Impact of N‐Terminal Domain Conformation and Domain Interactions on <scp>RfaH</scp> 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 ( β 3– β 4) located on the NTD, helps trigger domain dissociation. To this end, we use homology modeling to construct a structure, H 1 , which is similar to free RfaH but with a remodeled β 3– β 4 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 H 1 . For H 1 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 β 3– β 4 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.

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