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      Differential local stability governs the metamorphic fold-switch of bacterial virulence factor RfaH

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          Abstract

          RfaH, a two-domain protein from a universally conserved NusG/Spt5 family of regulators, is required for the transcription and translation of long virulence and conjugation operons in many Gram-negative bacterial pathogens. Escherichia coli RfaH action is controlled by a unique large-scale structural rearrangement triggered by recruitment to transcription elongation complexes through a specific DNA element. Upon recruitment, the C-terminal domain of RfaH refolds from an α -hairpin, which is bound to RNA polymerase binding site within the N-terminal domain, into an unbound β -barrel that interacts with the ribosome. Although structures of the autoinhibited ( α -hairpin) and active ( β -barrel) states and plausible refolding pathways have been reported, how this reversible switch is encoded within RfaH sequence and structure is poorly understood. Here, we combined hydrogen-deuterium exchange measurements by mass spectrometry and nuclear magnetic resonance with molecular dynamics to evaluate the differential local stability between both RfaH folds. Deuteron incorporation reveals that the tip of the C-terminal hairpin (residues 125–145) is stably folded in the autoinhibited state (∼20% deuteron incorporation), whereas the rest of this domain is highly flexible (>40% deuteron incorporation), and its flexibility only decreases in the β -folded state. Computationally predicted Δ G agree with these results by displaying similar anisotropic stability within the tip of the α -hairpin and on neighboring N-terminal domain residues. Remarkably, the β -folded state shows comparable structural flexibility than nonmetamorphic homologs. Our findings provide information critical for understanding the metamorphic behavior of RfaH and other chameleon proteins and for devising targeted strategies to combat bacterial infections.

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          Author and article information

          Journal
          Biophysical Journal
          Biophysical Journal
          Elsevier BV
          00063495
          November 2019
          November 2019
          Article
          10.1016/j.bpj.2019.11.014
          6950767
          31810657
          afd1bc57-b48a-4395-a5b1-c606a8e1fb42
          © 2019

          https://www.elsevier.com/tdm/userlicense/1.0/

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