Dynamic change in the distribution of alpha5beta1 integrin on isolated ventral membrane: effect of divalent cation species

We investigated the spatial distribution of alpha5beta1 integrin in isolated ventral plasma membranes (VPMs) of human foreskin fibroblasts in order to elucidate how the interaction of integrin with cytoskeletal and extracellular matrix proteins might affect the integrin distribution. Cells were exposed to the jet of buffer to remove the apical surface and most of cellular organelles. After this treatment VPMs, which adhered to the glass surface, possessed the cellular structures such as fibronectin (FN) fibrils and actin stress fibers. The isolated VPMs thus prepared were employed without fixation to investigate the change in the integrin distribution. In isolated VPMs, alpha5beta1 integrin, labeled with Cy3-tagged anti-integrin antibody, was found to accumulate not only at the tips of stress fibers but also along FN fibrils extending from there. When divalent cations were removed with EDTA, the accumulated integrin was dispersed, and the original pattern of distribution was recovered upon restoration of divalent cations. Talin, an integrin-actin cytoskeleton linker protein, was found to accumulate only at the tips of stress fibers in isolated VPMs, but alpha5beta1 integrin did not exhibit strong accumulation there, indicating that talin played little role in integrin distribution in isolated VPMs. The amount of alpha-actinin associated with stress fibers was found to drastically decrease in isolated VPMs, which was presumably related to the failure of localization of integrin at the tips of stress fibers. It was also shown that the association of stress fibers to isolated VPMs seemed to be independent of accumulation of integrin.     

Modulation of alpha5beta1 integrin functions by the phospholipid and cholesterol contents of cell membranes

Several modifications of the alpha5beta1 integrin, which alter its intracellular and extracellular interaction with fibronectin and other proteins, have been reported. However, the significance of the lateral mobility of integrin molecules in the plasma membrane, as a regulator of their distribution and function, is poorly understood. We examined this problem by increasing the cholesterol content of plasma membranes, and consequently modifying the fluidity of membrane phospholipids, in rat fibroblasts. Under these conditions, the clustering of alpha5beta1 integrin molecules in focal adhesions, their adhesion to the cell-binding domain of fibronectin, and their association with the cytoskeletal protein talin were significantly enhanced as compared to control cells. However, the activation of MAP-kinase pathways by the association of fibronectin with alpha5beta1 integrin, and its association with integrin-linked kinase (ilk), were suppressed. The treated cells also showed distinct changes in shape, and their actin stress fiber network was more dense and thick as compared to control cells. The changes in fluidity of phospholipids occurred differentially and fluidity of phosphatidyl-ethanolamine increased, while that of phosphatidyl-choline was reduced. Our results suggest that proteins in focal adhesions could be partitioned in specific lipid domains, which regulate specific aspects of alpha5beta1 integrin functions.     

Adhesion of human skin fibroblasts to Cyr61 is mediated through integrin alpha 6beta 1 and cell surface heparan sulfate proteoglycans

The angiogenic inducer Cyr61 is an extracellular matrix-associated heparin-binding protein that can mediate cell adhesion, stimulate cell migration, and enhance growth factor-stimulated DNA synthesis in both fibroblasts and endothelial cells in culture. In vivo, Cyr61 induces neovascularization and promotes tumor growth. Cyr61 is a prototypic member of a highly conserved family of secreted proteins that includes connective tissue growth factor, nephroblastoma overexpressed, Elm-1/WISP-1, Cop-1/WISP-2, and WISP-3. Encoded by an immediate early gene, Cyr61 synthesis is induced by serum growth factors in cultured fibroblasts and in dermal fibroblasts during cutaneous wound healing. We previously demonstrated that Cyr61 mediates adhesion of vascular endothelial cells and activation-dependent adhesion of blood platelets through direct interaction with integrins alpha(V)beta(3) and alpha(IIb)beta(3), respectively. In this study, we show that the adhesion of primary human skin fibroblasts to Cyr61 is mediated through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans (HSPGs), which most likely serve as co-receptors. Either destruction of cell surface HSPGs or prior occupancy of the Cyr61 heparin-binding site completely blocked cell adhesion to Cyr61. A heparin-binding defective mutant of Cyr61 was unable to mediate fibroblast adhesion through integrin alpha(6)beta(1) but still mediated endothelial cell adhesion through integrin alpha(V)beta(3), indicating that endothelial cell adhesion through integrin alpha(V)beta(3) is independent of the heparin-binding activity of Cyr61. These results identify Cyr61 as a novel adhesive substrate for integrin alpha(6)beta(1) and provide the first demonstration of the requirement for HSPGs in integrin-mediated cell attachment. In addition, these findings suggest that Cyr61 might elicit disparate biological effects in different cell types through interaction with distinct integrin receptors.     

Production of recombinant soluble human integrin alpha4beta1

Integrin alpha4beta1 is a major leukocyte adhesion receptor that is a key target for the development of anti-inflammatory therapeutics. With the dual long-term goals of developing a reagent for use in high-throughput inhibitor screening assays and for crystallisation trials and subsequent structure determination, we have generated a recombinant soluble alpha4beta1 receptor. Both subunits were truncated prior to the transmembrane domains by site-directed mutagenesis and expressed using baculovirus infection of insect cells. The molecular weights of the recombinant subunits were as expected for post-translationally unmodified protein. In addition, as observed for the native subunit, a proportion of the alpha4 subunit was proteolytically processed into two fragments. ELISA and solid phase ligand-binding assays were performed to investigate the folding and functionality of the soluble integrin. The data suggest that the receptor was correctly folded and that it bound recombinant ligands with similar kinetics to the native molecule.     

C-terminal opening mimics 'inside-out' activation of integrin alpha5beta1

Integrins are adhesion molecules that convey signals both to and from the cytoplasm across the plasma membrane. In resting cells, integrins in a low affinity state can be activated by 'inside-out signaling', in which signals affecting integrin heterodimer cytoplasmic domains cause a conformational change in the integrin ligand-binding headpiece connected to the membrane by two long, approximately 16 nm stalks. Here we demonstrate a mechanism for conveying a conformational change over the long distance from the plasma membrane to the headpiece. We prepared soluble, alpha5beta1 integrin heterodimer extracellular fragments in which interactions between alpha- and beta-subunit cytoplasmic domains were replaced with an artificial clasp. Release of this C-terminal clasp by specific protease cleavage resulted in an approximately 14 nm separation of the stalks coupled to increased binding to fibronectin. This activation did not require any associated molecules or clustering and was observed with physiological concentrations of divalent cations. These findings suggest that the overall mechanism for integrin inside-out activation involves the spatial separation of the cytoplasmic and/or transmembrane domains.     

The mesenchymal alpha11beta1 integrin attenuates PDGF-BB-stimulated chemotaxis of embryonic fibroblasts on collagens

alpha11beta1 constitutes the most recent addition to the integrin family and has been shown to display a binding preference for interstitial collagens found in mesenchymal tissues. We have previously observed that when alpha11beta1 integrin is expressed in cells lacking endogenous collagen receptors, it can mediate PDGF-BB-dependent chemotaxis on collagen I in vitro. To determine in which cells PDGF and alpha11beta1 might cooperate in regulating cell migration in vivo, we studied in detail the expression and distribution of alpha11 integrin chain in mouse embryos and tested the ability of PDGF isoforms to stimulate the alpha11beta1-mediated cell migration of embryonic fibroblasts. Full-length mouse alpha11 cDNA was sequenced and antibodies were raised to deduced alpha11 integrin amino acid sequence. In the embryonic mouse head, alpha11 protein and RNA were localized to ectomesenchymally derived cells. In the periodontal ligament, alpha11beta1 was expressed as the only detectable collagen-binding integrin, and alpha11beta1 is thus a major receptor for cell migration and matrix organization in this cell population. In the remainder of the embryo, the alpha11 chain was expressed in a subset of mesenchymal cells including tendon/ligament fibroblasts, perichondrial cells, and intestinal villi fibroblasts. Most of the alpha11-expressing cells also expressed the alpha2 integrin chain, but no detectable overlap was found with the alpha1 integrin chain. In cells expressing multiple collagen receptors, these might function to promote a more stable cell adhesion and render the cells more resistant to chemotactic stimuli. Wild-type embryonic fibroblasts activated mainly the PDGF beta receptor in response to PDGF-BB and migrated on collagens I, II, III, IV, V, and XI in response to PDGF-BB in vitro, whereas mutant fibroblasts that lacked alpha11beta1 in their collagen receptor repertoire showed a stronger chemotactic response on collagens when stimulated with PDGF-BB. In the cellular context of embryonic fibroblasts, alpha11beta1 is thus anti-migratory. We speculate that the PDGF BB-dependent cell migration of mesenchymal cells is tightly regulated by the collagen receptor repertoire, and disturbances of this repertoire might lead to unregulated cell migration that could affect normal embryonic development and tissue structure.     

Structure of integrin alpha5beta1 in complex with fibronectin

The membrane-distal headpiece of integrins has evolved to specifically bind large extracellular protein ligands, but the molecular architecture of the resulting complexes has not been determined. We used molecular electron microscopy to determine the three-dimensional structure of the ligand-binding headpiece of integrin alpha5beta1 complexed with fragments of its physiological ligand fibronectin. The density map for the unliganded alpha5beta1 headpiece shows a 'closed' conformation similar to that seen in the alphaVbeta3 crystal structure. By contrast, binding to fibronectin induces an 'open' conformation with a dramatic, approximately 80 degrees change in the angle of the hybrid domain of the beta subunit relative to its I-like domain. The fibronectin fragment binds to the interface between the beta-propeller and I-like domains in the integrin headpiece through the RGD-containing module 10, but direct contact of the synergy-region-containing module 9 to integrin is not evident. This finding is corroborated by kinetic analysis of real-time binding data, which shows that the synergy site greatly enhances k(on) but has little effect on the stability or k(off) of the complex.     

TGF-beta 1 modulated the expression of alpha 5 beta 1 integrin and integrin-mediated signaling in human hepatocarcinoma cells

Integrins are a family of cell surface adhesion molecules which mediate cell adhesion and initiate signaling pathways that regulate cell spreading, migration, differentiation, and proliferation. TGF-beta is a multifunctional factor that induces a wide variety of cellular processes. In this study, we show that, TGF-beta 1 treatment enhanced the amount of alpha 5 beta 1 integrin on cell surface, the mRNA level of alpha 5 subunit, and subsequently stimulated cell adhesion onto a fibronectin (Fn) and laminin (Ln) matrix in SMMC-7721 cells. TGF-beta 1 could also promote cell migration. Furthermore, our results showed that TGF-beta1 treatment stimulated the tyrosine phosphorylation level of FAK, which can be activated by the ligation and clustering of integrins. PTEN can directly dephosphorylate FAK, and the results that TGF-beta 1 could down-regulate PTEN at protein level suggested that TGF-beta 1 might stimulate FAK phosphorylation through increasing integrin signaling and reducing dephosphorylation of FAK. These studies indicated that TGF-beta 1 and integrin-mediated signaling act synergistically to enhance cell adhesion and migration and affect downstream signaling molecules of hepatocarcinoma cells.     

HIV-1 infection of human T lymphocytes results in enhanced alpha 5 beta 1 integrin expression.

Altered T cell adherence after human immunodeficiency virus 1 (HIV-1) infection may contribute to viral pathogenesis in the acquired immune deficiency syndrome. To address this hypothesis, we assessed mechanisms of T cell adherence to extracellular matrix proteins in vitro. We found that after HIV-1 infection, both chronically infected H9 CD4+ T cells and acutely infected primary peripheral blood lymphocytes acquired the ability to adhere to the extracellular matrix glycoprotein fibronectin, to a lesser extent to type IV collagen and laminin, but not to type I collagen. H9 cells chronically infected with two of the three HIV-1 strains studied showed approximately a sevenfold increase in attachment to fibronectin, while the same cells infected with the human retrovirus HIV-2 did not. Adhesion was accompanied by changes in morphology, including marked spreading and increased filopodia. These alterations were not blocked by the protein kinase C inhibitor H-7, which did inhibit TPA-induced T cell attachment to fibronectin. Monoclonal antibodies against both the alpha 5 and the beta 1 subunits of the classical fibronectin receptor as well as an Arg-Gly-Asp (RGD) peptide inhibited attachment, whereas anti-alpha 4 monoclonal antibodies and the CS1 peptide did not. Binding to collagen IV was also inhibited by the anti-beta 1 monoclonal antibody, but not the other antibodies. Cells metabolically labeled with [35S]methionine and analyzed by immunoprecipitation with polyclonal anti-beta 1 integrin antibody showed a 2.5-fold increase in integrin synthesis in infected cells compared to uninfected controls. This increase in synthesis was associated with an increase in cell surface expression of both alpha 5 and beta 1 integrins by FACS (registered trademark of Becton Dickinson for a fluorescence-activated cell sorter) analysis. Enhanced expression of integrins such as alpha 5 beta 1 may cause T cell adherence to a variety of tissues, where released viral gene products may induce some of the tissue-specific manifestations of HIV-1 infection.     

A regulated interaction between alpha5beta1 integrin and osteopontin

The extracellular matrix protein osteopontin (OPN) interacts with a number of integrins, namely alphavbeta1, alphavbeta3, alphavbeta5, alpha9beta1, alpha8beta1, and alpha4beta1. We have investigated the interaction of alpha5beta1 integrin with OPN using K562 cells, which only express alpha5beta1. alpha5beta1 is in a low activation state in this cell line, but can be stimulated to a higher activation state by the phorbol ester TPA. Treating K562 wild-type cells (K562-WT) with TPA stimulated an interaction between alpha5beta1 and OPN. No interaction was seen in the absence of TPA. alpha5beta1 selectively interacted with a GST fusion protein of the N-terminal fragment of OPN (aa17-168), which is generated in vivo by thrombin cleavage of OPN. Expression of the alpha4 integrin in K562 cells (K562-alpha4beta1) stimulated alpha5beta1-dependent binding to aa17-168 in the absence of TPA, suggesting that alpha4beta1 activates alpha5beta1 in K562 cells. Adhesion via alpha5beta1 is mediated by the Arg-Gly-Asp (RGD) motif of OPN, as mutating this sequence to Arg-Ala-Asp (RAD) blocked binding of both cell types. These data demonstrate that thrombin cleavage regulates the adhesive properties of OPN and that alpha5beta1 integrin can interact with thrombin-cleaved osteopontin when in a high activation state.     

Functions of alpha3beta1 integrin

alpha3beta1 integrin is a laminin receptor with apparently diverse functions. In epithelial cells it acts as a receptor for the basement membrane, whereas in neuronal and possibly tumor cells it mediates migration. Interactions of alpha3beta1 integrin with tetraspanin proteins may provide clues to how it transduces signals that affect cell behavior.     

Direct association between caspase 3 and alpha5beta1 integrin and its role during anoikis of rat fibroblasts

We have demonstrated a direct association between alpha5beta1 integrin and caspase 3, both pro- and mature enzyme, in various sub-cellular compartments of rat fibroblasts undergoing anoikis. Integrin associated caspase 3 showed high activity in the plasma membranes, whereas in the cytosol and microsomal fraction it showed little or no activity. Our results suggest a possible role for recycled un-ligated alpha5beta1 integrin molecules between cytosol and plasma membrane, in regulation of caspase-3 activity and induction of cell death in adhesion-deprived cells.     

A minimized human integrin alpha(5)beta(1) that retains ligand recognition

Two isolated recombinant fragments from human integrin alpha(5)beta(1) encompassing the FG-GAP repeats III to VII of alpha(5) and the insertion-type domain from beta(1), respectively, are structurally well defined in solution, based on CD evidence. Divalent cation binding induces a conformational adaptation that is achieved by Ca(2+) or Mg(2+) (or Mn(2+)) with alpha(5) and only by Mg(2+) (or Mn(2+)) with beta(1). Mn(2+) bound to beta(1) is highly hydrated ( approximately 3 water molecules), based on water NMR relaxation, in agreement with a metal ion-dependent adhesion site-type metal coordination. Each fragment saturated with Mg(2+) (or Mn(2+)) binds a recombinant fibronectin ligand in an RGD-dependent manner. A conformational rearrangement is induced on the fibronectin ligand upon binding to the alpha(5), but not to the beta(1) fragment, based on CD. Ligand binding results in metal ion displacement from beta(1). Both alpha(5) and beta(1) fragments form a stable heterodimer (alpha(5)beta(1) mini-integrin) that retains ligand recognition to form a 1:1:1 ternary complex, in the presence of Mg(2+), and induces a specific conformational adaptation of the fibronectin ligand. A two-site model for RGD binding to both alpha and beta integrin components is inferred from our data using low molecular weight RGD mimetics.     

Urokinase receptors promote beta1 integrin function through interactions with integrin alpha3beta1

The urokinase receptor (uPAR) is linked to cellular migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate cell signaling in response to urokinase (uPA) binding. The mechanisms for these activities remain incompletely defined, although uPAR was recently identified as a cis-acting ligand for the beta2 integrin CD11b/CD18 (Mac-1). Here we show that a major beta1 integrin partner for uPAR/uPA signaling is alpha3. In uPAR-transfected 293 cells uPAR complexed (>90%) with alpha3beta1 and antibodies to alpha3 blocked uPAR-dependent vitronectin (Vn) adhesion. Soluble uPAR bound to recombinant alpha3beta1 in a uPA-dependent manner (K(d) < 20 nM) and binding was blocked by a 17-mer alpha3beta1 integrin peptide (alpha325) homologous to the CD11b uPAR-binding site. uPAR colocalized with alpha3beta1 in MDA-MB-231 cells and uPA (1 nM) enhanced spreading and focal adhesion kinase phosphorylation on fibronectin (Fn) or collagen type I (Col) in a pertussis toxin- and alpha325-sensitive manner. A critical role of alpha3beta1 in uPA signaling was verified by studies of epithelial cells from alpha3-deficient mice. Thus, uPAR preferentially complexes with alpha3beta1, promoting direct (Vn) and indirect (Fn, Col) pathways of cell adhesion, the latter a heterotrimeric G protein-dependent mechanism of signaling between alpha3beta1 and other beta1 integrins.     

Down-regulation of the chicken alpha 5 beta 1 integrin fibronectin receptor during development.

We have characterized the diversity of the chicken beta 1 integrin family and studied the expression of individual receptors during development. The diversity of the beta 1 integrin family was investigated by affinity purifying the beta 1 integrins from a variety of adult and embryonic tissues. These purifications reveal the relative levels of expression and also the differential expression of the alpha subunits in those tissues. Monoclonal antibodies were generated against the prominent 'band 1' of the embryonic chicken integrins and used to characterize the expression of this alpha subunit in embryonic and adult tissues. This alpha subunit is shown to be the chicken homologue of human alpha 5 fibronectin receptor. The chicken alpha 5 beta 1 integrin is the most prominent beta 1 integrin in the embryo and is expressed on the majority of cell types through the day 17 stage. The distribution of this receptor in the embryo closely parallels the distribution of its ligand, fibronectin. In adult tissues, expression of this receptor is greatly diminished relative to the expression of other alpha subunits. The cell type distribution is highly restricted: limited primarily to the vasculature and to connective tissue regions. These studies reveal a prominent role for the alpha 5 beta 1 integrin in embryonic cell types and a down-regulation of this receptor on many cell types during development.     

The cAMP-Epac-Rap1 pathway regulates cell spreading and cell adhesion to laminin-5 through the alpha3beta1 integrin but not the alpha6beta4 integrin

Laminin-5 is an important constituent of the basal lamina. The receptors for laminin-5, the integrins alpha3beta1 and alpha6beta4, have been associated with epithelial wound migration and carcinoma invasion. The signal transduction mechanisms that regulate these integrins are not well understood. We report here that the small GTPase Rap1 regulates the adhesion of a number of cell lines to various extracellular matrix proteins including laminin-5. cAMP also mediates cell adhesion and spreading on laminin-5, a process that is independent of protein kinase A but rather dependent on Epac1, a cAMP-dependent exchange factor for Rap. Interestingly, although both alpha3beta1 and alpha6beta4 mediate adhesion to laminin-5, only alpha3beta1-dependent adhesion is dependent on Rap1. These results provide evidence for a function of the cAMP-Epac-Rap1 pathway in cell adhesion and spreading on different extracellular matrix proteins. They also define different roles for the laminin-binding integrins in regulated cell adhesion and subsequent cell spreading.     

Affinity of integrin alpha 1 beta 1 from liver sinusoidal membranes for type IV collagen.

Hepatic sinusoidal membranes isolated from adult rats were extracted with detergent and fractionated on a wheat germ agglutinin affinity column. Bound glycoproteins were eluted with N-acetyl glucosamine and chromatographed on a type IV collagen affinity column. Recovery of the bound fraction by EDTA and analysis by SDS-PAGE revealed two glycoproteins with apparent molecular weights of 180,000 and 117,000. These were identified immunologically by Western blotting as the alpha and beta subunits of integrin alpha 1 beta 1.     

Human fibroblasts with mutations in COL5A1 and COL3A1 genes do not organize collagens and fibronectin in the extracellular matrix, down-regulate alpha2beta1 integrin, and recruit alphavbeta3 Instead of alpha5beta1 integrin

Dermal fibroblasts derived from types I and IV Ehlers-Danlos syndrome (EDS) patients, carrying mutations in COL5A1 and COL3A1 genes, respectively, synthesize aberrant types V and III collagen (COLL) and show defective organization of these proteins into the extracellular matrix (ECM) and high reduction of their functional receptor, the alpha(2)beta(1) integrin, compared with control fibroblasts. EDS cells also show reduced levels of fibronectin (FN) in the culture medium and lack an FN fibrillar network. Finally, EDS cells prevalently organize alpha(v)beta(3) integrin instead of alpha(5)beta(1) integrin. The alpha(v)beta(3) integrin, distributed on the whole EDS cell surface, shows FN binding and assembly properties when the cells are treated with purified FN. Treatment of EDS cells with purified COLLV or COLLIII, but not with FN, restores the control phenotype (COLL(+), FN(+), alpha(v)beta(3)(-), alpha(5)beta(1)(+), alpha(2)beta(1)(+)). Function-blocking antibodies to COLLV, COLLIII, or alpha(2)beta(1) integrin induce in control fibroblasts an EDS-like phenotype (COLL(-), FN(-), alpha(v)beta(3)(+), alpha(5)beta(1)(-), alpha(2)beta(1)(-)). These results show that in human fibroblasts alpha(2)beta(1) integrin organization and function are controlled by its ligand, and that the alpha(2)beta(1)-COLL interaction, in turn, regulates FN integrin receptor recruitment: high alpha(2)beta(1) integrin levels induce alpha(5)beta(1) integrin organization, while low alpha(2)beta(1) integrin levels lead to alpha(v)beta(3) integrin organization.     

Distinct mechanisms of alpha 5beta 1 integrin activation by Ha-Ras and R-Ras

To investigate the possible roles of the Ras/Rho family members in the inside-out signals to activate integrins, we examined the ability of Ras/Rho small GTPases to stimulate avidity of alpha(5)beta(1) (VLA-5) to fibronectin in bone marrow-derived mast cells. We found that both Ha-Ras(Val-12) and R-Ras(Val-38) had strong stimulatory effects on adhesion and ligand binding activity of VLA-5 to fibronectin. However, only Ha-Ras(Val-12)-, but not R-Ras(Val-38)-induced adhesion was inhibited by wortmannin, which suggests that Ha-Ras(Val-12) is dependent on phosphatidylinositol (PI) 3-kinase on adhesion whereas R-Ras(Val-38) has another PI 3-kinase independent pathway to induce adhesion. The effector loop mutant Ha-Ras(Val-12)E37G, but not Y40C retained the ability to stimulate adhesion of mast cells to fibronectin. Consistently, PI 3-kinase p110delta, predominantly expressed in mast cells, interacted with Ha-Ras(Val-12) E37G, but not Y40C, which was also correlated with the levels of Akt phosphorylation in mast cells. Furthermore, marked adhesion was induced by a membrane-targeted version of p110delta. These results indicate that Ha-Ras(Val-12) activated VLA-5 through PI 3-kinase p110delta. The mutational effects of the R-Ras effector loop region on adhesion were not correlated with PI 3-kinase activities, consistent with our contention that R-Ras has a distinct pathway to modulate avidity of VLA-5.     

Jararhagin-derived RKKH peptides induce structural changes in alpha1I domain of human integrin alpha1beta1

Integrin alpha(1)beta(1) is one of four collagen-binding integrins in humans. Collagens bind to the alphaI domain and in the case of alpha(2)I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In alpha(2)I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound "open" conformation of alpha(2)I differs significantly from the "closed" conformation seen in the structure of apo-alpha(2)I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to alpha(1)I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of alpha(1)I with Mg(2+) and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-alpha(1)I ("closed" conformation) and a model structure of the alpha(1)I ("open" conformation) based on the closely related structure of alpha(2)I reveals that the binding site is partially blocked to ligands by Glu(255) and Tyr(285) in the "closed" structure, whereas in the "open" structure helix C is unwound and these residues are shifted, and the "RKKH" peptides fit well when docked. The "open" conformation of alpha(2)I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of alpha(1)I and shown experimentally for alpha(1)I using a fluorescent hydrophobic probe.