Distinct Roles of β1 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 α2β1

Dimitra Valdramidou,Martin J Humphries,A Paul Mould

doi:10.1074/jbc.m802066200

Abstract

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.

Highlights

Crystallographic studies of liganded, non-␣I domain containing ␣V␤3 and ␣IIb␤3 integrin segments [13, 14] have revealed the basis for the absolute dependence on divalent cations for ligand binding: a ligand carboxyl group coordinates to the cation present at the metal-ion dependent adhesion site (MIDAS) of ␤I
The LIMBS is a candidate for a Ca2ϩ-binding stimulatory site, it was not possible to test this suggestion for ␣5␤1 because mutation of this site led to a complete loss of ligand recognition [22]
To elucidate the roles of the MIDAS, ADMIDAS, and LIMBS we mutated critical residues for cation binding in the ␤I domain and tested for ligand binding ability and regulation by divalent cations in solid phase assays

Summary

Introduction

Crystallographic studies of liganded, non-␣I domain containing ␣V␤3 and ␣IIb␤3 integrin segments [13, 14] have revealed the basis for the absolute dependence on divalent cations for ligand binding: a ligand carboxyl group coordinates to the cation present at the MIDAS of ␤I. To elucidate the roles of the MIDAS, ADMIDAS, and LIMBS we mutated critical residues for cation binding in the ␤I domain and tested for ligand binding ability and regulation by divalent cations in solid phase assays. In this system, an activating mutation in the ␣2 I domain was found to partially overcome the effect of LIMBS mutations, thereby allowing us to test whether the LIMBS is a stimulatory site. Our results demonstrate that the three sites have distinct roles in the regulation of collagen binding to the ␣2 I domain, and that the LIMBS is essential for the stimulation of Mg2ϩsupported ligand binding by Ca2ϩ

Results

Discussion

Conclusion