Structure-Function Analysis of Arg-Gly-Asp Helix Motifs in αvβ6 Integrin Ligands

Abstract

Data relating to the structural basis of ligand recognition by integrins are limited. Here we describe the physical requirements for high affinity binding of ligands to ␣v␤6. By combining a series of structural analyses with functional testing, we show that 20-mer peptide ligands, derived from high affinity ligands of ␣v␤6 (foot-and-mouth-disease virus, latency associated peptide), have a common structure comprising an Arg-Gly-Asp motif at the tip of a hairpin turn followed immediately by a C-terminal helix. This arrangement allows two conserved Leu/ Ile residues at Asp ؉1 and Asp ؉4 to be presented on the outside face of the helix enabling a potential hydrophobic interaction with the ␣v␤6 integrin, in addition to the Arg-Gly-Asp interaction. The extent of the helix determines peptide affinity for ␣v␤6 and potency as an ␣v␤6 antagonist. A major role of this C-terminal helix is likely to be the correct positioning of the Asp ؉1 and Asp ؉4 residues. These data suggest an explanation for several biological functions of ␣v␤6 and provide a structural platform for design of ␣v␤6 antagonists.Understanding of the molecular basis of the binding interface of integrins with their ligands still is relatively poor. The most detailed information available comes from x-ray crystallography of RGD 4 peptide binding to ␣v␤3, where the RGD motif bridged the ␣ and ␤ subunits, the arginine associating with the ␣v subunit and the aspartate coordinating with the bivalent metal ion on the ␤3 subunit (1). However, the RGD motif occurs in many extracellular matrix ligands so specificity is modified by other residues, often flanking the RGD site (2), although distant residues also can affect ligand binding (3). Detailed comparison of the different ligands of one integrin could illuminate the essential elements that determine specificity and affinity, improve biological understanding of integrinligand interactions, and allow rational design of targeting peptides and peptidomimetics (4 -6); we have made just such a comparison for ␣v␤6.The integrin ␣v␤6 is an epithelial specific integrin that is expressed at low or undetectable levels in adult tissues but can be up-regulated during tissue remodeling. Thus increased ␣v␤6 expression occurs during wound healing, development and inflammation (7), and in more severe pathologies, including chronic skin wounds (8) and cancer (9). The ability of ␣v␤6 to promote migration and invasion, in part through protease upregulation (10 -12), may explain why ␣v␤6 expression is an independent indicator of colon cancer aggressiveness (13). These data suggest that reagents designed to specifically antagonize ␣v␤6 could have clinical utility in colon cancer and possibly other diseases.␣v␤6 binds to the arginine-glycine-aspartate (RGD) motif in its ligands, which include fibronectin, tenascin, the latency associated peptides (LAP) of TGF␤1 (14) and TGF␤3 (15), and the VP1 coat protein of foot-and-mouth disease virus (FMDV) (16). In addition, the motif DLXXL was identified by phage display of 7-and 12-residue peptid...

Molecular Basis of the Ligand Binding Specificity of αvβ8 Integrin

“…Nephronectin also contains an auxiliary sequence LFEIFEIER required for the high affinity binding of nephronectin to the ␣8␤1 integrin, which functions in concert with an RGD motif (24). DiCara et al (25) reported that the interaction of latent TGF-␤1 with ␣v␤6 integrin is determined by an LXXI sequence immediately C-terminal to the RGD motif, in which two hydrophobic residues Leu-218 and Ile-221 are required for the high affinity binding of latent TGF-␤1 to the ␣v␤6 integrin. Our results show that Leu-218 is critically required for latent TGF-␤1 recognition by ␣v␤8 integrin, but Ile-221 is dispensable for recognition, because the substitution of Ile-221 with Ala did not affect the high affinity binding of latent TGF-␤1 to ␣v␤8 integrin.…”

“…Integrin-To explore the molecular basis of the restricted ligand specificity of ␣v␤8 integrin, we focused on the LXXI sequence immediately following the RGD motif, because the LXXI sequence was required for the high affinity binding of latent TGF-␤1 to ␣v␤6 integrin in concert with the RGD motif (25,26). We constructed latent TGF-␤1 mutants, in which the Leu-218 and/or Ile-221 residues of the LXXI sequence were substituted with Ala, and we assessed their ability to bind to the ␣v␤8 integrin (Fig.…”

“…␣8␤1 integrin selectively binds to nephronectin via a bipartite interaction with the RGD motif and LFEIFEIER sequence, the latter located at the C-terminal ϳ10 amino acids from the RGD motif (24). The high affinity binding of ␣v␤6 integrin to its ligands, foot-and-mouth disease virus and latent TGF-␤1, requires the RGD motif and an LXX(L/I) sequence, of which the latter forms an ␣-helix to align the two conserved hydrophobic residues along the length of the helix (25,26). Thus, the ligand-binding specificity of RGD-binding integrins might be defined by the bipartite recognition site comprising an RGD motif and residues flanking the RGD motif or those in neighboring domains that come into close proximity with the RGD motif in an intact ligand protein.…”

Production of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin αvβ6 binding peptides for NMR

“…Structural analysis of 20-mer peptides from the VP1 coat protein of FMDV and the latency associated peptide of TGFb1 revealed that secondary structure is also important for binding potency, in particular, an a-helix immediately proximal to the RGD hairpin that brings the two Leu residues in the consensus sequence together on the same face of the helix. 9 The potency of different peptides was also found to be related to the length and propensity of helix formation, and furthermore, NMR experiments revealed that the two Leu residues are in close contact with the integrin when the peptide is bound which suggests that the helix is present in the bound conformation of the peptide. 9 To enable further NMR-based studies into the structure, dynamics and binding interactions of peptide ligands for avb6, it was desirable to generate 15 N and 13 C isotopically labelled peptides.…”

“…9 The potency of different peptides was also found to be related to the length and propensity of helix formation, and furthermore, NMR experiments revealed that the two Leu residues are in close contact with the integrin when the peptide is bound which suggests that the helix is present in the bound conformation of the peptide. 9 To enable further NMR-based studies into the structure, dynamics and binding interactions of peptide ligands for avb6, it was desirable to generate 15 N and 13 C isotopically labelled peptides. Such an approach allows the use of more sophisticated multi-dimensional NMR and nucleus-selective editing techniques to provide more detailed information than possible with homonuclear 1 H NMR alone.…”