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

AbstractA 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 α-hairpin, which is bound to the RNA polymerase binding site within the N-terminal domain of RfaH, into an unbound β-barrel that interacts with the ribosome to enable translation. 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), while the rest of this domain is highly flexible (>40% deuteron incorporation) and its flexibility only decreases in the β-folded state. Computationally-predicted ΔGs 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 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.SignificanceInfections 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.

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Unveiling azole resistance mechanisms in Candida glabrata clinical isolates encoding wild-type or gain-of-function CgPdr1 alleles

Access Microbiology ◽

10.1099/acmi.cc2021.po0099 ◽

2021 ◽

Vol 3 (12) ◽

Author(s):

Sara B. Salazar ◽

Noémi Valez ◽

Danielle Sotti-Novais ◽

Rita Simões ◽

José António Souza ◽

...

Keyword(s):

Large Scale ◽

Target Genes ◽

Efflux Pump ◽

Resistance Mechanisms ◽

Azole Resistance ◽

Gain Of Function ◽

Resistance Phenotype ◽

Rapid Acquisition ◽

The One

The relevance of C. glabrata as a human pathogen is linked with its poor susceptibility to azoles as well as its extreme genomic plasticity that allows the rapid acquisition of resistance. Extensive characterization of azole-resistant C. glabrata strains unveiled the central role of the transcriptional regulator CgPdr1 in the resistance phenotype, with many strains encoding hyperactive (or gain-of-function; GOF) CgPdr1 alleles. Large scale profiling of a collection of clinical C. glabrata isolates recovered in hospitals of the Lisbon area, in Portugal, led to the identification of 11 strains exhibiting resistance to fluconazole and voriconazole, while 2 were only resistant to fluconazole. Among these strains, 10 were found to encode alleles of the CgPDR1 gene harbouring multiple non-synonymous SNPs that were not found in the alleles encoded by susceptible strains, including K274Q, I392M and I803T not previously described as GOF mutations. The isolates encoding these alleles were found to over-express several CgPdr1 target genes including the azole efflux pump CgCDR1 sustaining the idea that these represent new gain-of-function CgPdr1 alleles. Only one of the identified azole-resistant strains was found to encode a CgPDR1 allele fully identical to the one encoded by susceptible strains. To better understand the resistance phenotype of this strain, its transcriptome was compared with the one of a susceptible strain and of strains encoding CgPdr1 GOF alleles. The results of this comparative transcriptomic analysis will be discussed shedding light into the different azole-resistance mechanisms evolved by C. glabrata, including those independent of CgPdr1 GOF strains.

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Molecular dynamics simulations of nucleotide release from the circadian clock protein KaiC reveal atomic-resolution functional insights

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1812555115 ◽

2018 ◽

Vol 115 (49) ◽

pp. E11475-E11484 ◽

Cited By ~ 6

Author(s):

Lu Hong ◽

Bodhi P. Vani ◽

Erik H. Thiede ◽

Michael J. Rust ◽

Aaron R. Dinner

Keyword(s):

Molecular Dynamics ◽

Conformational Changes ◽

Bound States ◽

Large Scale ◽

Nucleotide Exchange ◽

Terminal Domain ◽

Nucleotide Exchange Factor ◽

Nucleotide Release ◽

Dynamics Simulations

The cyanobacterial clock proteins KaiA, KaiB, and KaiC form a powerful system to study the biophysical basis of circadian rhythms, because an in vitro mixture of the three proteins is sufficient to generate a robust ∼24-h rhythm in the phosphorylation of KaiC. The nucleotide-bound states of KaiC critically affect both KaiB binding to the N-terminal domain (CI) and the phosphotransfer reactions that (de)phosphorylate the KaiC C-terminal domain (CII). However, the nucleotide exchange pathways associated with transitions among these states are poorly understood. In this study, we integrate recent advances in molecular dynamics methods to elucidate the structure and energetics of the pathway for Mg·ADP release from the CII domain. We find that nucleotide release is coupled to large-scale conformational changes in the KaiC hexamer. Solvating the nucleotide requires widening the subunit interface leading to the active site, which is linked to extension of the A-loop, a structure implicated in KaiA binding. These results provide a molecular hypothesis for how KaiA acts as a nucleotide exchange factor. In turn, structural parallels between the CI and CII domains suggest a mechanism for allosteric coupling between the domains. We relate our results to structures observed for other hexameric ATPases, which perform diverse functions.

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

10.1101/2021.03.18.436019 ◽

2021 ◽

Author(s):

Pablo Galaz-Davison ◽

Ernesto A Román ◽

Cesar A. Ramirez-Sarmiento

Keyword(s):

Molecular Dynamics ◽

Virulence Factors ◽

Elongation Factor ◽

Active Role ◽

Structural Rearrangement ◽

Umbrella Sampling ◽

Protein Fold ◽

Native State ◽

Terminal Domain ◽

Rnap Binding

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 β-barrel while RfaH CTD is forming an α-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 β-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 α-folded CTD, whereas the NTD interferes with βCTD folding, becoming trapped in a β-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 α-folded RfaH by specifically orienting the αCTD upon interdomain binding and also by favoring β-barrel rupture into an intermediate from which fold-switching proceeds.

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

PLoS Computational Biology ◽

10.1371/journal.pcbi.1008882 ◽

2021 ◽

Vol 17 (9) ◽

pp. e1008882

Author(s):

Pablo Galaz-Davison ◽

Ernesto A. Román ◽

César A. Ramírez-Sarmiento

Keyword(s):

Molecular Dynamics ◽

Virulence Factors ◽

Elongation Factor ◽

Active Role ◽

Structural Rearrangement ◽

Umbrella Sampling ◽

Protein Fold ◽

Terminal Domain ◽

Rnap Binding

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) paused at a DNA signal. 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 β-barrel while RfaH CTD is forming an α-hairpin blocking such site. Upon recognition of the specific DNA exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a β-barrel structurally identical to NusG CTD. Although RfaH transformation has been extensively characterized computationally, little attention has been given to the role of the NTD in the fold-switching process, as its structure remains unchanged. 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 α-folded CTD, whereas the NTD interferes with βCTD folding, becoming trapped in a β-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 α-folded RfaH by specifically orienting the αCTD upon interdomain binding and by favoring β-barrel rupture into an intermediate from which fold-switching proceeds.

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Combustion Driven by Fragment-based Ab Initio Molecular Dynamics Simulation

10.26434/chemrxiv.11462160.v1 ◽

2019 ◽

Author(s):

Liqun Cao ◽

Jinzhe Zeng ◽

Mingyuan Xu ◽

Chih-Hao Chin ◽

Tong Zhu ◽

...

Keyword(s):

Molecular Dynamics ◽

Ab Initio ◽

Large Scale ◽

Methane Combustion ◽

Combustion Method ◽

Dynamics Simulation ◽

Ab Initio Molecular Dynamics ◽

Computational Time ◽

Combustion Mechanism ◽

Daily Lives

Combustion is a kind of important reaction that affects people's daily lives and the development of aerospace. Exploring the reaction mechanism contributes to the understanding of combustion and the more efficient use of fuels. Ab initio quantum mechanical (QM) calculation is precise but limited by its computational time for large-scale systems. In order to carry out reactive molecular dynamics (MD) simulation for combustion accurately and quickly, we develop the MFCC-combustion method in this study, which calculates the interaction between atoms using QM method at the level of MN15/6-31G(d). Each molecule in systems is treated as a fragment, and when the distance between any two atoms in different molecules is greater than 3.5 Å, a new fragment involved two molecules is produced in order to consider the two-body interaction. The deviations of MFCC-combustion from full system calculations are within a few kcal/mol, and the result clearly shows that the calculated energies of the different systems using MFCC-combustion are close to converging after the distance thresholds are larger than 3.5 Å for the two-body QM interactions. The methane combustion was studied with the MFCC-combustion method to explore the combustion mechanism of the methane-oxygen system.

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Evolution of Metastable Structures in Bimetallic Catalysts from Microscopy and Machine-Learning Molecular Dynamics

10.26434/chemrxiv.11811660.v1 ◽

2020 ◽

Author(s):

Jin Soo Lim ◽

Jonathan Vandermause ◽

Matthijs A. van Spronsen ◽

Albert Musaelian ◽

Christopher R. O’Connor ◽

...

Keyword(s):

Machine Learning ◽

Molecular Dynamics ◽

Large Scale ◽

Materials Science ◽

Complete Characterization ◽

Layer By Layer ◽

Surface Restructuring ◽

Metastable Structures ◽

Mechanistic Investigation ◽

Underlying Mechanisms

Restructuring of interface plays a crucial role in materials science and heterogeneous catalysis. Bimetallic systems, in particular, often adopt very different composition and morphology at surfaces compared to the bulk. For the first time, we reveal a detailed atomistic picture of the long-timescale restructuring of Pd deposited on Ag, using microscopy, spectroscopy, and novel simulation methods. Encapsulation of Pd by Ag always precedes layer-by-layer dissolution of Pd, resulting in significant Ag migration out of the surface and extensive vacancy pits. These metastable structures are of vital catalytic importance, as Ag-encapsulated Pd remains much more accessible to reactants than bulk-dissolved Pd. The underlying mechanisms are uncovered by performing fast and large-scale machine-learning molecular dynamics, followed by our newly developed method for complete characterization of atomic surface restructuring events. Our approach is broadly applicable to other multimetallic systems of interest and enables the previously impractical mechanistic investigation of restructuring dynamics.

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EGCG binds intrinsically disordered N-terminal domain of p53 and disrupts p53-MDM2 interaction

Nature Communications ◽

10.1038/s41467-021-21258-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jing Zhao ◽

Alan Blayney ◽

Xiaorong Liu ◽

Lauren Gandy ◽

Weihua Jin ◽

...

Keyword(s):

Large Scale ◽

Molecular Mechanisms ◽

Epigallocatechin Gallate ◽

Cancer Drug ◽

Dynamic Interactions ◽

Cancer Drug Discovery ◽

Intrinsically Disordered ◽

Terminal Domain ◽

Cancerous Cells

AbstractEpigallocatechin gallate (EGCG) from green tea can induce apoptosis in cancerous cells, but the underlying molecular mechanisms remain poorly understood. Using SPR and NMR, here we report a direct, μM interaction between EGCG and the tumor suppressor p53 (KD = 1.6 ± 1.4 μM), with the disordered N-terminal domain (NTD) identified as the major binding site (KD = 4 ± 2 μM). Large scale atomistic simulations (>100 μs), SAXS and AUC demonstrate that EGCG-NTD interaction is dynamic and EGCG causes the emergence of a subpopulation of compact bound conformations. The EGCG-p53 interaction disrupts p53 interaction with its regulatory E3 ligase MDM2 and inhibits ubiquitination of p53 by MDM2 in an in vitro ubiquitination assay, likely stabilizing p53 for anti-tumor activity. Our work provides insights into the mechanisms for EGCG’s anticancer activity and identifies p53 NTD as a target for cancer drug discovery through dynamic interactions with small molecules.

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