Interleukin 32 (IL-32) contains a typical α-helix bundle structure that resembles focal adhesion targeting region of focal adhesion kinase-1

IL-32 can be expressed in several isoforms. The amino acid sequences of the major IL-32 isoforms were used to predict the secondary and tertiary protein structure by I-TASSER software. The secondary protein structure revealed coils and α-helixes, but no β sheets. Furthermore, IL-32 contains an RGD motif, which potentially activates procaspase-3 intracellular and or binds to integrins. Mutation of the RGD motif did not result in inhibition of the IL-32β- or IL-32γ-induced cytotoxicity mediated through caspase-3. Although IL-32α interacted with the extracellular part of αVβ3 and αVβ6 integrins, only the αVβ3 binding was inhibited by small RGD peptides. Additionally, IL-32β was able to bind to αVβ3 integrins, whereas this binding was not inhibited by small RGD peptides. In addition to the IL-32/integrin interactions, we observed that IL-32 is also able to interact with intracellular proteins that are involved in integrin and focal adhesion signaling. Modeling of IL-32 revealed a distinct α-helix protein resembling the focal adhesion targeting region of focal adhesion kinase (FAK). Inhibition of FAK resulted in modulation of the IL-32β- or IL-32γ-induced cytotoxicity. Interestingly, IL-32α binds to paxillin without the RGD motif being involved. Finally, FAK inhibited IL-32α/paxillin binding, whereas FAK also could interact with IL-32α, demonstrating that IL-32 is a member of the focal adhesion protein complex. This study demonstrates for the first time that IL-32 binds to the extracellular domain of integrins and to intracellular proteins like paxillin and FAK, suggesting a dual role for IL-32 in integrin signaling.     

Cyclic RGD peptides interfere with binding of the Helicobacter pylori protein CagL to integrins αVβ3 and α5β1

The human pathogen Helicobacter pylori that may cause different gastric diseases exploits integrins for infection of gastric cells. The H. pylori protein CagL present on the outer region of the type IV secretion pilus contains an RGD sequence (-Arg-Gly-Asp-) that enables binding to cells presenting integrins α5β1 and αVβ3. This interaction can be inhibited with conformationally designed cyclic RGD peptides derived from the CagL epitope -Ala-Leu-Arg-Gly-Asp-Leu-Ala-. The inhibition of the CagL-αVβ3 interaction by different RGD peptides strongly suggests the importance of the RGD motif for CagL binding. CagL point mutants (RAD, RGA) show decreased affinity to integrin αVβ3. Furthermore, structure-activity relationship studies with cyclic RGD peptides in a spatial screening approach show the distinct influence of the three-dimensional arrangement of RGD motif on the ability to interfere with this interaction. Resulting from these studies, similar structural requirements for the CagL epitope as previously suggested for other ligands of integrin αVβ3 are proposed.     

Adhesion of Cultured Human Kidney Mesangial Cells to Native Entactin: Role of Integrin Receptors

Entactin is an extracellular matrix glycoprotein which binds to laminin and is found in most renal basement membranes and in the glomerular mesangial matrix. In the present study, we have characterized specific integrin receptors on cultured human mesangial cells (CHMC) responsible for adhesion to native entactin. The integrin receptors α2,β1, α3,β1, α5,β1, αv,β3, αv,β5, and α6 complexed with either β1 or β4 could be immune precipitated from detergent extracts of metabolically labeled CHMC. Adhesion assays with inhibitory anti integrin monoclonal antibodies (mab) demonstrated that CHMC use both αv,β3 and a β1-containing integrin to bind surfaces coated with native entactin. Optimal binding of CHMC to native entactin required the participation of cations. Using wild type and mutant recombinant entactin fragments, the binding site for the αv,β3 receptor was localized to the RGD sequence on the rod or E domain of entactin. CHMC adhesion to mutant full length recombinant entactin ligands lacking the E domain RGD sequence confirmed the presence of ligand binding site(s) for β1 integrin receptor(s). Differences in CHMC binding characteristics to recombinant and full length entactin compared to native bovine basement membrane entactin were observed. This suggests that tertiary molecular structure may contribute to entactin ligand binding properties. Primary amino acid residue sequences and tertiary structure of entactin may play roles in forming functional cell attachment sites in native basement membrane entactin.     

Tenascin-C: Its functions as an integrin ligand

This review summarizes the experimental evidence of tenascin-C/integrin interactions, emphasizing the identification of integrin binding sites and the effects of specific interactions on cell behavior. At least four integrins appear to bind to the third fibronectin-type 3 domain of tenascin-C: α9β1, αVβ3, α8β1 and αVβ6. The α9β1 integrin recognizes a highly conserved IDG motif in this domain, while the others recognize an RGD motif. There is also significant evidence that the collagen receptor α2β1 can bind to tenascin-C, but the interacting site is unknown. Tenascin-C interactions with α9β1 and αVβ3 can promote cell proliferation and interactions with αVβ3 can also inhibit apoptosis. Interactions with α7β1 integrin, which may bind to the alternatively spliced domain of tenascin-C, and α9β1 integrin are able to influence the differentiation of mesenchymal stem cells into the neuronal lineage. This illustrates the potential for using our knowledge of tenascins and their integrin receptors in stem cell-based therapies.     

Blockade of osteoclast-mediated bone resorption through occupancy of the integrin receptor: A potential approach to the therapy of osteoporosis

Bone resorption requires the tight attachment of the bone-resorbing cells, the osteoclasts, to the bone mineralized matric. Integrins, a class of cell surface adhesion glycoproteins, play a key role in the attachment process. Most integrins bind to their ligands via the arginyl-glycyl-aspartyl (R-G-D) tripeptide present within the ligand sequence. The interaction between integrins and ligaands results in bidirectional transfer of signals across the plasma Membrane. Tyrosine phosphorylaation occurs within cells as a result of integrin binding to ligaands and probably plays a role in the formation of the osteoclast clear zone, a specialized region of the osteoclast membraane maintained by cytoskeletal structure and involved in bone resorption. Human osteoclasts express α₂β₃ and α_vβ₃ integrins on their surface. Such signaling may also lead to “inside-out” effects, like increased expression of integrin receptors on the cell surface, or increased affinity of the integrin to its ligand. The α_vβ₃ integrin, a vitronectin receptor, plays an essential role in bone resorption. Antibodies to this integrin and short synthetic RGD-containing peptides are able to block bone resorption in vitro. Echistatin, an RGD-containing protein from a snake venom, binds to the α_vβ₃ integrin and blocks bone resssorption both in vitro and in vivo. Peptides containing the RGD motif are potential competitive “antagonists” a of the osteoclast integrins and may have utility in the blockage of bone resorption. Agonists may be identified by stimulation of intracellularsignaling. In theory, tissue spacificity can be achieved by (1) introducing specific amino acids in positions adjacent to the RGD sequence, (2) identifying non-RGD integrin binding domains, or (3) modulating the affinity of integrins for their endogenous ligands.     

Mapping of heparin/heparan sulfate binding sites on αvβ3 integrin by molecular docking

Heparin/heparan sulfate interact with growth factors, chemokines, extracellular proteins, and receptors. Integrins are αβ heterodimers that serve as receptors for extracellular proteins, regulate cell behavior, and participate in extracellular matrix assembly. Heparin binds to RGD‐dependent integrins (αIIbβ3, α5β1, αvβ3, and αvβ5) and to RGD‐independent integrins (α4β1, αXβ2, and αMβ2), but their binding sites have not been located on integrins. We report the mapping of heparin binding sites on the ectodomain of αvβ3 integrin by molecular modeling. The surface of the ectodomain was scanned with small rigid probes mimicking the sulfated domains of heparan sulfate. Docking results were clustered into binding spots. The best results were selected for further docking simulations with heparin hexasaccharide. Six potential binding spots containing lysine and/or arginine residues were identified on the ectodomain of αvβ3 integrin. Heparin would mostly bind to the top of the genu domain, the Calf‐I domain of the α subunit, and the top of the β subunit of RGD‐dependent integrins. Three spots were close enough from each other on the integrin surface to form an extended binding site that could interact with heparin/heparan sulfate chains. Because heparin does not bind to the same integrin site as protein ligands, no steric hindrance prevents the formation of ternary complexes comprising the integrin, its protein ligand, and heparin/heparan sulfate. The basic amino acid residues predicted to interact with heparin are conserved in the sequences of RGD‐dependent but not of RGD‐independent integrins suggesting that heparin/heparan sulfate could bind to different sites on these two integrin subfamilies. Copyright © 2013 John Wiley & Sons, Ltd.     

Staphylococcus epidermidis serine–aspartate repeat protein G (SdrG) binds to osteoblast integrin alpha V beta 3

Staphylococcus epidermidis is the leading etiologic agent of orthopaedic implant infection. Contamination of the implanted device during insertion allows bacteria gain entry into the sterile bone environment leading to condition known as osteomyelitis. Osteomyelitis is characterised by weakened bones associated with progressive bone loss. The mechanism through which S. epidermidis interacts with bone cells to cause osteomyelitis is poorly understood. We demonstrate here that S. epidermidis can bind to osteoblasts in the absence of matrix proteins. S. epidermidis strains lacking the cell wall protein SdrG had a significantly reduced ability to bind to osteoblasts. Consistent with this, expression of SdrG in Lactococcus lactis resulted in significantly increased binding to the osteoblasts. Protein analysis identified that SdrG contains a potential integrin recognition motif. αVβ3 is a major integrin expressed on osteoblasts and typically recognises RGD motifs in its ligands. Our results demonstrate that S. epidermidis binds to recombinant purified αVβ3, and that a mutant lacking SdrG failed to bind. Blocking αVβ3 on osteoblasts significantly reduced binding to S. epidermidis. These studies are the first to identify a mechanism through which S. epidermidis binds to osteoblasts and potentially offers a mechanism through which implant infection caused by S. epidermidis leads to osteomyelitis.     

Site‐specific inhibition of integrin αvβ3‐vitronectin association by a ser‐asp‐val sequence through an Arg‐Gly‐Asp‐binding site of the integrin

A functional proteomic technology using protein chip and molecular simulation was used to demonstrate a novel biomolecular interaction between P11, a peptide containing the Ser‐Asp‐Val (SDV) sequence and integrin αvβ3. P11 (HSDVHK) is a novel antagonistic peptide of integrin αvβ3 screened from hexapeptide library through protein chip system. An in silico docking study and competitive protein chip assay revealed that the SDV sequence of P11 is able to create a stable inhibitory complex onto the vitronectin‐binding site of integrin αvβ3. The Arg‐Gly‐Asp (RGD)‐binding site recognition by P11 was site specific because the P11 was inactive for the complex formation of a denatured form of integrin–vitronectin. P11 showed a strong antagonism against αvβ3‐GRGDSP interaction with an IC₅₀ value of 25.72±3.34 nM, whereas the value of GRGDSP peptide was 1968.73±444.32 nM. The binding‐free energies calculated from the docking simulations for each P11 and RGD peptide were ?3.99 and ?3.10 kcal/mol, respectively. The free energy difference between P11 and RGD corresponds to approximately a 4.5‐fold lower K_i value for the P11 than the RGD peptide. The binding orientation of the docked P11 was similar to the crystal structure of the RGD in αvβ3. The analyzed docked poses suggest that a divalent metal–ion coordination was a common driving force for the formation of both SDV/αvβ3 and RGD/αvβ3 complexes. This is the first report on the specific recognition of the RGD‐binding site of αvβ3 by a non‐RGD containing peptide using a computer‐assisted proteomic approach.     

Structural specializations of α(4)β(7), an integrin that mediates rolling adhesion

The lymphocyte homing receptor integrin α(4)β(7) is unusual for its ability to mediate both rolling and firm adhesion. α(4)β(1) and α(4)β(7) are targeted by therapeutics approved for multiple sclerosis and Crohn's disease. Here, we show by electron microscopy and crystallography how two therapeutic Fabs, a small molecule (RO0505376), and mucosal adhesion molecule-1 (MAdCAM-1) bind α(4)β(7). A long binding groove at the α(4)-β(7) interface for immunoglobulin superfamily domains differs in shape from integrin pockets that bind Arg-Gly-Asp motifs. RO0505376 mimics an Ile/Leu-Asp motif in α(4) ligands, and orients differently from Arg-Gly-Asp mimics. A novel auxiliary residue at the metal ion-dependent adhesion site in α(4)β(7) is essential for binding to MAdCAM-1 in Mg(2+) yet swings away when RO0505376 binds. A novel intermediate conformation of the α(4)β(7) headpiece binds MAdCAM-1 and supports rolling adhesion. Lack of induction of the open headpiece conformation by ligand binding enables rolling adhesion to persist until integrin activation is signaled.     

Dynamics and functional differences between dendroaspin and rhodostomin: Insights into protein scaffolds in integrin recognition

Dendroaspin (Den) and rhodostomin (Rho) are snake venom proteins containing a PRGDMP motif. Although Den and Rho have different 3D structures, they are highly potent integrin inhibitors. To study their structure, function, and dynamics relationships, we expressed Den and Rho in Pichia pastoris. The recombinant Den and Rho inhibited platelet aggregation with the K_I values of 149.8 and 83.2 nM. Cell adhesion analysis showed that Den was 3.7 times less active than Rho when inhibiting the integrin αIIbβ3 and 2.5 times less active when inhibiting the integrin αvβ3. In contrast, Den and Rho were similarly active when inhibiting the integrin α5β1 with the IC₅₀ values of 239.8 and 256.8 nM. NMR analysis showed that recombinant Den and Rho have different 3D conformations for their arginyl‐glycyl‐aspartic acid (RGD) motif. However, the comparison with Rho showed that the docking of Den into integrin αvβ3 resulted in a similar number of contacts. Analysis of the dynamic properties of the RGD loop in Den and Rho showed that they also had different dynamic properties. These results demonstrate that protein scaffolds affect the function, structure, and dynamics of their RGD motif.     

Crystal structure of trimestatin, a disintegrin containing a cell adhesion recognition motif RGD

Disintegrins are a family of small proteins containing an Arg-Gly-Asp (RGD) sequence motif that binds specifically to integrin receptors. Since the integrin is known to serve as the final common pathway leading to aggregation via formation of platelet-platelet bridges, disintegrins act as fibrinogen receptor antagonists. Here, we report the first crystal structure of a disintegrin, trimestatin, found in snake venom. The structure of trimestatin at 1.7A resolution reveals that a number of turns and loops form a rigid core stabilized by six disulfide bonds. Electron densities of the RGD sequence are visible clearly at the tip of a hairpin loop, in such a manner that the Arg and Asp side-chains point in opposite directions. A docking model using the crystal structure of integrin alphaVbeta3 suggests that the Arg binds to the propeller domain, and Asp to the betaA domain. This model indicates that the C-terminal region is another potential binding site with integrin receptors. In addition to the RGD sequence, the structural evidence of a C-terminal region (Arg66, Trp67 and Asn68) important for disintegrin activity allows understanding of the high affinity and selectiveness of snake venom disintegrin for integrin receptors. The crystal structure of trimestatin should provide a useful framework for designing and developing more effective drugs for controlling platelet aggregation and anti-angiogenesis cancer.     

RGD cadherins and α2β1 integrin in cancer metastasis: A dangerous liaison

We propose a new cadherin family classification comprising epithelial cadherins (cadherin 17 [CDH17], cadherin 16, VE-cadherin, cadherin 6 and cadherin 20) containing RGD motifs within their sequences. Expression of some RGD cadherins is associated with aggressive forms of cancer during the late stages of metastasis, and CDH17 and VE-cadherin have emerged as critical actors in cancer metastasis. After binding to α2β1 integrin, these cadherins promote integrin β1 activation, and thereby cell adhesion, invasion and proliferation, in liver and lung metastasis. Activation of α2β1 integrin provokes an affinity increase for type IV collagen, a major component of the basement membrane and a critical partner for cell anchoring in liver and other metastatic organs. Activation of α2β1 integrin by RGD motifs breaks an old paradigm of integrin classification and supports an important role of this integrin in cancer metastasis. Recently, synthetic peptides containing the RGD motif of CDH17 elicited highly specific and selective antibodies that block the ability of CDH17 RGD to activate α2β1 integrin. These monoclonal antibodies inhibit metastatic colonization in orthotopic mouse models of liver and lung metastasis for colorectal cancer and melanoma, respectively. Hopefully, blocking the cadherin RGD ligand capacity will give us control over the integrin activity in solid tumors metastasis, paving the way for development of new agents of cancer treatment.     

Correlation between biological activity and binding energy in systems of integrin with cyclic RGD-containing binders: a QM/MM molecular dynamics study

We here report a combined quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) study on the binding interactions between the α(V)β(3) integrin and eight cyclic arginine-glycine-aspartate (RGD) containing peptides. The initial conformation of each peptide within the binding site of the integrin was determined by docking the ligand to the reactive site of the integrin crystal structure with the aid of docking software FRED. The subsequent QM/MM MD simulations of the complex structures show that these eight cyclic RGD-peptides have a generally similar interaction mode with the binding site of the integrin to the cyclo(RGDf-N[M]V) analog found in the crystal structure. Still, there are subtle differences in the interactions of peptide ligands with the integrin, which contribute to the different inhibition activities. The averaged QM/MM protein-ligand interaction energy (IE) is remarkably correlated to the biological activity of the ligand. The IE, as well as a three-variable model which is somewhat interpretable, thus can be used to predict the bioactivity of a new ligand quantitatively, at least within a family of analogs. The present study establishes a helpful protocol for advancing lead compounds to potent inhibitors.     

RGD-containing ankyrin externalized onto the cell surface triggers αVβ3 integrin-mediated erythrophagocytosis

The RGD motif on the extracellular matrix or cell surface, together with its integrin receptors, constitutes a major recognition system for cell adhesion. There are several erythrocyte major membrane skeletal proteins, e.g., α spectrin, ankyrin, and protein 4.2, that bear an RGD motif. However, it is not known whether the RGD/integrin recognition system is utilized in the erythrocyte-macrophage adhesion during erythrophagocytosis. Here we report that the RGD motif of ankyrin, but not others, is recognized by the α_vβ₃ integrin receptor. In addition, the RGD motif of ankyrin, a peripheral membrane protein, can be externalized onto the cell surface when erythrocytes are incubated with calcium and sheared both at physiological levels. Furthermore, the erythrocyte-macrophage adhesion can be specifically inhibited by ankyrin and/or α_vβ₃. Thus, externalization of ankyrin followed by RGD/integrin recognition may be a novel mechanism by which erythrocytes adhere to macrophages preceding phagocytosis.     

Non-RGD domains of osteopontin promote cell adhesion without involving αv integrins

Osteopontin (OPN) is an integrin-binding secreted protein that contains an Arg-Gly-Asp (RGD) amino acid sequence and binds to various cell types via RGD-mediated interaction with the αvβ3 integrin. We have identified a cell line whose binding to OPN does not require RGD or αv interactions. We compared the ability of two murine cell lines, L929 fibroblastic cells and B16-BL6 melanoma cells, to interact with OPN (from human milk, and recombinant human and mouse OPN) as well as recombinant OPN prepared to include either the N-terminal or C-terminal halves but lacking the RGD sequence. Both cell lines adhered to GRGDS peptides coupled to BSA, and these interactions were inhibited by addition of GRGDS (but not GRGES) peptides or a monoclonal antibody specific to the αv integrin subunit. Adhesion of L929 cells to OPN was also dependent on the RGD sequence and the αv integrin subunit. However, the binding of B16-BL6 cells was not inhibited by either GRGDS peptides or the anti-αv antibody. B16-BL6 (but not L929) cells were also able to adhere to and spread on both N-terminal and C-terminal OPN proteins that lack the RGD sequence, and these interactions were not inhibited by either GRGDS peptides or anti-αv antibody. Together these results indicate that B16-BL6 cells can adhere to OPN by interactions that are independent of either the RGD sequence or the αv integrin subunit, and suggest that some cells can interact with additional, non-RGD binding sites in OPN. © 1996 Wiley-Liss, Inc.     

Structural basis for distinctive recognition of fibrinogen gammaC peptide by the platelet integrin alphaIIbbeta3

Hemostasis and thrombosis (blood clotting) involve fibrinogen binding to integrin alpha(IIb)beta(3) on platelets, resulting in platelet aggregation. alpha(v)beta(3) binds fibrinogen via an Arg-Asp-Gly (RGD) motif in fibrinogen's alpha subunit. alpha(IIb)beta(3) also binds to fibrinogen; however, it does so via an unstructured RGD-lacking C-terminal region of the gamma subunit (gammaC peptide). These distinct modes of fibrinogen binding enable alpha(IIb)beta(3) and alpha(v)beta(3) to function cooperatively in hemostasis. In this study, crystal structures reveal the integrin alpha(IIb)beta(3)-gammaC peptide interface, and, for comparison, integrin alpha(IIb)beta(3) bound to a lamprey gammaC primordial RGD motif. Compared with RGD, the GAKQAGDV motif in gammaC adopts a different backbone configuration and binds over a more extended region. The integrin metal ion-dependent adhesion site (MIDAS) Mg(2+) ion binds the gammaC Asp side chain. The adjacent to MIDAS (ADMIDAS) Ca(2+) ion binds the gammaC C terminus, revealing a contribution for ADMIDAS in ligand binding. Structural data from this natively disordered gammaC peptide enhances our understanding of the involvement of gammaC peptide and integrin alpha(IIb)beta(3) in hemostasis and thrombosis.     

Polydom/SVEP1 is a ligand for integrin α9β1

A variety of proteins, including tenascin-C and osteopontin, have been identified as ligands for integrin α9β1. However, their affinities for integrin α9β1 are apparently much lower than those of other integrins (e.g. α3β1, α5β1, and α8β1) for their specific ligands, leaving the possibility that physiological ligands for integrin α9β1 still remain unidentified. In this study, we found that polydom (also named SVEP1) mediates cell adhesion in an integrin α9β1-dependent manner and binds directly to recombinant integrin α9β1 with an affinity that far exceeds those of the known ligands. Using a series of recombinant polydom proteins with N-terminal deletions, we mapped the integrin-binding site to the 21st complement control protein domain. Alanine-scanning mutagenesis revealed that the EDDMMEVPY sequence (amino acids 2636-2644) in the 21st complement control protein domain was involved in the binding to integrin α9β1 and that Glu(2641) was the critical acidic residue for the integrin binding. The importance of this sequence was further confirmed by integrin binding inhibition assays using synthetic peptides. Immunohistochemical analyses of mouse embryonic tissues showed that polydom colocalized with integrin α9 in the stomach, intestine, and other organs. Furthermore, in situ integrin α9β1 binding assays using frozen mouse tissues showed that polydom accounts for most, but not all, of the integrin α9β1 ligands in tissues. Taken together, the present findings indicate that polydom is a hitherto unknown ligand for integrin α9β1 that functions as a physiological ligand in vivo.     

Identification of the principal binding site for RGD-containing ligands in the alpha(V)beta(3) integrin: a photoaffinity cross-linking study

By superimposing data obtained by photo-cross-linking RGD-containing ligands to the human alpha(V)beta(3) integrin onto the crystal structure of the ectopic domain of this receptor (Xiong et al. (2001) Science 294, 339-345), we have identified the binding site for the RGD triad within this integrin. We synthesized three novel analogues of the 49-amino acid disintegrin, echistatin: [Bpa(21),Leu(28)]-, [Bpa(23),Leu(28)]-, and [Bpa(28)]echistatin. Each contains a photoreactive p-benzoyl-phenylalanyl (Bpa) residue in close proximity to the RGD motif which spans positions 24-26; together, the photoreactive positions flank the RGD motif. The analogues bind with high affinity to the purified recombinant alpha(V)beta(3) integrin, but very poorly to the closely related human alpha(IIb)beta(3) platelet integrin. While echistatin analogues containing Bpa in either position 23 or 28 cross-link specifically and almost exclusively to the beta(3) subunit of alpha(V)beta(3), [Bpa(21),Leu(28)]echistatin cross-links to both the alpha(V) and the beta(3) subunits, with cross-linking to the former favored. [Bpa(23),Leu(28)]echistatin cross-links 10-30 times more effectively than the other two analogues. We identified beta(3)[109-118] as the domain that encompasses the contact site for [Bpa(28)]echistatin. This domain is included in beta(3)[99-118] (Bitan et al. (2000) Biochemistry 39, 11014-11023), a previously identified contact domain for a cyclic RGD-containing heptapeptide with a benzophenone moiety in a position that is similar to the placement of the benzophenone in [Bpa(28)]echistatin relative to the RGD triad. Recently, we identified beta(3)[209-220] as the contact site for an echistatin analogue with a photoreactive group in position 45, near the C-terminus of echistatin (Scheibler et al. (2001) Biochemistry 40, 15117-14126). Taken together, these results support the hypothesis that the very high binding affinity of echistatin to alpha(V)beta(3) results from two distinct epitopes in the ligand, a site including the RGD triad and an auxiliary epitope at the C-terminus of echistatin. Combining our results from photoaffinity cross-linking studies with data now available from the recently published crystal structure of the ectopic domain of alpha(V)beta(3), we characterize the binding site for the RGD motif in this receptor.     

Effect of P to A mutation of the N-terminal residue adjacent to the Rgd motif on rhodostomin: importance of dynamics in integrin recognition

Rhodostomin (Rho) is an RGD protein that specifically inhibits integrins. We found that Rho mutants with the P48A mutation 4.4-11.5 times more actively inhibited integrin α5β1. Structural analysis showed that they have a similar 3D conformation for the RGD loop. Docking analysis also showed no difference between their interactions with integrin α5β1. However, the backbone dynamics of RGD residues were different. The values of the R(2) relaxation parameter for Rho residues R49 and D51 were 39% and 54% higher than those of the P48A mutant, which caused differences in S(2), R(ex), and τ(e). The S(2) values of the P48A mutant residues R49, G50, and D51 were 29%, 14%, and 28% lower than those of Rho. The R(ex) values of Rho residues R49 and D51 were 0.91 s(-1) and 1.42 s(-1); however, no R(ex) was found for those of the P48A mutant. The τ(e) values of Rho residues R49 and D51 were 9.5 and 5.1 times lower than those of P48A mutant. Mutational study showed that integrin α5β1 prefers its ligands to contain (G/A)RGD but not PRGD sequences for binding. These results demonstrate that the N-terminal proline residue adjacent to the RGD motif affect its function and dynamics, which suggests that the dynamic properties of the RGD motif may be important in Rho's interaction with integrin α5β1.     

Solution structures and integrin binding activities of an RGD peptide with two isomers

The Arg-Gly-Asp (RGD) sequence serves as the primary integrin recognition site in extracellular matrix proteins, and peptides containing this sequence can mimic the activities of the matrix proteins. Depending on the context of the RGD sequence, an RGD-containing peptide may bind to all of the RGD-directed integrins, to a few, or to only a single one. We have previously isolated from a phage-displayed peptide library a cyclic peptide that binds avidly to the alpha(v)beta3 and alpha(v)beta5 integrins but does not bind to other closely related integrins. This peptide, ACDCRGDCFCG, exists in two natural configurations depending on internal disulfide bonding. The peptide with the 1-4; 2-3 disulfide bond arrangement accounts for most of the alpha(v) integrin binding activity, whereas the 1-3; 2-4 peptide is about 10-fold less potent. Solution structure analysis by nuclear magnetic resonance reveals an entirely different presentation of the RGD motif in the two isomers of RGD-4C. These results provide new insight into the ligand recognition specificity of integrins.