Coevolution‐derived native and non‐native contacts determine the emergence of a novel fold in a universally conserved family of transcription factors

Abstract

The NusG protein family is structurally and functionally conserved in all domains of life. Its members directly bind RNA polymerases and regulate transcription processivity and termination. RfaH, a divergent sub‐family in its evolutionary history, is known for displaying distinct features than those in NusG proteins, which allows them to regulate the expression of virulence factors in enterobacteria in a DNA sequence‐dependent manner. A striking feature is its structural interconversion between an active fold, which is the canonical NusG three‐dimensional structure, and an autoinhibited fold, which is distinctively novel. How this novel fold is encoded within RfaH sequence to encode a metamorphic protein remains elusive. In this work, we used publicly available genomic RfaH protein sequences to construct a complete multiple sequence alignment, which was further augmented with metagenomic sequences and curated by predicting their secondary structure propensities using JPred. Coevolving pairs of residues were calculated from these sequences using plmDCA and GREMLIN, which allowed us to detect the enrichment of key metamorphic contacts after sequence filtering. Finally, we combined our coevolutionary predictions with molecular dynamics to demonstrate that these interactions are sufficient to predict the structures of both native folds, where coevolutionary‐derived non‐native contacts may play a key role in achieving the compact RfaH novel fold. All in all, emergent coevolutionary signals found within RfaH sequences encode the autoinhibited and active folds of this protein, shedding light on the key interactions responsible for the action of this metamorphic protein.

Evolutionary selection of proteins with two folds

“…These studies, though suggestive, focus on a couple of specific systems and infer fold switching from experimental observation of a few variants (XCL1) or inconsistent secondary structure predictions (NusG). Weak coevolutionary couplings of a fold-switching NusG have also been predicted, though the couplings had high proportions of noise: 38% at best 38 .…”

“…The MRF construction offers several advantages: (i) it converges to a global minimum as MSA depth increases, (ii) it can generate reasonable predictions from fairly shallow MSAs, and (iii) the MRF formalism accounts for noncausal correlations that arise when two residues interact with a third but not with one another 39-41 . Among the numerous MRF-based methods 4,42-44 , we selected GREMLIN (Generative REgularized ModeLs of proteINs) because of its superior performance 38,45 . The second technique, MSA transformer, infers coevolved amino acid pairs using a language model that focuses on both evolutionary patterns of amino acids within an MSA (column-wise attention) and properties of the individual sequences (row-wise attention), often with better accuracy than GREMLIN for single-fold proteins 35 .…”

Prediction of multiple conformational states by combining sequence clustering with AlphaFold2

“…This line of research indicates that coevolutionary signals can be present for multiple conformational states, but computational methods must first deconvolve the signal from multiple states. Methods proposed to deconvolve signal when prior knowledge about one or more states is known include ablating signal from a known dominant state 25 and supplementing the original multiple sequence alignment (MSA) with proteins known to occupy a rarer state 26 . However, simply subdividing a MSA and making predictions for portions of the MSA has also been used to detect variations in coevolutionary signal within a protein family 24,27,28 .…”

Fluid protein fold space and its implications

“…[70] Two independent coevolutionary analyses of RfaH proteins identified conservation of both the α-helical and the β-sheet CTD folds. [36,71] Furthermore, a recent preprint performed coevolutionary analysis on 56 foldswitching proteins from different families and found dual-fold coevolution in all 56 cases. [38] This study strongly suggests that both folds of many fold-switching proteins have been preserved by natural selection, likely conferring selective advantage.…”