Effects of conformational activation of integrin alpha 1I and alpha 2I domains on selective recognition of laminin and collagen subtypes

Collagen receptor integrins alpha 1 beta 1 and alpha 2 beta 1 can selectively recognize different collagen subtypes. Here we show that their alpha I domains can discriminate between laminin isoforms as well: alpha 1I and alpha 2I recognized laminin-111, -211 and -511, whereas their binding to laminin-411 was negligible. Residue Arg-218 in alpha1 was found to be instrumental in high-avidity binding. The gain-of-function mutation E318W makes the alpha 2I domain to adopt the "open" high-affinity conformation, while the wild-type alpha 2I domain favors the "closed" low-affinity conformation. The E318W mutation markedly increased alpha 2I domain binding to the laminins (-111, -211 and -511), leading us to propose that the activation state of the alpha 2 beta 1 integrin defines its role as a laminin receptor. However, neither wild-type nor alpha 2IE318W domain could bind to laminin-411. alpha 2IE318W also bound tighter to all collagens than alpha 2I wild-type, but it showed reduced ability to discriminate between collagens I, IV and IX. The corresponding mutation, E317A, in the alpha 1I domain transformed the domain into a high-avidity binder of collagens I and IV. Thus, our results indicate that conformational activation of integrin alpha 1I and alpha 2I domains leads to high-avidity binding to otherwise disfavored collagen subtypes.     

An Activating Mutation Reveals a Second Binding Mode of the Integrin α2 I Domain to the GFOGER Motif in Collagens

The GFOGER motif in collagens (O denotes hydroxyproline) represents a high-affinity binding site for all collagen-binding integrins. Other GxOGER motifs require integrin activation for maximal binding. The E318W mutant of the integrin α2β1 I domain displays a relaxed collagen specificity, typical of an active state. E318W binds more strongly than the wild-type α2 I domain to GMOGER, and forms a 2:1 complex with a homotrimeric, collagen-like, GFOGER peptide. Crystal structure analysis of this complex reveals two E318W I domains, A and B, bound to a single triple helix. The E318W I domains are virtually identical to the collagen-bound wild-type I domain, suggesting that the E318W mutation activates the I domain by destabilising the unligated conformation. E318W I domain A interacts with two collagen chains similarly to wild-type I domain (high-affinity mode). E318W I domain B makes favourable interactions with only one collagen chain (low-affinity mode). This observation suggests that single GxOGER motifs in the heterotrimeric collagens V and IX may support binding of activated integrins.     

Integrin activation state determines selectivity for novel recognition sites in fibrillar collagens

Only three recognition motifs, GFOGER, GLOGER, and GASGER, all present in type I collagen, have been identified to date for collagen-binding integrins, such as alpha(2)beta(1). Sequence alignment was used to investigate the occurrence of related motifs in other human fibrillar collagens, and located a conserved array of novel GER motifs within their triple helical domains. We compared the integrin binding properties of synthetic triple helical peptides containing examples of such sequences (GLSGER, GMOGER, GAOGER, and GQRGER) or the previously identified motifs. Recombinant inserted (I) domains of integrin subunits alpha(1), alpha(2) and alpha(11) all bound poorly to all motifs other than GFOGER and GLOGER. Similarly, alpha(2)beta(1) -containing resting platelets adhered well only to GFOGER and GLOGER, while ADP-activated platelets, HT1080 cells and two active alpha(2)I domain mutants (E318W, locked open) bound all motifs well, indicating that affinity modulation determines the sequence selectivity of integrins. GxO/SGER peptides inhibited platelet adhesion to collagen monomers with order of potency F >/= L >/= M > A. These results establish GFOGER as a high affinity sequence, which can interact with the alpha(2)I domain in the absence of activation and suggest that integrin reactivity of collagens may be predicted from their GER content.     

Selective binding of collagen subtypes by integrin alpha 1I, alpha 2I, and alpha 10I domains

Four integrins, namely alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1), form a special subclass of cell adhesion receptors. They are all collagen receptors, and they recognize their ligands with an inserted domain (I domain) in their alpha subunit. We have produced the human integrin alpha(10)I domain as a recombinant protein to reveal its ligand binding specificity. In general, alpha(10)I did recognize collagen types I-VI and laminin-1 in a Mg(2+)-dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When alpha(10)I was tested together with the alpha(1)I and alpha(2)I domains, all three I domains seemed to have their own collagen binding preferences. The integrin alpha(2)I domain bound much better to fibrillar collagens (I-III) than to basement membrane type IV collagen or to beaded filament-forming type VI collagen. Integrin alpha(1)I had the opposite binding pattern. The integrin alpha(10)I domain was similar to the alpha(1)I domain in that it bound very well to collagen types IV and VI. Based on the previously published atomic structures of the alpha(1)I and alpha(2)I domains, we modeled the structure of the alpha(10)I domain. The comparison of the three I domains revealed similarities and differences that could potentially explain their functional differences. Mutations were introduced into the alphaI domains, and their binding to types I, IV, and VI collagen was tested. In the alpha(2)I domain, Asp-219 is one of the amino acids previously suggested to interact directly with type I collagen. The corresponding amino acid in both the alpha(1)I and alpha(10)I domains is oppositely charged (Arg-218). The mutation D219R in the alpha(2)I domain changed the ligand binding pattern to resemble that of the alpha(1)I and alpha(10)I domains and, vice versa, the R218D mutation in the alpha(1)I and alpha(10)I domains created an alpha(2)I domain-like ligand binding pattern. Thus, all three collagen receptors appear to differ in their ability to recognize distinct collagen subtypes. The relatively small structural differences on their collagen binding surfaces may explain the functional specifics.     

Two synergistic activation mechanisms of alpha2beta1 integrin-mediated collagen binding

Activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces ligand-independent aggregation of a cell surface collagen receptor, alpha2beta1 integrin. Concomitantly, TPA increases the avidity of alpha2beta1 for collagen and the number of conformationally activated alpha2beta1 integrins. The structural change was shown using a monoclonal antibody 12F1 that recognizes the "open" (active) conformation of the inserted domain in the alpha2 subunit (alpha2I). Amino acid residue Glu-336 in alpha2 subunit is proposed to mediate the interaction between alpha2I domain and beta1 subunit. Glu-336 seems to regulate a switch between open and "closed" conformations, since the mutation alpha2E336A inhibited the TPA-related increase in the number of 12F1 positive integrins. E336A also reduced cell adhesion to collagen. However, E336A did not prevent the TPA-related increase in adhesion to collagen or alpha2beta1 aggregation. Thus, alpha2beta1 integrin avidity is regulated by two synergistic mechanisms, first an alpha2E336-dependent switch to the open alpha2I conformation, and second an alpha2E336-independent mechanism temporally associated with receptor aggregation.     

Mapping the collagen-binding site in the I domain of the glycoprotein Ia/IIa (integrin alpha(2)beta(1))

The I domain present within the alpha2 chain of the integrin alpha(2)beta(1) (GPIa/IIa) contains the principal collagen-binding site. Based on the crystal structure of the alpha2-I domain, a hypothetical model was proposed in which collagen binds to a groove on the upper surface of the I domain (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517). We have introduced point mutations into 13 residues on the upper surface of the domain. Recombinant mutant proteins were assayed for binding to monoclonal antibodies 6F1 and 12F1, to collagen under static conditions, and for the ability to retain adhesive activity under flow conditions. The mutations to residues surrounding the metal ion-dependent adhesion site that caused the greatest loss of collagen binding under both static and flow conditions are N154S in the betaA-alpha1 turn, N190D in the betaB-betaC turn, D219R in the alpha3-alpha4 turn, and E256V and H258V in the betaD-alpha5 turn. Mutation in one of the residues that coordinate the metal binding, S155A, completely lost the adhesive activity under flow but bound normally under static conditions, whereas the mutation Y285F had the converse effect. We conclude that the upper surface of the domain, including the metal ion-dependent adhesion site motif, defines the collagen recognition site.     

Studies on the metal binding sites in the catalytic domain of beta1,4-galactosyltransferase

The catalytic domain of bovine beta1,4-galactosyltransferase (beta4Gal-T1) has been shown to have two metal binding sites, each with a distinct binding affinity. Site I binds Mn(2+) with high affinity and does not bind Ca(2+), whereas site II binds a variety of metal ions, including Ca(2+). The catalytic region of beta4Gal-T1 has DXD motifs, associated with metal binding in glycosyltransferases, in two separate sequences: D(242)YDYNCFVFSDVD(254) (region I) and W(312)GWGGEDDD(320) (region II). Recently, the crystal structure of beta4Gal-T1 bound with UDP, Mn(2+), and alpha-lactalbumin was determined in our laboratory. It shows that in the primary metal binding site of beta4Gal-T1, the Mn(2+) ion, is coordinated to five ligands, two supplied by the phosphates of the sugar nucleotide and the other three by Asp254, His347, and Met344. The residue Asp254 in the D(252)VD(254) sequence in region I is the only residue that is coordinated to the Mn(2+) ion. Region II forms a loop structure and contains the E(317)DDD(320) sequence in which residues Asp318 and Asp319 are directly involved in GlcNAc binding. This study, using site-directed mutagenesis, kinetic, and binding affinity analysis, shows that Asp254 and His347 are strong metal ligands, whereas Met344, which coordinates less strongly, can be substituted by alanine or glutamine. Specifically, substitution of Met344 to Gln has a less severe effect on the catalysis driven by Co(2+). Glu317 and Asp320 mutants, when partially activated by Mn(2+) binding to the primary site, can be further activated by Co(2+) or inhibited by Ca(2+), an effect that is the opposite of what is observed with the wild-type enzyme.     

Use of synthetic peptides to locate novel integrin alpha2beta1-binding motifs in human collagen III

A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg(2+)-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg(2+). We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.     

Monkey rotavirus binding to alpha2beta1 integrin requires the alpha2 I domain and is facilitated by the homologous beta1 subunit

Rotaviruses utilize integrins during virus-cell interactions that lead to infection. Cell binding and infection by simian rotavirus SA11 were inhibited by antibodies (Abs) to the inserted (I) domain of the alpha2 integrin subunit. To determine directly which integrins or other proteins bind rotaviruses, cell surface proteins precipitated by rotaviruses were compared with those precipitated by anti-alpha2beta1 Abs. Two proteins precipitated by SA11 and rhesus rotavirus RRV from MA104 and Caco-2 cells migrated indistinguishably from alpha2beta1 integrin, and SA11 precipitated beta1 from alpha2beta1-transfected CHO cells. These viruses specifically precipitated two MA104 cell proteins only, but an additional 160- to 165-kDa protein was precipitated by SA11 from Caco-2 cells. The role of the alpha2 I domain in rotavirus binding, infection, and growth was examined using CHO cell lines expressing wild-type or mutated human alpha2 or alpha2beta1. Infectious SA11 and RRV, but not human rotavirus Wa, specifically bound CHO cell-expressed human alpha2beta1 and, to a lesser extent, human alpha2 combined with hamster beta1. Binding was inhibited by anti-alpha2 I domain monoclonal Abs (MAbs), but not by non-I domain MAbs to alpha2, and required the presence of the alpha2 I domain. Amino acid residues 151, 221, and 254 in the metal ion-dependent adhesion site of the alpha2 I domain that are necessary for type I collagen binding to alpha2beta1 were not essential for rotavirus binding. Rotavirus-alpha2beta1 binding led to increased virus infection and RRV growth. SA11 and RRV require the alpha2 I domain for binding to alpha2beta1, and their binding to this integrin is distinguishable from that of collagen.     

Integrin alpha(2)I domain recognizes type I and type IV collagens by different mechanisms

The collagens are recognized by the alphaI domains of the collagen receptor integrins. A common structural feature in the collagen-binding alphaI domains is the presence of an extra helix, named helix alphaC. However, its participation in collagen binding has not been shown. Here, we have deleted the helix alphaC in the alpha(2)I domain and tested the function of the resultant recombinant protein (DeltaalphaCalpha(2)I) by using a real-time biosensor. The DeltaalphaCalpha(2)I domain had reduced affinity for type I collagen (430 +/- 90 nM) when compared with wild-type alpha(2)I domain (90 +/- 30 nM), indicating both the importance of helix alphaC in type I collagen binding and that the collagen binding surface in alpha(2)I domain is located near the metal ion-dependent adhesion site. Previous studies have suggested that the charged amino acid residues, surrounding the metal ion-dependent adhesion site but not interacting with Mg(2+), may play an important role in the recognition of type I collagen. Direct evidence indicating the participation of these residues in collagen recognition has been missing. To test this idea, we produced a set of recombinant alpha(2)I domains with mutations, namely D219A, D219N, D219R, E256Q, D259N, D292N, and E299Q. Mutations in amino acids Asp(219), Asp(259), Asp(292), and Glu(299) resulted in weakened affinity for type I collagen. When alpha(2) D219N and D292N mutations were introduced separately into alpha(2)beta(1) integrin expressed on Chinese hamster ovary cells, no alterations in the cell spreading on type I collagen were detected. However, Chinese hamster ovary cells expressing double mutated alpha(2) D219N/D292N integrin showed remarkably slower spreading on type I collagen, while spreading on type IV collagen was not affected. The data indicate that alpha(2)I domain binds to type I collagen with a different mechanism than to type IV collagen.     

Crystal structure of the alpha1beta1 integrin I domain in complex with an antibody Fab fragment

The alpha1beta1 (VLA-1) integrin is a cell-surface receptor for collagen and laminin and has been implicated in biological pathways involved in several pathological processes. These processes may be inhibited by the monoclonal antibody AQC2, which binds with high affinity to human alpha1beta1 integrin. To understand the structural basis of the inhibition we determined the crystal structure of the complex of a chimeric rat/human I domain of the alpha1beta1 integrin and the Fab fragment of humanized AQC2 antibody. The structure of the complex shows that the antibody blocks the collagen binding site of the I domain. An aspartate residue, from the CDR3 loop of the antibody heavy chain, coordinates the MIDAS metal ion in a manner similar to that of a glutamate residue from collagen. Substitution of the aspartate residue by alanine or arginine results in significant reduction of antibody binding affinity. Interestingly, although the mode of metal ion coordination resembles that of the open conformation, the I domain maintains an overall closed conformation previously observed only for unliganded I domains.     

The binding site in {beta}2-glycoprotein I for ApoER2' on platelets is located in domain V

The antiphospholipid syndrome is caused by autoantibodies directed against beta(2)-glycoprotein I (beta(2)GPI). Dimerization of beta(2)GPI results in an increased platelet deposition to collagen. We found that apolipoprotein E receptor 2' (apoER2'), a member of the low density lipoprotein receptor family, is involved in activation of platelets by dimeric beta(2)GPI. To identify which domain of dimeric beta(2)GPI interacts with apoER2', we have constructed domain deletion mutants of dimeric beta(2)GPI, lacking domain I (DeltaI), II (DeltaII), or V (DeltaV), and a mutant with a W316S substitution in the phospholipid (PL)-insertion loop of domain V. DeltaI and DeltaII prolonged the clotting time, as did full-length dimeric beta(2)GPI; DeltaV had no effect on the clotting time. Second, DeltaI and DeltaII bound to anionic PL, comparable with full-length dimeric beta(2)GPI. DeltaV and the W316S mutant bound with decreased affinity to anionic PL. Platelet adhesion to collagen increased significantly when full-length dimeric beta(2)GPI, DeltaI, or DeltaII (mean increase 150%) were added to whole blood. No increase was found with plasma beta(2)GPI, DeltaV, or the W316S mutant. Immunoprecipitation indicated that full-length dimeric beta(2)GPI, DeltaI, DeltaII, and the W316S mutant can interact with apoER2' on platelets. DeltaV did not associate with apoER2'. We conclude that domain V is involved in both binding beta(2)GPI to anionic PL and in interaction with apoER2' and subsequent activation of platelets. The binding site in beta(2)GPI for interaction with apoER2' does not overlap with the hydrophobic insertion loop in domain V.     

Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.     

Effects of substitution of tryptophan 412 in the substrate activation pathway of yeast pyruvate decarboxylase.

Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at W412, located on the putative substrate activation pathway and linking E91 on the alpha domain with W412 on the gamma domain of the enzyme. While C221 on the beta domain is the residue at which substrate activation is triggered [Baburina, I., et al. (1994) Biochemistry 33, 5630-5635; Baburina, I., et al. (1996) Biochemistry 35, 10249-10255], that information, via the substrate bound at C221, is transmitted to H92 on the alpha domain, across the domain divide from C221 [Baburina, I., et al. (1998) Biochemistry 37, 1235-1244; Baburina, I., et al. (1998) Biochemistry 37, 1245-1255], thence to E91 on the alpha domain [Li, H., and Jordan, F. (1999) Biochemistry 38, 9992-10003], and then on to W412 on the gamma domain and to the active site thiamin diphosphate located at the interface of the alpha and gamma domains [Arjunan, D., et al. (1996) J. Mol. Biol. 256, 590-600]. Substitution at W412 with F and A was carried out, resulting in active enzymes with specific activities about 4- and 10-fold lower than that of the wild-type enzyme. Even though W412 interacts with E91 and H115 via a main chain hydrogen bond donor and acceptor, respectively, there is clear evidence for the importance of the indole side chain of W412 from a variety of experiments: thermostability, fluorescence quenching, and the binding constants of the thiamin diphosphate, and circular dichroism spectroscopy, in addition to conventional steady-state kinetic measurements. While the substrate activation is still prominent in the W412F variant, its level is very much reduced in the W412A variant, signaling that the size of the side chain is also important in positioning the amino acids surrounding the active center to achieve substrate activation. The fluorescence studies demonstrate that W412 is a relatively minor contributor to the well-documented fluorescence of apopyruvate decarboxylase in its native state. The information about the W412 variants provides strong additional support for the putative substrate activation pathway from C221 --> H92 --> E91 --> W412 --> G413 --> thiamin diphosphate. The accumulating evidence for the central role of the beta domain in stabilizing the overall structure is summarized.     

The collagen-binding A-domains of integrins alpha(1)beta(1) and alpha(2)beta(1) recognize the same specific amino acid sequence, GFOGER, in native (triple-helical) collagens

We have previously assigned an integrin alpha(2)beta(1)-recognition site in collagen I to the sequence, GFOGERGVEGPOGPA (O = Hyp), corresponding to residues 502-516 of the alpha(1)(I) chain and located in the fragment alpha(1)(I)CB3 (Knight, C. G., Morton, L. F., Onley, D. J., Peachey, A. R., Messent, A. J., Smethurst, P. A., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (1998) J. Biol. Chem. 273, 33287-33294). In this study, we show that recognition is entirely contained within the six-residue sequence GFOGER. This sequence, when in triple-helical conformation, readily supports alpha(2)beta(1)-dependent cell adhesion and exhibits divalent cation-dependent binding of isolated alpha(2)beta(1) and recombinant alpha(2) A-domain, being at least as active as the parent collagen. Replacement of E by D causes loss of recognition. The same sequence binds integrin alpha(1) A-domain and supports integrin alpha(1)beta(1)-mediated cell adhesion. Triple-helical GFOGER completely inhibits alpha(2) A-domain binding to collagens I and IV and alpha(2)beta(1)-dependent adhesion of platelets and HT 1080 cells to these collagens. It also fully inhibits alpha(1) A-domain binding to collagen I and strongly inhibits alpha(1)beta(1)-mediated adhesion of Rugli cells to this collagen but has little effect on either alpha1 A-domain binding or adhesion of Rugli cells to collagen IV. We conclude that the sequence GFOGER represents a high-affinity binding site in collagens I and IV for alpha(2)beta(1) and in collagen I for alpha(1)beta(1). Other high-affinity sites in collagen IV mediate its recognition of alpha(1)beta(1).     

Functional analysis of a recombinant glycoprotein Ia/IIa (Integrin alpha(2)beta(1)) I domain that inhibits platelet adhesion to collagen and endothelial matrix under flow conditions

The interaction of platelets with collagen plays an important role in primary hemostasis. Glycoprotein Ia/IIa (GPIa/IIa, integrin alpha(2)beta(1)) is a major platelet receptor for collagen. The binding site for collagen has been mapped to the I domain within the alpha(2) subunit (GPIa). In order to assess the role of the alpha(2)-I domain structure in GPIa/IIa binding to collagen, a recombinant I domain (amino acids 126-337) was expressed in Escherichia coli. The alpha(2)-I protein bound human types I and III collagen in a saturable and divalent cation-dependent manner and was blocked by the alpha(2)beta(1) function blocking antibody 6F1. The alpha(2)-I protein inhibited collagen-induced platelet aggregation (IC(50) = 600 nM). Unexpectedly, 6F1, an antibody that fails to inhibit platelet aggregation in platelet-rich plasma, blocked the inhibitory effect of the alpha(2)-I protein. The alpha(2)-I protein was able to prevent platelet adhesion to a collagen surface exposed to flowing blood under low shear stress. Interestingly, it inhibited platelet adhesion to extracellular matrix at high shear stress. These results, taken together, provide firm evidence that GPIa/IIa directly mediates the first contact of platelets with collagen under both stirring and flow conditions.     

Identification of a negatively charged peptide motif within the catalytic domain of progelatinases that mediates binding to leukocyte beta 2 integrins

The alpha M beta 2 integrin of leukocytes can bind a variety of ligands. We screened phage display libraries to isolate peptides that bind to the alpha M I domain, the principal ligand binding site of the integrin. Only one peptide motif, (D/E)(D/E)(G/L)W, was obtained with this approach despite the known ligand binding promiscuity of the I domain. Interestingly, such negatively charged sequences are present in many known beta 2 integrin ligands and also in the catalytic domain of matrix metalloproteinases (MMPs). We show that purified beta 2 integrins bind to pro-MMP-2 and pro-MMP-9 gelatinases and that that the negatively charged sequence of the MMP catalytic domain is an active beta 2 integrin-binding site. Furthermore, a synthetic DDGW-containing phage display peptide inhibited the ability of beta 2 integrin to bind progelatinases but did not inhibit the binding of cell adhesion-mediating substrates such as intercellular adhesion molecule-1, fibrinogen, or an LLG-containing peptide. Immunoprecipitation and cell surface labeling demonstrated complexes of pro-MMP-9 with both the alpha M beta 2 and alpha L beta 2 integrins in leukocytes, and pro-MMP-9 colocalized with alpha M beta 2 in cell surface protrusions. The DDGW peptide and the gelatinase-specific inhibitor peptide CTTHWGFTLC blocked beta 2 integrin-dependent leukocyte migration in a transwell assay. These results suggest that leukocytes may move in a progelatinase-beta 2 integrin complex-dependent manner.     

The leukocyte integrin alpha D beta 2 binds VCAM-1: evidence for a binding interface between I domain and VCAM-1.

The trafficking of leukocytes through tissues is supported by an interaction between the beta 2 (CD18) integrins CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) and their ligand ICAM-1. The most recently identified and fourth member of the beta 2 integrins, alpha D beta 2, selectively binds ICAM-3 and does not appear to bind ICAM-1. We have reported recently that alpha D beta 2 can support eosinophil adhesion to VCAM-1. Here we demonstrate that expression of alpha D beta 2 in a lymphoid cell that does not express alpha 4 integrins confers efficient binding to VCAM-1. In addition, a soluble form of alpha D beta 2 binds VCAM-1 with greater efficiency relative to ICAM-3. The I domain of alpha D contains a binding site for VCAM-1 since recombinant alpha D I domain binds specifically to VCAM-1. In addition, alpha D mAb that block cellular binding to VCAM-1 bind the alpha D I domain. Using VCAM-1 mutants we have determined that the binding site on VCAM-1 for alpha D beta 2 overlaps with that of alpha 4++ integrins. Substitution of VCAM-1 aspartate at position 40, D40, within the conserved integrin binding site, diminishes binding to alpha D beta 2 and abrogates binding to the alpha D I domain. The corresponding integrin binding site residue in ICAM-3 is also essential to alpha D beta 2 binding. Finally, we demonstrate that alpha D beta 2 can support lymphoid cell adhesion to VCAM-1 under flow conditions at levels equivalent to those mediated by alpha 4 beta 1. These results indicate that VCAM-1 can bind to an I domain and that the binding of alpha D beta 2 to VCAM-1 may contribute to the trafficking of a subpopulation of leukocytes that express alpha D beta 2.     

Crystal structure of EMS16 in complex with the integrin alpha2-I domain

Snake venoms contain a number of heterodimeric C-type lectin-like proteins (CLPs) that interact specifically with components of the haemostatic system. EMS16 from the venom of Echis multisquamatus binds to the collagen receptor, integrin alpha2beta1, also known as glycoprotein (GP) Ia/IIa, and specifically inhibits collagen binding. Here we report the crystal structure of EMS16 in complex with recombinant integrin alpha2-I domain that plays a central role in collagen binding. The structure of the complex at 1.9 Angstrom resolution reveals that the collagen-binding site of the alpha2-I domain is covered completely by the bound EMS16. This blockage by EMS16 appears to spatially inhibit collagen binding to the alpha2-I domain. The bound alpha2-I domain adopts a closed conformation, which is seen in the absence of ligand, suggesting that EMS16 stabilizes a closed conformation corresponding to the less active structure of the alpha2-I domain. EMS16 does not directly bind to the manganese ion and residues of the metal ion-dependent adhesion site (MIDAS) of the alpha2-I domain, suggesting that EMS16 may have the potential to bind specifically to the alpha2-I domain in a metal ion-independent fashion.     

Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase. Keywords: collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase