Interdomain Contacts and RNA Polymerase Control Native State Interconversion of the Transformer Protein RfaH on a Dual-Funneled Landscape

RfaH is a bacterial virulence factor that belongs to the universally conserved NusG family of transcription factors and is commonly referred as the first example of a transformer protein. In contrast to other NusG proteins, the C-terminal domain (CTD) of RfaH folds into an α-helical hairpin that is stabilized by interactions with the N-terminal domain (NTD), which in turn masks an RNA polymerase (RNAp) binding site and thus constitutes an autoinhibited state.This autoinhibition is relieved when binding to a specific DNA element in the transcription elongation complex triggers domain dissociation, allowing the NTD to interact with RNAp to facilitate transcription while the CTD dramatically refolds into a NusG-like β-barrel that interacts with the ribosome to activate translation, thus changing both the structure and function of RfaH.Since this phenomenon is experimentally challenging, their structural details remain to be elucidated. Here, we explore the mechanism of the conformational switching of RfaH in the full-length protein using a dual-basin structure-based model. Our simulations capture several features described experimentally, such as the requirement of disruption of interdomain contacts to trigger the dramatic α-to-β transformation of RfaH, confirms the roles of previously indicated residues E48 and R138, and suggests a new important role for F130, in the stability of the interdomain interaction. These native basins are connected through an intermediate state that builds up upon binding to the NTD and shares features from both folds. We also examine the competitive binding between RfaH-CTD and RNAp for the NTD, showing that RNAp binding favors the β fold.Our study shows that native-biased models are appropriate for interrogating detailed mechanisms of the structural rearrangements during the native state interconversion of RfaH. FUNDING: Fondecyt 11140601.