/I domains, even when the main chain conformation is conserved, consistent with their being the principal determinants of ligand binding specificity. The βA-α1 loop, which includes the metal-coordinating DxSxS consensus sequence, has a 2-residue deletion in the α2-I domain, at the beginning of the α1 helix, but is otherwise conserved. The α3-α4 loop has a 1-residue insertion in the α2-I domain, which creates space for the glutamate (in DESNS), as already noted. The βD-α5 loop is similar in α2-I, αL-I, and vWF-A3, but different in αM-I, while αL-I lacks most of helix α5. The βE-α6 loop is the site of the principal insertion in the α2-I domain that creates the protruding C-helix. Its conformation is similar in αL-I and αM-I, but very different in α2-I and vWF-A3. It undergoes substantial rearrangement in the two structures of αM-I, creating an acidic pocket in the active conformer, and we suggest that this loop is a major determinant of ligand specificity. Integrin α2β1 binds several types of fibrillar collagens (types I-VI and XI), and recombinant α2-I domain exhibits specific binding to some, but not all of these (reviewed in Ref. 12Tuckwell D. Humphries M. Cell Dev. Biol. 1996; 7: 649-657Crossref Scopus (38) Google Scholar). Two groups have shown that collagen binding to the α2-I domain is cation-dependent, being supported by Mg2+ and Mn2+ but not by Ca2+ (7Tuckwell D.S. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 108: 1629-1637Crossref PubMed Google Scholar,11Dickeson S.K. Walsh J.J. Santoro S.A. J. Biol. Chem. 1997; 272: 7661-7668Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The MIDAS residues Asp151, Thr221, and Asp254 are all essential for collagen binding to the α2β1 integrin, and Thr221 is also critical in the recombinant α2-I domain (8Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar, 32Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar). While the sequence motifs in collagen to which α2β1 bind have yet to be defined, the triple helical structure is required, and Asp/Glu and Arg (but not Lys) have been shown to be important for the binding of α1β1 to collagen IV (34Eble J.A. Golbik R. Mann K. Kühn K. EMBO J. 1993; 12: 4795-4802Crossref PubMed Scopus (179) Google Scholar). The new C-helix on top of the MIDAS face creates a groove about 25 Å long and 20 Å wide centered on the metal ion, with a tyrosine residue (Tyr285) projecting prominently into the groove. Into this groove we manually docked a collagen triple helix, which was derived from the crystal structure of a collagen-like peptide (30Bella J. Eaton M. Brodsky B. Berman H.M. Science. 1994; 266: 75-81Crossref PubMed Scopus (903) Google Scholar) and modified to contain a glutamate residue to coordinate the MIDAS metal ion (Fig.5). The shape of the groove places strong constraints on the position of the collagen helix; in particular, the projecting C-helix, the top of helix α6, and their connecting loop, severely restrict rotations of the collagen about an axis parallel to the glutamate-metal bond. The amino acid side chains in natural collagen sequences would restrict rotations still further. Replacement of the glutamate by the shorter aspartate creates steric clashes in all possible orientations of the collagen. The model predicts that the following I domain residues make contact with the collagen: from the βA-α1 turn (Asn154), from the α3-α4 turn (Asp219 and Leu220), from the βD-α5 turn (Glu256 and His258), and from the C-helix, α6 and C-α6 turn (Tyr285, Asn289, Leu291, Asn295 and Lys298). The "footprint" on the collagen is about 10 residues long. Mutagenesis data (32Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar) show that individual alanine mutants of two of these residues, Glu256 and Asn295, do not detectably affect collagen binding, but given the large number of potential contacts this may not be surprising. The epitopes for two blocking antibodies have been mapped to Asp160 and Tyr216 (8Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar, 35Bergelson J.M. Chan B.M.C. Finberg R.W. Hemler M.E. J. Clin. Invest. 1993; 92: 232-239Crossref PubMed Scopus (48) Google Scholar, 36Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1996; 271: 19008Abstract Full Text Full Text PDF Scopus (0) Google Scholar). These residues lie on the surface of the domain to the side of the MIDAS face, 12–15 Å from the MIDAS motif, consistent with their forming parts of epitopes for blocking antibodies. Takada's group (32Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar) have reported conflicting data on collagen binding, showing that in their system binding is cation-independent. They did, however, also show that binding was completely abrogated by alanine mutagenesis of the MIDAS residue Thr221. A possible resolution of these conflicting data has been provided by Tuckwellet al. (7Tuckwell D.S. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 108: 1629-1637Crossref PubMed Google Scholar), who showed that although most binding was cation-dependent, there was nonetheless a cation-independent fraction which correlated with the degree of protein modification. Takada's group modified their protein by iodination. Our crystal structure reveals a tyrosine residue on the MIDAS face very close to the metal ion, and our modeling studies suggest that this tyrosine is intimately involved in collagen binding. It is therefore plausible that iodination of this tyrosine affects I domain-collagen binding, either by providing an additional hydrophobic element to binding, or by directly inducing a conformational change to a high affinity state, abrogating the need for a metal ion. The α1-I domain has a similar but not identical ligand-binding range as α2-I, binding collagen types I and III-VI, and laminins (17Kern A. Briesewitz R. Bank I. Marcantonio E.E. J. Biol. Chem. 1994; 269: 22811-22816Abstract Full Text PDF PubMed Google Scholar). The α1 and α2 sequences form a subfamily distinct from the leukocyte integrins, and the sequences can be aligned with no gaps, so that the α2-I domain crystal structure provides an excellent main chain model for α1-I. The MIDAS motif is strictly conserved in α1-I, as is the aspartic acid at the top of helix α7. The new C-helix is structurally conserved, although divergent in sequence (Tyr285 is replaced by a serine, while Leu286 is replaced by a tyrosine). The βA-α1 and βD-α5 loops are strictly conserved around the MIDAS motif, while the α3-α4 loop is divergent. The vWF-A1 and A3 domains have been shown to bind collagens type I and III. Binding to A3 is independent of cation, and the crystal structure of vWF-A3 does not contain a bound metal (24Bienkowska J. Cruz M. Atiemo A. Handin R. Liddington R. J. Biol. Chem. 1997; 272: 25162-25167Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In addition, mutation of the MIDAS residues do not affect collagen binding. The βE-α6 loop, which contains the C-helix insert in α2-I, is truncated in vWF-A3, so that it is even shorter than in αL-I and αM-I. The result is that the vestigial MIDAS face is quite featureless, suggesting that α2-I and vWF-A3 bind collagen in different ways. The α2β1 integrin is the receptor for the human pathogen echovirus-1 (4Bergelson J.M. Shepley M.P. Chan B.M.C. Hemler M.E. Finberg R.W. Science. 1992; 255: 1718-1720Crossref PubMed Scopus (250) Google Scholar), and the α2-I domain binds directly to the virus (25King S.L. Cunningham J.A. Finberg R.W. Bergelson J.M. J. Virol. 1995; 69: 3237-3239Crossref PubMed Google Scholar). Virus binding is cation-independent (35Bergelson J.M. Chan B.M.C. Finberg R.W. Hemler M.E. J. Clin. Invest. 1993; 92: 232-239Crossref PubMed Scopus (48) Google Scholar), is not affected by mutations of the MIDAS motif (37King S.L. Kamata T. Cunningham J.A. Emsley J. Liddington R.C. Takada Y. Bergelson J.M. J. Biol. Chem. 1997; 272: 28518-28522Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), and does not require activation of the integrin (35Bergelson J.M. Chan B.M.C. Finberg R.W. Hemler M.E. J. Clin. Invest. 1993; 92: 232-239Crossref PubMed Scopus (48) Google Scholar). King et al. (37King S.L. Kamata T. Cunningham J.A. Emsley J. Liddington R.C. Takada Y. Bergelson J.M. J. Biol. Chem. 1997; 272: 28518-28522Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) have recently shown that residues 199–201 and 212–216 are involved in virus binding. These residues map to the loops flanking both ends of helix α3, forming part of a flat surface (∼20 Å × 30 Å) at one end of the β-sheet, adjacent to but not overlapping the MIDAS face. This location is consistent with biochemical data suggesting that the collagen and echovirus-binding sites are distinct (35Bergelson J.M. Chan B.M.C. Finberg R.W. Hemler M.E. J. Clin. Invest. 1993; 92: 232-239Crossref PubMed Scopus (48) Google Scholar). Several monoclonal antibodies block both virus and collagen binding, supporting the idea that the binding sites are in close proximity (35Bergelson J.M. Chan B.M.C. Finberg R.W. Hemler M.E. J. Clin. Invest. 1993; 92: 232-239Crossref PubMed Scopus (48) Google Scholar). Part of the epitope for one such antibody, 5E8, has been mapped; it includes residue Tyr216 (36Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1996; 271: 19008Abstract Full Text Full Text PDF Scopus (0) Google Scholar), on the loop between helix α3 and the MIDAS motif. We thank Remy Loris and Carlo Petosa for assistance in refinement and modeling.
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