NMR analysis of the alphaIIb beta3 cytoplasmic interaction suggests a mechanism for integrin regulation.
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abstract
The integrin IIb3 is a transmembrane (TM) heterodimeric adhesion receptor that exists in equilibrium between resting and active ligand binding conformations. In resting IIb3, the TM and cytoplasmic domains of IIb and 3 form a heterodimer that constrains IIb3 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 hydrogen-deuterium exchange of this complex in micelles showed that the IIb cytoplasmic domain is largely disordered, but it interacts with and influences the conformation of the 3 cytoplasmic domain. The 3 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 3 cytoplasmic domain tethered to phospholipid bilayers. Hydrogen-deuterium exchange mass spectrometry, as well as circular dichroism spectroscopy, demonstrated that the 3 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.
Metcalf, D. G., Moore, D. T., Wu, Y., Kielec, J. M., Molnar, K., Valentine, K. G., ... DeGrado, W. F.
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Metcalf, Douglas G||Moore, David T||Wu, Yibing||Kielec, Joseph M||Molnar, Kathleen||Valentine, Kathleen G||Wand, A Joshua||Bennett, Joel S||DeGrado, William F
