INVESTIGATING THE CONFORMATIONAL CHANGES OF THE C-TERMINAL DOMAIN OF THE TRANSCRIPTION ANTITERMINATION PROTEIN RfaH

Metamorphic proteins are characterized by their ability to adopt more than one native conformation. A example is the Escherichia coli transcription antitermination protein RfaH, which regulates toxin gene expression by switching its C-terminal domain (CTD) between a two α-helix hairpin and a five β-strand barrel. However, the structural pathways describing this switch-folding remain poorly understood. In this study, we performed molecular dynamics simulations of the RfaH-CTD domain using the AWSEM (Associative Memory, Water Mediated, Structure and Energy Model) force field to investigate its switch-folding mechanism. To map conformational changes across trajectories, we utilized the Energy Landscape Visualization Method (ELViM) developed by our group to project the protein phase space into 2D and 3D representations. Landscape analysis revealed distinct structural pathways across four simulated conditions. The α→β transition initiates through the nucleation of the three central β-strands of the barrel, whereas the β→α transition proceeds via an intermediate with helical structures. Unfolded→β simulations corroborate the α→β folding pathway while populating transient helical structures. Finally, unfolded→α simulations indicate that the longer α-helix forms first, followed by the assembly of the shorter helix. Overall, this study provides results detailing possible conformational changes presented by the CTD domain.

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (#2025/27919-1)