In Silico and In Vitro Analysis of ITGB1 Binding to its Partners ITGA5 and ITGA6

Abstract

Integrins are cell adhesion molecules that mediate cell-cell and cell-matrix interactions, forming heterodimers which affect their interaction specificity. In particular, the integrin (ITG) β1 subunit heterodimerizes with the α5 subunit in various extracellular matrix, blood, and cell surface proteins but not in epithelial cells, where the α6 subunit is its preferred binding partner. The importance of α6β1 and α5β1 in maintaining normal cell activity is observed in cases of abnormal levels α6β1 and α5β1 heterodimer expression that lead to metastasis. As the type of integrin expressed (i.e. ITGα5β1 or ITGα6β1) depends on the ITGβ1 binding partner (i.e α5 or α6), the current study focuses on investigating factors that affect binding partner preference. In-silico structural analysis investigated the electrostatic potential of the integrin heterodimer (e.g. α5β1) interface, quantified the bonds and accessible surfaces present, and analyzed the contributions of electrostatic complementarity to binding. Steered molecular dynamics (SMD) simulations were used to compare the relative binding affinities of the different heterodimer pairings. In vitro analysis will be conducted to verify the in silico predictions. Current progress with in vitro studies has allowed the recombinant expression and purification of the β-propeller domain of ITGα6. The putative recombinant protein was purified by fast protein liquid chromatography (FPLC) using a HisTrap metal affinity column. SDS-PAGE and Anti-His Western Blot analysis indicated the presence of the successfully expressed, His-tagged recombinant ITGα6 ligand binding domain. For both ITGα6β1 and ITGα5β1, equilibrium molecular dynamics are in progress to determine factors that stabilize their interactions.