Apostol Arnold J, Bragagnolo Nicholas J, Rodriguez Christina S, Audette Gerald F
Department of Chemistry, York University, 4700 Keele St, Toronto, ON, Canada, M3J 1P3.
Centre for Research on Biomolecular Interactions, York University, Canada.
Curr Res Struct Biol. 2024 Jul 14;8:100156. doi: 10.1016/j.crstbi.2024.100156. eCollection 2024.
Bacteria have evolved elaborate mechanisms to thrive in stressful environments. F-like plasmids in gram-negative bacteria encode for a multi-protein Type IV Secretion System (T4SS) that is functional for bacterial proliferation and adaptation through the process of conjugation. The periplasmic protein TrbB is believed to have a stabilizing chaperone role in the T4SS assembly, with TrbB exhibiting disulfide isomerase (DI) activity. In the current report, we demonstrate that the deletion of the disordered N-terminus of TrbB, resulting in a truncation construct TrbB, does not affect its catalytic activity compared to the wild-type protein (p = 0.76). Residues W37-K161, which include the active thioredoxin motif, are sufficient for DI activity. The N-terminus of TrbB is disordered as indicated by a structural model of GST-TrbB based on ColabFold-AlphaFold2 and Small Angle X-Ray Scattering data and H-N Heteronuclear Single Quantum Correlation (HSQC) spectroscopy of the untagged protein. This disordered region likely contributes to the protein's dynamicity; removal of this region results in a more stable protein based on H-N HSQC and Circular Dichroism Spectroscopies. Lastly, size exclusion chromatography analysis of TrbB in the presence of TraW, a T4SS assembly protein predicted to interact with TrbB, does not support the inference of a stable complex forming . This work advances our understanding of TrbB's structure and function, explores the role of structural disorder in protein dynamics in the context of a T4SS accessory protein, and highlights the importance of redox-assisted protein folding in the T4SS.
细菌已经进化出复杂的机制以在压力环境中生存。革兰氏阴性菌中的F-样质粒编码一种多蛋白IV型分泌系统（T4SS），该系统通过接合过程对细菌增殖和适应发挥作用。周质蛋白TrbB被认为在T4SS组装中具有稳定伴侣的作用，TrbB具有二硫键异构酶（DI）活性。在本报告中，我们证明，删除TrbB无序的N端产生的截短构建体TrbB，与野生型蛋白相比，其催化活性不受影响（p = 0.76）。包括活性硫氧还蛋白基序的W37-K161残基足以产生DI活性。基于ColabFold-AlphaFold2的GST-TrbB结构模型、小角X射线散射数据以及未标记蛋白的H-N异核单量子相关（HSQC）光谱表明，TrbB的N端是无序的。该无序区域可能有助于蛋白质的动态性；基于H-N HSQC和圆二色光谱，去除该区域会产生更稳定的蛋白质。最后，在存在TraW（一种预测与TrbB相互作用的T4SS组装蛋白）的情况下对TrbB进行尺寸排阻色谱分析，并不支持形成稳定复合物的推断。这项工作增进了我们对TrbB结构和功能的理解，探索了结构无序在T4SS辅助蛋白背景下蛋白质动态性中的作用，并强调了氧化还原辅助蛋白质折叠在T4SS中的重要性。
