NMR analysis of the αIIbβ3 cytoplasmic interaction suggests a mechanism for integrin regulation

Abstract

The integrin αIIbβ3 is a transmembrane (TM) heterodimeric adhesion receptor that exists in equilibrium between resting and active ligand binding conformations. In resting αIIbβ3, the TM and cytoplasmic domains of αIIb and β3 form a heterodimer that constrains αIIbβ3 in its resting conformation. To study the structure and dynamics of the cytoplasmic domain heterodimer, we prepared a disulfide-stabilized complex consisting of portions of the TM domains and the full cytoplasmic domains. NMR and hydrogendeuterium exchange of this complex in micelles showed that the αIIb cytoplasmic domain is largely disordered, but it interacts with and influences the conformation of the β3 cytoplasmic domain. The β3 cytoplasmic domain consists of a stable proximal helix contiguous with the TM helix and two distal amphiphilic helices. To confirm the NMR structure in a membrane-like environment, we studied the β3 cytoplasmic domain tethered to phospholipid bilayers. Hydrogen-deuterium exchange mass spectrometry, as well as circular dichroism spectroscopy, demonstrated that the β3 cytoplasmic domain becomes more ordered and helical under these conditions, consistent with our NMR results. Further, these experiments suggest that the two distal helices associate with lipid bilayers but undergo fluctuations that would allow rapid binding of cytoplasmic proteins regulating integrin activation, such as talin and kindlin-3. Thus, these results provide a framework for understanding the kinetics and thermodynamics of protein interactions involving integrin cytoplasmic domains and suggest that such interactions act in a concerted fashion to influence integrin stalk separation and exposure of extracellular ligand binding sites.platelets | fibrinogen receptors | receptor regulation

Affinity of talin-1 for the β3-integrin cytosolic domain is modulated by its phospholipid bilayer environment

“…Thus, there is likely little reorganization energy associated with the interaction of these subdomains with bilayers. By contrast, the β3 CT, tethered to a bilayer, is in a rapid equilibrium that favors a conformation in which the peptide interacts with the membrane in a way that masks residues important for talin-1 binding and that would otherwise be located 10-30 Å into the cytoplasm (27). A second minor conformation is more exposed to the aqueous surroundings, presumably allowing rapid kinetic access to the FERM domain.…”

“…Accordingly, we immobilized the β3 CT on phospholipid bilayers by reacting a β3 CT construct containing Cys at residue 719 with maleimide-functionalized phosphatidylethanolamine on the surface of LUVs composed of POPC and either 1% PtdInsð4;5ÞP 2 , 20% PS, or 20% PS + 1% PtdInsð4;5ÞP 2 (27). Titration of F0-F3 into a suspension of LUVs containing 1% PtdInsð4;5ÞP 2 in the absence of immobilized β3 CT or into buffer containing the free β3 CT generated little or no enthalpy.…”

“…Previous studies reported on the interaction of the talin-1 FERM domain and the β3 CT with micelles and phospholipid vesicles (15,27,(38)(39)(40). Here, we studied these interactions using a β3 CT construct tethered to phospholipid bilayers to understand the energetics of the overall process.…”

“…S2D), encompassing residues Thr720 through the C terminus at Thr762, was previously described (27). For the current experiments, residue Ile719 was replaced with Cys to enable the construct to be immobilized on the surface of bilayers or to be labeled via maleimide chemistry.…”

“…S1D) (25,26). This positions talin to modulate interaction between membrane-proximal cytosolic and transmembrane segments of integrin α and β subunits (25)(26)(27).…”