Abstract

--- - "Author summary Proteins commonly adopt a single three-dimensional structure that is required for biological function. Nevertheless, proteins are not isolated in the cell, and the presence of binding partners can give rise to alternate structural configurations. Metamorphic proteins represent an extreme case of the latter, by folding into at least two well-defined configurations that are both structurally and functionally different. For RfaH, a virulence factor in enterobacteria, two distinct folds are found: an autoinhibited state in which its two protein domains strongly interact, and an active state in which these domains dissociate due to a specific DNA signal on RNA polymerases. This activation is accompanied by the refolding of the C-terminal domain (CTD) from an alpha-helical structure to a beta-barrel. Our work employs computational simulations to explore the role of the N-terminal domain (NTD) in regulating the metamorphic behavior of RfaH, determining that this domain has a major part in orienting and binding to the CTD in its alpha-helical fold, and in stabilizing an intermediate state instead of the fully folded beta-barrel. These results suggest that the NTD not only participates in stabilizing the autoinhibited state, but also aids in fold-switching back to it after active RfaH is released from RNA polymerase." - The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) paused at a DNA signal. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a beta-barrel while RfaH CTD is forming an alpha-hairpin blocking such site. Upon recognition of the specific DNA exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a beta-barrel structurally identical to NusG CTD. Although RfaH transformation has been extensively characterized computationally, little attention has been given to the role of the NTD in the fold-switching process, as its structure remains unchanged. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards alpha-folded CTD, whereas the NTD interferes with beta CTD folding, becoming trapped in a beta-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the alpha-folded RfaH by specifically orienting the alpha CTD upon interdomain binding and by favoring beta-barrel rupture into an intermediate from which fold-switching proceeds.