Structural insights into the disulfide isomerase and chaperone activity of TrbB of the F plasmid type IV secretion system

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_F) 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_F assembly, with TrbB exhibiting disulfide isomerase (DI) activity. In the current report, we demonstrate that the deletion of the disordered N-terminus of TrbB_WT, resulting in a truncation construct TrbB_37-161, does not affect its catalytic in vitro 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_WT is disordered as indicated by a structural model of GST-TrbB_WT based on ColabFold-AlphaFold2 and Small Angle X-Ray Scattering data and ¹H–¹⁵N 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 ¹H–¹⁵N HSQC and Circular Dichroism Spectroscopies. Lastly, size exclusion chromatography analysis of TrbB_WT in the presence of TraW, a T4SS_F assembly protein predicted to interact with TrbB_WT, does not support the inference of a stable complex forming in vitro. 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_F accessory protein, and highlights the importance of redox-assisted protein folding in the T4SS_F.