Biology subjects

Galaz-Davison, P.

Publications and source records attributed to Galaz-Davison, P..

4 recordsLinked to original sources

bioRxiv2026

Exploration of the structural and functional diversity in the metamorphic RfaH subfamily

RfaH, a member of the universally conserved NusG/Spt5 family, activates transcription and translation of virulence genes by bridging the transcribing RNA polymerase to the translating ribosome. Unlike its monomorphic, constitutively active paralog NusG, RfaH fold-switches from an autoinhibited state, wherein its C-terminal domain forms an -helical hairpin bound to the N-terminal domain, into an active NusG-like state through C-terminal domain dissociation and refolding into a {beta}-barrel. RfaH has been proposed to evolve from NusG via gene duplication by first activating genes adjacent to its production site, before developing autoinhibition to selectively control distant genes. Using AlphaFold2, we predicted the structures of thousands of RfaH homologs and identified a subset ([~]14%) that appear predominantly folded in the active state. In vivo assays confirmed that these putative monomorphic homologs exhibit constitutive activity comparable to known RfaH mutants. Phylogenetic and genomic analyses revealed that these monomorphic proteins form a distinct clade and are preferentially located within or next to long virulence-associated operons. Together, these results further support a stepwise evolutionary model of RfaH specialization through structural transformation.

biochemistry↗

bioRxiv2023

The Evolution of Local Energetic Frustration in Protein Families

Energetic local frustration offers a biophysical perspective to interpret the effects of sequence variability on protein families. Here we present a methodology to analyze local frustration patterns within protein families that allows us to uncover constraints related to stability and function, and identify differential frustration patterns in families with a common ancestry. We have analyzed these signals in very well studied cases such as PDZ, SH3, and {beta} globins and RAS families. Recent advances in protein structure prediction make it possible to analyze a vast majority of the protein space. An automatic and unsupervised proteome-wide analysis on the SARS-CoV-2 virus demonstrates the potential of our approach to enhance our understanding of the natural phenotypic diversity of protein families beyond single protein instances. We have applied our method to modify biophysical properties of natural proteins based on their family properties, as well as perform unsupervised analysis of large datasets to shed light on the physicochemical signatures of poorly characterized proteins such as emergent pathogens.

bioinformatics↗

bioRxiv2021

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

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) stalled at a DNA signal known as ops. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a {beta}-barrel while RfaH CTD is forming an -hairpin blocking such site. Upon recognition of the ops exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a {beta}-barrel structurally identical to NusG CTD. Although RfaH transformation has been extensively characterized computationally, most studies employ tertiary biases towards each native state, hampering the analysis of sequence-encoded interactions on fold-switching. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards -folded CTD, whereas the NTD interferes with {beta}CTD folding, becoming trapped in a {beta}-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the -folded RfaH by specifically orienting the CTD upon interdomain binding and also by favoring {beta}-barrel rupture into an intermediate from which fold-switching proceeds.

biophysics↗

bioRxiv2019

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

A regulatory factor RfaH, present in many Gram-negative bacterial pathogens, is required for transcription and translation of long operons encoding virulence determinants. Escherichia coli RfaH action is controlled by a unique large-scale structural rearrangement triggered by recruitment to transcription elongation complexes through a specific DNA sequence within these operons. Upon recruitment, the C-terminal domain of this two-domain protein refolds from an -hairpin, which is bound to the RNA polymerase binding site within the N-terminal domain of RfaH, into an unbound {beta}-barrel that interacts with the ribosome to enable translation. Although structures of the autoinhibited (-hairpin) and active ({beta}-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), while the rest of this domain is highly flexible (>40% deuteron incorporation) and its flexibility only decreases in the {beta}-folded state. Computationally-predicted {Delta}Gs agree with these results by displaying similar anisotropic stability within the tip of the -hairpin and on neighboring N-terminal domain residues. Remarkably, the {beta}-folded state shows comparable stability to non-metamorphic 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 diseases.\n\nSignificanceInfections caused by Gram-negative bacteria are a worldwide health threat due to rapid acquisition of antibiotic resistance. RfaH, a protein essential for virulence in several Gram-negative pathogens, undergoes a large-scale structural rearrangement in which one RfaH domain completely refolds. Refolding transforms RfaH from an inactive state that restricts RfaH recruitment to a few target genes into an active state that binds to, and couples, transcription and translation machineries to elicit dramatic activation of gene expression. However, the molecular basis of this unique conformational change is poorly understood. Here, we combine molecular dynamics and structural biology to unveil the hotspots that differentially stabilize both states of RfaH. Our findings provide novel insights that will guide design of inhibitors blocking RfaH action.

biophysics↗