Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA.
Proc Natl Acad Sci U S A. 2010 Dec 28;107(52):22481-6. doi: 10.1073/pnas.1015545107. Epub 2010 Dec 14.
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 hydrogen-deuterium 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.
整合素 αIIbβ3 是一种跨膜 (TM) 异二聚体粘附受体，存在于静息和活性配体结合构象之间的平衡状态。在静息的 αIIbβ3 中，αIIb 和 β3 的 TM 和细胞质结构域形成异二聚体，将 αIIbβ3 限制在其静息构象中。为了研究细胞质结构域异二聚体的结构和动力学，我们制备了一种由 TM 结构域的部分和完整的细胞质结构域组成的二硫键稳定的复合物。该复合物在胶束中的 NMR 和氘氢交换表明，αIIb 细胞质结构域大部分是无序的，但它与 β3 细胞质结构域相互作用并影响其构象。β3 细胞质结构域由与 TM 螺旋连续的稳定近端螺旋和两个远端两亲性螺旋组成。为了在类似膜的环境中确认 NMR 结构，我们研究了与磷脂双层连接的 β3 细胞质结构域。氘氢交换质谱和圆二色光谱表明，在这些条件下，β3 细胞质结构域变得更加有序和螺旋，这与我们的 NMR 结果一致。此外，这些实验表明，两个远端螺旋与脂质双层结合，但会发生波动，从而允许快速结合调节整合素激活的细胞质蛋白，如 talin 和 kindlin-3。因此，这些结果为理解涉及整合素细胞质结构域的蛋白质相互作用的动力学和热力学提供了一个框架，并表明这种相互作用以协同方式发挥作用，影响整合素柄部分离和细胞外配体结合位点的暴露。
