Impact of N‐Terminal Domain Conformation and Domain Interactions on RfaH Fold Switching

Abstract

RfaH is a two‐domain metamorphic protein involved in transcription regulation and translation initiation. To carry out its dual functions, RfaH relies on two coupled structural changes: Domain dissociation and fold switching. In the free state, the C‐terminal domain (CTD) of RfaH adopts an all‐α fold and is tightly associated with the N‐terminal domain (NTD). Upon binding to RNA polymerase (RNAP), the domains dissociate and the CTD transforms into an all‐β fold while the NTD remains largely, but not entirely, unchanged. We test the idea that a change in the conformation of an extended β‐hairpin (β3–β4) located on the NTD, helps trigger domain dissociation. To this end, we use homology modeling to construct a structure, H1, which is similar to free RfaH but with a remodeled β3–β4 hairpin. We then use an all‐atom physics‐based model enhanced with a dual basin structure‐based potential to simulate domain separation driven by the thermal unfolding of the CTD with NTD in a fixed, folded conformation. We apply our model to both free RfaH and H1. For H1 we find, in line with our hypothesis, that the CTD exhibits lower stability and the domains dissociate at a lower temperature T, as compared to free RfaH. We do not, however, observe complete refolding to the all‐β state in these simulations, suggesting that a change in β3–β4 orientation aids in, but is not sufficient for, domain dissociation. In addition, we study the reverse fold switch in which RfaH returns from a domain‐open all‐β state to its domain‐closed all‐α state. We observe a T‐dependent transition rate; fold switching is slow at low T, where the CTD tends to be kinetically trapped in its all‐β state, and at high‐T, where the all‐α state becomes unstable. Consequently, our simulations suggest an optimal T at which fold switching is most rapid. At this T, the stabilities of both folds are reduced. Overall, our study suggests that both inter‐domain interactions and conformational changes within NTD may be important for the proper functioning of RfaH.