Solution characterization of TrhW, a type 4 secretion system regulatory protein from the R27 plasmid.
- 1Department of Chemistry & Centre for Research on Biomolecular Interactions, York University, 4700 Keele St., Toronto, Ontario M3J 1P3, Canada.
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.
Related
- An evolutionary strategy for highly efficient secretion of multidomain Staphylococcal protein A: N- to C-terminal stability gradient
- The impact of evolutionary dynamics and expression plasmid stability on long-term heterologous protein production by yeast.
- Functional dissection of Drosophila Myc cis-regulatory modules (Myc-CRMs) reveals developmentally active DNA-protein interactions
- Engineered protein degradation of farnesyl pyrophosphate synthase is an effective regulatory mechanism to increase monoterpene production in Saccharomyces cerevisiae
- Structural basis for the CsrA-dependent modulation of translation initiation by an ancient regulatory protein.
BibTeX
@article{rodriguez2026solution,
title = {{Solution characterization of TrhW, a type 4 secretion system regulatory protein from the R27 plasmid.}},
author = {Christina S Rodriguez and Gerald F Audette},
journal = {Structural dynamics (Melville, N.Y.)},
year = {2026},
month = sep,
doi = {10.1063/4.0001225},
url = {https://europepmc.org/article/MED/42904001},
note = {CC BY},
}
RIS
TY - JOUR
TI - Solution characterization of TrhW, a type 4 secretion system regulatory protein from the R27 plasmid.
AU - Christina S Rodriguez
AU - Gerald F Audette
PY - 2026
DA - 2026/09/01
JO - Structural dynamics (Melville, N.Y.)
DO - 10.1063/4.0001225
UR - https://europepmc.org/article/MED/42904001
AB - 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.
SN - 2329-7778
ER -