Structure-function studies of integrin-ligand recognition

• Main Author: McCleverty, Clare
• Published: University of Leicester 2001
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.696940

Integrins are a large family of αβ heterodimeric cell surface receptors that interact with the extracellular matrix and/or counter-receptors on other cells. These interactions control the adhesion and migration of cells as well as regulating numerous signal transduction pathways. Integrins exist in low and high affinity states, subject to allosteric regulation. Integrin-ligand recognition is divalent cation-dependent and mediated by the α subunit N-terminal repeats, the β subunit I domain and in certain integrins, the α subunit I domain. Two competing models based upon structure predictions, the β-propeller and EF-hand-like models, were tested to determine which model represents the structural components of the ligand binding α subunit N-terminal repeats. Recombinant fragments of α4 repeats IV-V, VI-VII and IV-VII, corresponding to the EF-hand-like model, were insoluble, thus preventing further analysis. A recombinant fragment of all seven α4 repeats contains a predominant secondary structure content of anti-parallel β-sheet, compatible with the β-propeller model. The interactions of the αM I domain with fragment D from fibrinogen and the extracellular domains of ICAM-1 were studied using surface plasmon resonance. Optimal binding conditions and equilibrium dissociation constants were established for these interactions. Co-crystallisation studies were pursued with the αM I domain and its ligands, fragment D and ICAM-1, but a co-crystal was not obtained due to the presence of a subpopulation of low affinity I domain molecules. Disulphide bonds were then introduced to lock the αM I domain in the open and closed conformations, corresponding to the high and low affinity states, respectively. Equilibrium dissociation constants for the open and closed mutants reveal a marked increase and decrease in ligand binding affinity, respectively. Stabilisation of the closed mutant via a disulphide bond is verified in the crystal structure. The affinity state of both mutants is fully reversible by reduction of the disulphide bond. These mutants provide useful tools for future studies to understand integrin allostery and will simplify ligand binding studies in the isolated I domain and intact receptor.