Structural and Functional Studies on the Interaction of GspC and GspD in the Type II Secretion System

Type II secretion systems (T2SSs) are critical for secretion of many proteins from Gram-negative bacteria. In the T2SS, the outer membrane secretin GspD forms a multimeric pore for translocation of secreted proteins. GspD and the inner membrane protein GspC interact with each other via periplasmic domains. Three different crystal structures of the homology region domain of GspC (GspCHR) in complex with either two or three domains of the N-terminal region of GspD from enterotoxigenic Escherichia coli show that GspCHR adopts an all-β topology. N-terminal β-strands of GspC and the N0 domain of GspD are major components of the interface between these inner and outer membrane proteins from the T2SS. The biological relevance of the observed GspC–GspD interface is shown by analysis of variant proteins in two-hybrid studies and by the effect of mutations in homologous genes on extracellular secretion and subcellular distribution of GspC in Vibrio cholerae. Substitutions of interface residues of GspD have a dramatic effect on the focal distribution of GspC in V. cholerae. These studies indicate that the GspCHR–GspDN0 interactions observed in the crystal structure are essential for T2SS function. Possible implications of our structures for the stoichiometry of the T2SS and exoprotein secretion are discussed.II型分泌系统（Type II secretion systems, T2SSs）是革兰氏阴性菌多种蛋白分泌过程的关键介导因子。在T2SS通路中，外膜分泌素GspD（outer membrane secretin GspD）会组装形成多聚体孔道，以介导分泌蛋白的跨膜转运。GspD与内膜蛋白GspC（inner membrane protein GspC）可通过周质结构域发生相互作用。针对产肠毒素大肠杆菌来源的GspD N端区域的2或3个结构域与GspC同源区域结构域（GspCHR）所形成的复合物，研究解析了3种不同的晶体结构，结果显示GspCHR采取全β折叠拓扑结构。GspC的N端β链与GspD的N0结构域，是该T2SS内膜与外膜蛋白间相互作用界面的主要组成部分。通过双杂交实验对突变蛋白的分析，以及霍乱弧菌（Vibrio cholerae）中同源基因突变对胞外分泌与GspC亚细胞分布的影响，证实了所观测到的GspC-GspD相互作用界面的生物学相关性。GspD界面残基的替换会显著改变霍乱弧菌中GspC的局灶分布。本研究表明，晶体结构中解析的GspCHR-GspDN0相互作用对T2SS的功能至关重要。本文最后还讨论了这些结构对T2SS化学计量比与外蛋白分泌过程的潜在意义。