Identification of novel integrin binding partners for CIB1: structural and thermodynamic basis of CIB1 promiscuity

The short cytoplasmic tails of the α and β chains of integrin adhesion receptors regulate integrin activation and cell signaling. Significantly less is known about proteins that bind to α-integrin cytoplasmic tails (CTs) than β-CTs to regulate integrins. CIB1 was previously identified as an αIIb binding partner that inhibits agonist-induced activation of the platelet-specific integrin, αIIbβ3. A sequence alignment of all α-integrin CTs revealed that key residues in the CIB1 binding site on αIIb are well-conserved, and was used to delineate a consensus binding site (I/L-x-x-x-L/M-W/Y-K-x-G-F-F). Because the CIB1 binding site on αIIb is conserved in all α-integrins, and CIB1 expression is ubiquitous, we asked if CIB1 could interact with other α-integrin CTs. We predicted that multiple α-integrin CTs were capable of binding to the same hydrophobic binding pocket on CIB1 with docking models generated by all-atom replica exchange discrete molecular dynamics. After demonstrating novel in vivo interactions between CIB1 and other whole integrin complexes with co-immunopreceipitations, we validated the modeled predictions with solid-phase competitive binding assays showing that other α-integrin CTs compete with the αIIb CT for binding to CIB1 in vitro . Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site. These new mechanistic details of CIB1-integrin binding imply that CIB1 could bind to all integrin complexes and act as a broad regulator of integrin function.