Department of Cell & Developmental Biology, Northwestern University Chicago, Illinois, USA.
Xilio Therapeutics, Waltham, Massachusetts, USA.
Protein Sci. 2022 May;31(5):e4282. doi: 10.1002/pro.4282.
The bacterial pathogen Vibrio cholerae use a type III secretion system to inject effector proteins into a host cell. Recently, a putative Toxic GTPase Activating Protein (ToxGAP) called Vibrio outer protein E (VopE) was identified as a T3SS substrate and virulence factor that affected host mitochondrial dynamics and immune response. However, biophysical and structural characterization has been absent. Here, we describe solution NMR structure of the putative GTPase-activating protein (GAP) domain (73-204) of VopE. Using size exclusion chromatography coupled with small-angle x-ray scattering and residual dipolar coupling data, we restrained the MD process to efficiently determine the overall fold and improve the quality of the output calculated structures. Comparing the structure of VopE with other ToxGAP's revealed a similar overall fold with several features unique to VopE. Specifically, the "Bulge 1," α1 helix, and noteworthy "backside linker" elements on the N-terminus are dissimilar to the other ToxGAP's. By using NMR relaxation dispersion experiments, we demonstrate that these regions undergo motions on a > 6 s timescale. Based on the disposition of these mobile regions relative to the putative catalytic arginine residue, we hypothesize that the protein may undergo structural changes to bind cognate GTPases.
细菌病原体霍乱弧菌使用 III 型分泌系统将效应蛋白注入宿主细胞。最近，一种被称为霍乱弧菌外膜蛋白 E（VopE）的假定毒性 GTP 酶激活蛋白（ToxGAP）被鉴定为 T3SS 底物和毒力因子，影响宿主线粒体动力学和免疫反应。然而，生物物理和结构特征尚不清楚。在这里，我们描述了 VopE 的假定 GTP 酶激活蛋白 (GAP) 结构域 (73-204) 的溶液 NMR 结构。通过尺寸排阻色谱与小角 X 射线散射和残差偶极耦合数据相结合，我们限制了 MD 过程，以有效地确定整体折叠并提高计算结构的质量。将 VopE 的结构与其他 ToxGAP 进行比较，揭示了相似的整体折叠，并有几个特征是 VopE 所特有的。具体来说，“凸起 1”、α1 螺旋和引人注目的“背面接头”元素在 N 端与其他 ToxGAP 不同。通过使用 NMR 弛豫分散实验，我们证明这些区域在 > 6 s 的时间尺度上发生运动。根据这些运动区域相对于假定的催化精氨酸残基的位置，我们假设该蛋白可能发生结构变化以结合同源 GTP 酶。
