Integrins are ubiquitously expressed α/β heterodimers that mediate cell-cell and cell-extracellular matrix interactions. The platelet integrin αIIbβ3 binds soluble fibrinogen following platelet activation, an event necessary for the formation of platelet aggregates. Integrins reside on plasma membranes in a highly regulated and dynamic equilibrium between inactive resting states and active ligand binding conformations. An essential feature of this equilibrium is the association and dissociation of integrin transmembrane (TM) and cytoplasmic domains. Thus, when integrins are inactive, the TM and cytoplasmic domains of their α and β subunits are in proximity; the domains separate when integrins assume their active conformations. Agonist-induced activation of αIIbβ3 in platelets (inside-out activation) requires binding of the FERM domain of the cytoskeletal protein talin to highly conserved membrane proximal and distal sequences in the β3 subunit cytoplasmic domain. FERM domain binding to the β3 cytoplasmic domain likely disrupts the αIIbβ3 TM domain-cytoplasmic domain heterodimer. As the β3 cytoplasmic domain exists in close apposition to the inner leaflet of the plasma membrane and the talin FERM domain is known to interact with the negatively-charged phospholipids that are common in eukaryotic membranes, we have investigated the effects of the membrane environment on β3 and talin structure and the talin-β;3 protein-protein interaction that regulates αIIbβ3 activation. A water soluble form of the β3 cytoplasmic domain was expressed, purified, and chemically conjugated to phospholipid bilayers, mimicking the native environment of the αIIb and β3 cytoplasmic domains as they exit the membrane. Using circular dichroism (CD) spectroscopy, the β3 cytoplasmic domain was found to have a significant increase in secondary structure and overall helicity when attached to the bilayer. Similarly, using hydrogen-deuterium exchange (HDX) mass spectrometry, we found that there is an overall decrease in amide backbone flexibility, particularly in the membrane proximal talin-interacting region. We also found significant changes in talin secondary structure in detergents by CD and by limited proteolysis mapping, supporting dramatic conformational changes in talin upon membrane binding. Using isothermal titration calorimetry (ITC) and integrin-conjugated lipid bilayers, we investigated the effects of lipid composition on talin-bilayer and talin-β3 cytoplasmic domain interactions. We found that the talin FERM domain likely has two binding sites for the membrane-conjugated β3 cytoplasmic domain, one in the canonical F3 subdomain and a second that depends on the F2 subdomain. Moreover, the talin head domain does not bind to β3 on neutral bilayers and requires a significant excess of phosphatidylserine (PS) relative to β3 to allow saturation. Interestingly phosphatidylinositol (4, 5)-bisphosphate containing bilayers have significantly higher affinity and mitigate the need for excess PS. Our results point toward a model in which β3 cytoplasmic domain structure is dependent on the lipid environment and its interaction with talin. Thus, αIIbβ3 activation is significantly regulated by lipid which may play a role in constraining αIIbβ3 activation by talin in the crowded platelet intracellular environment.
No relevant conflicts of interest to declare.