The C‐terminal domain of transcription factor RfaH: Folding, fold switching and energy landscape

Abstract

We simulate the folding and fold switching of the C-terminal domain (CTD) of the transcription factor RfaH using an all-atom physics-based model augmented with a dual-basin structure-based potential energy term. We show that this hybrid model captures the essential thermodynamic behavior of this metamorphic domain, that is, a change in the global free energy minimum from an α-helical hairpin to a 5-stranded β-barrel upon the dissociation of the CTD from the rest of the protein. Using Monte Carlo sampling techniques, we then analyze the energy landscape of the CTD in terms of progress variables for folding toward the two folds. We find that, below the folding transition, the energy landscape is characterized by a single, dominant funnel to the native β-barrel structure. The absence of a deep funnel to the α-helical hairpin state reflects a negligible population of this fold for the isolated CTD. We observe, however, a higher α-helix structure content in the unfolded state compared to results from a similar but fold switch-incompetent version of our model. Moreover, in folding simulations started from an extended chain conformation we find transiently formed α-helical structure, occurring early in the process and disappearing as the chain progresses toward the thermally stable β-barrel state. | INTRODUCTIONProteins are increasingly being discovered with a remarkable ability to switch between folds with widely different structures. [1][2][3] While it is not uncommon for proteins to undergo large-scale motions after their initial folding, such as domain-swapping [4] or other hinge-like motions, [5] fold switching is a distinct phenomenon. It involves a reorganization of the protein at the most basic structural level at play in folding, that is, secondary structure (α-helices and β-sheets). Despite the remarkable complexity of these molecular transformations, fold switching is reversible and thereby controlled by the system's free energy. In this sense, it can be said that metamorphic proteins adhere to Anfinsen's thermodynamic principle (or hypothesis) of protein folding. [6] Clearly, however, fold switching fundamentally challenges the idea of a unique native conformation, which was a central aspect of the classic view of folding since emerging from the pioneering refolding experiments on ribonuclease A. [7] It is important to note that fold switching typically occurs only when triggered by specific changes to the local environment (or milieu) of the protein, such as salt concentration, [8] redox condition [9] or oligomerization state. [10] In the absence of such a trigger, metamorphic proteins typically fold to an apparently unique structure, which masks their fold switching capabilities. As a result, metamorphic proteins often go unrecognized. [11] Metamorphic proteins thus encode two different folds within a single amino acid sequence even though, as mentioned, they typically adopt a single fold for a given (constant) local milieu. It is natural, then, to ask: what impact does this dual encoding have on their fol...

The N-terminal domain of RfaH plays an active role in protein fold-switching

“…Our simulations using the AWSEM MD and force field package correctly model RfaH in all its conformations and recapitulate its thermodynamic behavior in solution, evidenced as the switching of the energetic minimum between αCTD and βCTD when breaking interdomain interactions. This switch has also been observed in previous computational works on full-length RfaH using various simulation strategies [18,37].…”

“…Meanwhile, a second peak in heat capacity is observed at 1.15T f and corresponds to the transition between the β-intermediate and the unfolded state. In the first of these landscapes, the αCTD minimum is shown as a high and broad free energy minimum similarly to its basin observed in Fig 2B, a characteristic that likely arises from the structuredness of the helices, which have been ascertained in both simulations [18,37] and experiments [38].…”

Secondary-structure switch regulates the substrate binding of a YopJ family acetyltransferase

“…A random coil is revealed during the fold-switching process from a long α-helix to a β-hairpin. A similar process was also reported in the C-terminal domain of the transcription factor RfaH, in which an α-helical hairpin is refolded into a 5-stranded β-barrel through an unfolded state 38,39 . It would be worth investigating the universality of this process in…”

The N-terminal domain of RfaH plays an active role in protein fold-switching

“…In contrast, the non-metamorphic E. coli NusG only transiently forms interdomain interactions and exists in solution as a two-domain protein [5,23]. Our simulations using the AWSEM molecular dynamics and force field package correctly model RfaH in all its conformations and recapitulate its behavior in solution, evidenced as the switching of the energetic minimum between αCTD and βCTD when breaking interdomain interactions that has also been observed in previous computational works on full-length RfaH using various simulation strategies [20,27].…”

“…Also, despite the full-length RfaH is deeply stabilized as αRfaH, the free-energy minima of the βCTD and its intermediate are still observed albeit at a higher energy. Lastly, the native basin for the αCTD in the full-length protein is broad enough to account for fluctuations in the structuredness of the helices, which have been ascertained in both simulations [20,27] and experiments [28].…”