Identification of Novel Integrin Binding Partners for Calcium and Integrin Binding Protein 1 (CIB1): Structural and Thermodynamic Basis of CIB1 Promiscuity

The short cytoplasmic tails of the alpha- and beta-chains of integrin adhesion receptors regulate integrin activation and cell signaling. Significantly less is known about proteins that bind to alpha-integrin cytoplasmic tails (CTs) as opposed to beta-CTs to regulate integrins. Calcium and integrin binding protein 1 (CIB1) was previously identified as an alpha IIb binding partner that inhibits agonist-induced activation of the platelet-specific integrin, alpha IIb beta 3. A sequence alignment of all a-integrin CTs revealed that key residues in the CIB1 binding site of alpha IIb 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 of alpha IIb is conserved in all alpha-integrins and CIB1 expression is ubiquitous, we asked if CIB1 could interact with other alpha-integrin CTs. We predicted that multiple alpha-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-immunoprecipitations, we validated the modeled predictions with solid-phase competitive binding assays, which showed that other alpha-integrin CTs compete with the alpha IIb 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.