Protein‐protein interactions between CIB1 and novel integrin binding partners: structural and thermodynamic basis of CIB1 promiscuity (979.5)

The cytoplasmic protein CIB1 (calcium‐and‐integrin‐binding protein 1) has been shown to promote cancer cell growth by affecting the RAS/RAF/MEK/ERK and the PI3K/AKT pathways, and was previously identified as an αIIb binding partner that inhibits agonist‐induced activation of the platelet‐specific integrin, αIIbβ3. To better understand the nature of the protein‐protein interactions between CIB1 and other proteins, we characterized the interaction between CIB1 and several α‐integrins. 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). A hydrophobic binding pocket on CIB1 was shown to be the major binding site for multiple α‐integrins by all‐atom replica exchange discrete molecular dynamics, competitive binding assays, and mutational analysis. We also demonstrated that CIB1 can bind to other whole integrin complexes in vivo using co‐immunopreceipitation assays. 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. Moreover, this work provides new insight into the nature of the protein‐protein interactions between CIB1 and its binding partners. Grant Funding Source : Supported byNHLBI 5R01HL092544 and NIGMS 5K12GM000678