Computational and experimental assessment of key interdomain residues controlling the fold-switch of RfaH.

Escherichia coli RfaH, a member of the universally conserved NusG family of transcription factors, regulates its function by undergoing a structural rearrangement of its C-terminal domain (CTD) upon recruitment to RNA polymerase (RNAP) paused at the DNA signal known as operon polarity suppressor (ops) element. While it is known that the fold-switch of RfaH CTD from an α-helical hairpin (autoinhibited state) into a β-barrel (active state) is controlled by interactions between the N-terminal domain (NTD) and CTD, which are broken apart upon NTD binding to RNAP, a comprehensive analysis of residues that stabilize the autoinhibited state is lacking. Here, we utilize a combination of molecular dynamics (MD), protein structure prediction, and in vivo functional assays as a workflow to determine key interdomain (ID) residues controlling the fold-switch of RfaH. First, MD simulations employing structure-based models identified eight CTD residues with high ID contact probability, therefore expected to play a crucial role in stabilizing the autoinhibited state of RfaH. In silico alanine scanning mutagenesis followed by structure prediction using AlphaFold2 showed that four of these mutants (F126A, E136A, R138A, and L142A) led to several models with mixed α/β secondary structure for the CTD in comparison to the known fold-switching mutant E48A. Lastly, experimental alanine scanning mutagenesis and RfaH-dependent in vivo luminescence assays confirmed that I129 and L142 contribute to the stabilization of the autoinhibited state. These results deepen our understanding of the fold-switch of RfaH, with tools that are applicable to other metamorphic proteins.