Oleksy Arkadiusz, Barton Holly, Devedjiev Yancho, Purdy Michael, Derewenda Urszula, Otlewski Jacek, Derewenda Zygmunt S
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908-0736, USA.
Acta Crystallogr D Biol Crystallogr. 2004 Apr;60(Pt 4):740-2. doi: 10.1107/S0907444904002318. Epub 2004 Mar 23.
PDZ-containing RhoGEF (PDZ-RhoGEF) is a multidomain protein composed of 1522 amino acids that belongs to the guanine nucleotide exchange factors family (GEF) active on Rho GTPases. It is highly specific for RhoA and is thought to transduce signals from Galpha(12/13)-coupled receptors to the RhoA-dependent regulatory cascades. The protein shows high sequence homology to LARG, p115-RhoGEF and Drosophila DRhoGEF2. The exchange reaction is catalyzed by a DH domain, which is directly downstream of a PH domain in all known Rho-specific GEFs. The DH/PH tandem of PDZ-RhoGEF and C-terminally truncated RhoA were overexpressed in Escherichia coli as TEV protease-cleavable fusion proteins containing GST and a hexahistidine tag at the N-termini, respectively. The nucleotide-free DH/PH-RhoA complex was purified by gel filtration and crystallized. The crystals belong to space group P2(1), with unit-cell parameters a = 88.6, b = 119.0, c = 91.5 A, beta = 114.7 degrees.
含PDZ结构域的Rho鸟嘌呤核苷酸交换因子（PDZ-RhoGEF）是一种由1522个氨基酸组成的多结构域蛋白，属于对Rho GTP酶有活性的鸟嘌呤核苷酸交换因子（GEF）家族。它对RhoA具有高度特异性，被认为可将来自Gα(12/13)偶联受体的信号转导至RhoA依赖性调节级联反应。该蛋白与LARG、p115-RhoGEF和果蝇DRhoGEF2具有高度序列同源性。交换反应由一个DH结构域催化，在所有已知的Rho特异性GEF中，该结构域直接位于PH结构域的下游。PDZ-RhoGEF的DH/PH串联结构和C末端截短的RhoA在大肠杆菌中分别作为N末端含有GST和六组氨酸标签的可被TEV蛋白酶切割的融合蛋白进行过表达。通过凝胶过滤纯化无核苷酸的DH/PH-RhoA复合物并使其结晶。晶体属于空间群P2(1)，晶胞参数a = 88.6，b = 119.0，c = 91.5 Å，β = 114.7°。
