Structural characterization of the interaction between α M I‐domain of the integrin Mac‐1 (α M β 2 ) and the cytokine pleiotrophin

Abstract

Integrin receptors are involved in numerous physiological processes, including inflammation, cell migration, extracellular matrix recognition, survival, and growth. Integrin Mac-1 (αMβ2) is a monocyte adhesion receptor that can undergo rapid activation yielding conformational changes that subsequently allow increased affinity for its ligands and enable cellular responses such as rolling, adhesion, and transmigration of phagocytes to the inflammatory sites. αMβ2 is also the most promiscuous integrin receptor because it recognizes a large number of distinct ligands that share no homologous recognition motif. In particular, αMI-domain is known to bind cationic proteins/peptides depleted in acidic residues. This contradicts the canonical ligand-binding mechanism of αI-domains, which requires an acidic amino acid in the ligand to coordinate the divalent cation within the metal ion-dependent adhesion site (MIDAS) of αI-domains. The lack of acidic amino acids in the αMI-domain-binding sequences suggests the existence of an as-yet uncharacterized interaction mechanism. In the present study, we analyzed interactions of αMI-domain with a representative Mac-1 ligand, the cationic cytokine pleiotrophin (PTN). Through nuclear magnetic resonance (NMR) chemical shift perturbations, cross saturation, and nuclear Overhauser effect spectroscopy (NOESY), we found the interaction between αMI-domain and PTN is divalent cation-independent and mediated mostly by hydrophobic contacts between the N-terminal domain of PTN and residues in the α5-β5 loop of αMI-domain. The mutation I265 in the α5-β5 region confirms this area as a binding site of αMI-domain for PTN. The observation that both increased ionic strength and mutations of acidic residues in the α5-β5 loop weaken the interaction between the proteins indicates electrostatic interaction may also play a significant role in the binding. Based on results from these experiments, we formulated a model of the interaction between αMI-domain and PTN. This unique mechanism of interaction between PTN and αMI-domain should explain ligand recognition specificity of αMβ2.