Derewenda Urszula, Oleksy Arkadiusz, Stevenson Andra S, Korczynska Justyna, Dauter Zbigniew, Somlyo Andrew P, Otlewski Jacek, Somlyo Avril V, Derewenda Zygmunt S
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA.
Structure. 2004 Nov;12(11):1955-65. doi: 10.1016/j.str.2004.09.003.
Calcium sensitization in smooth muscle is mediated by the RhoA GTPase, activated by hitherto unspecified nucleotide exchange factors (GEFs) acting downstream of Galphaq/Galpha(12/13) trimeric G proteins. Here, we show that at least one potential GEF, the PDZRhoGEF, is present in smooth muscle, and its isolated DH/PH fragment induces calcium sensitization in the absence of agonist-mediated signaling. In vitro, the fragment shows high selectivity for the RhoA GTPase. Full-length fragment is required for the nucleotide exchange, as the isolated DH domain enhances it only marginally. We crystallized the DH/PH fragment of PDZRhoGEF in complex with nonprenylated human RhoA and determined the structure at 2.5 A resolution. The refined molecular model reveals that the mutual disposition of the DH and PH domains is significantly different from other previously described complexes involving DH/PH tandems, and that the PH domain interacts with RhoA in a unique mode. The DH domain makes several specific interactions with RhoA residues not conserved among other Rho family members, suggesting the molecular basis for the observed specificity.
平滑肌中的钙敏化由RhoA GTP酶介导，RhoA GTP酶由作用于Gαq / Gα(12/13)三聚体G蛋白下游的迄今未明确的核苷酸交换因子（GEF）激活。在此，我们表明，至少一种潜在的GEF，即PDZRhoGEF，存在于平滑肌中，并且其分离的DH / PH片段在没有激动剂介导的信号传导的情况下诱导钙敏化。在体外，该片段对RhoA GTP酶表现出高选择性。核苷酸交换需要全长片段，因为分离的DH结构域仅略微增强它。我们使PDZRhoGEF的DH / PH片段与未异戊二烯化的人RhoA形成复合物结晶，并在2.5埃分辨率下确定其结构。优化的分子模型表明，DH和PH结构域的相互位置与其他先前描述的涉及DH / PH串联的复合物有显著差异，并且PH结构域以独特模式与RhoA相互作用。DH结构域与RhoA残基进行了一些其他Rho家族成员中不保守的特异性相互作用，这表明了观察到的特异性的分子基础。
