Solution characterization of TrhW, a type 4 secretion system regulatory protein from the R27 plasmid

Abstract

Type IV secretion systems (T4SSs) are multiprotein nanomachines that mediate conjugative DNA transfer and contribute to the dissemination of antimicrobial resistance genes. The IncHI1 plasmid R27 encodes TrhW, a periplasmic T4SS-associated protein homologous to the F-plasmid proteins TraW and TrbC, which are expressed as separate polypeptides in many F-like systems but are fused into a single protein in IncHI1 plasmids. Despite its proposed role in conjugation, the structural organization of TrhW has remained unknown. Here, we employed complementary computational and solution-based biophysical approaches to characterize the oligomeric state, architecture, and conformational properties of TrhW. Size-exclusion chromatography (SEC) coupled with multi-angle light scattering demonstrated that TrhW exists as a monomer in solution, while circular dichroism spectroscopy revealed a structured protein containing substantial non-canonical secondary structure consistent with flexible regions. SEC-SAXS (small-angle x-ray scattering) showed that TrhW adopts an elongated, non-globular architecture with a radius of gyration of 44.6 Å and a maximum particle dimension of 160 Å. Ab initio reconstructions, BilboMD, and ensemble optimization modeling demonstrated that the experimental scattering data are best described by an ensemble of interconverting conformations rather than a single static structure. Although AlphaFold accurately predicts the overall domain organization of TrhW, the solution-state analyses reveal substantial conformational heterogeneity arising from both the N-terminal region and inter-domain motion. Together, these findings establish TrhW as a dynamic multidomain protein and provide the first experimental structural characterization of this conserved T4SS component, offering a framework for future studies investigating its role in IncHI1 conjugation.

Abstract of "Solution characterization of TrhW, a type 4 secretion system regulatory protein from the R27 plasmid" by Rodriguez CS, Audette GF, https://doi.org/10.1063/4.0001225, used under its Creative Commons Attribution license (CC-BY); markup removed, otherwise unmodified.