Shimaoka Motomu, Lu Chafen, Salas Azucena, Xiao Tsan, Takagi Junichi, Springer Timothy A
Center for Blood Research, Departments of Pathology, Pediatrics, and Anesthesia, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16737-41. doi: 10.1073/pnas.252633099. Epub 2002 Dec 4.
Conformational movement of the C-terminal alpha7 helix in the integrin inserted (I) domain, a major ligand-binding domain that adopts an alpha/beta Rossmann fold, has been proposed to allosterically regulate ligand-binding activity. Disulfide bonds were engineered here to reversibly lock the position of the alpha7 helix in one of two alternative conformations seen in crystal structures, termed open and closed. Our results show that pairs of residues with Cbeta atoms farther apart than optimal for disulfide bond stereochemistry can be successfully replaced by cysteine, suggesting that backbone movement accommodates disulfide formation. We also find more success with substituting partially exposed than buried residues. Disulfides stabilizing the open conformation resulted in constitutively active alphaMbeta2 heterodimers and isolated alphaM inserted domains, which were reverted to an inactive form by dithiothreitol reduction. By contrast, a disulfide stabilizing the closed conformation resulted in inactive alphaMbeta2 that was resistant to activation but became activatable after dithiothreitol treatment.
整联蛋白插入（I）结构域中的C末端α7螺旋的构象运动被认为可通过变构调节配体结合活性，该结构域是一个主要的配体结合结构域，采用α/β罗斯曼折叠。在此设计了二硫键，以可逆地锁定α7螺旋在晶体结构中所见的两种替代构象之一的位置，分别称为开放构象和闭合构象。我们的结果表明，具有比二硫键立体化学最佳距离更远的Cβ原子的残基对可以成功地被半胱氨酸取代，这表明主链运动适应了二硫键的形成。我们还发现，取代部分暴露的残基比取代埋藏的残基更成功。稳定开放构象的二硫键导致组成型活性αMβ2异二聚体和分离的αM插入结构域，通过二硫苏糖醇还原可将其恢复为无活性形式。相比之下，稳定闭合构象的二硫键导致无活性的αMβ2，其对激活具有抗性，但在二硫苏糖醇处理后可被激活。
