Biology subjects

Gonzalez-Gutierrez, G.

Publications and source records attributed to Gonzalez-Gutierrez, G..

11 recordsLinked to original sources

bioRxiv2026

Extracellular Vacuole-derived bodies (EVacs) mediate RNA secretion in plants

Extracellular RNAs are found in the plant extracellular space, but how they are exported from cells remains unclear. We found that the plant vacuole is a major source of extracellular RNA and identified a class of large extracellular vacuole-derived bodies, which we termed EVacs, that are key mediators of this transport. EVacs are marked by the vacuolar membrane (tonoplast) proteins {gamma}-TIP and V-ATPase and originate as intravacuolar structures formed by inward folding of the tonoplast, encapsulating intact cytoplasmic material, including both RNAs and proteins. These intravacuolar bodies then escape the vacuole and are subsequently released from the plasma membrane of mesophyll cells into the apoplast. These findings provide a novel mechanism for the unconventional secretion of macromolecules in plants.

plant biology↗

bioRxiv2026

Evolution of allostery without shape shifting: Internal dynamics drives functional diversification of a transcriptional repressor superfamily

Allostery enables proteins to couple environmental signals to functional outputs, yet how allosteric mechanisms diversify during evolution remains poorly understood. Here, we address this question in the ubiquitous and functionally diverse arsenic repressor (ArsR) superfamily by integrating information-theoretic bioinformatics, structural characterization of DNA recognition and NMR measurements of fast internal dynamics. We identify conserved residues that define the structural scaffold of ArsR proteins and subfamily-specific positions that encode inducer and DNA specificity. In the persulfide sensor SqrR, the crystal structure of the DNA-bound complex reveals how operator specificity is encoded by a limited set of residues, consistent with sequence-derived predictions functionally validated by in vitro transcription assays across divergent ArsR regulators. We further show that allosteric inhibition of DNA binding in SqrR occurs without large-scale conformational rearrangements and is instead associated with changes in internal dynamics, as previously observed for the zinc sensor CzrA. Together, these results support a model in which conformational entropy preserves allosteric connectivity while relaxing sequence constraints, thereby enabling functional diversification within a protein superfamily.

biochemistry↗

bioRxiv2026

A Fragment Screen Identifies Acrylamide Covalent Inhibitors of the TEAD/YAP Protein-Protein Interaction

TEA domain (TEAD) proteins bind co-activator Yes-associated protein (YAP) to regulate the expression of target genes of the Hippo pathway. The TEAD*YAP protein-protein interaction is not druggable, but TEADs possess a unique and deep palmitate pocket with a highly conserved cysteine located outside the TEAD*YAP protein-protein interaction interface. Here, we screen a fragment library of acrylamide electrophiles and identify a fragment that forms an adduct with the conserved palmitate pocket cysteine and inhibits TEAD4 binding to YAP. Synthesis of a focused set of derivatives and time- and concentration-dependent studies with four TEADs provide reaction rates and binding constants. Co-crystal structures of fragments bound to TEAD2 and TEAD3 reveal reaction at the conserved palmitate pocket cysteine but also at another less conserved cysteine located in the palmitate pocket of TEAD2 closer to the TEAD*YAP interface. These fragments provide a starting point for the development of allosteric acrylamide small-molecule covalent TEAD*YAP inhibitors.

biochemistry↗

bioRxiv2026

Self-S-sulfonation in a bacterial persulfide dioxygenase mediates thiol persulfide detoxification

A ubiquitous class of non-heme Fe(II) enzymes, the persulfide dioxygenases (PDOs), provide protection against hydrogen sulfide (H2S) poisoning. The PDO in humans is a single-domain enzyme, while bacterial PDOs, such as CstB of Staphylococcus aureus, are often fused to a sulfurtransferase (rhodanese) module. Canonical PDOs cleave the S-S bond of glutathione persulfide (GSSH) to produce GSH and sulfite (SO32-). In contrast, CstB, via an unknown mechanism, converts two RSSH to thiosulfate (S2O32-) without the release of sulfite. Six crystallographic structures of S. aureus CstB reveal that a Cys-Gly sequence (C201-G202) in a CstB-unique dynamic loop functions as a glutathione mimic, occupying one face of the hemifacial octahedral Fe(II) coordination site. We establish that CstB self-S-sulfonates C201 in a thiol persulfide, Fe(II) and O2-dependent manner, which is then shuttled to a persulfidated C408 in the rhodanese domain {approx}27 [A] away via electrostatic steering to generate thiosulfate as the sole oxidation product. Both C201A and C408A CstBs are inactive in O2-consumption. Self-S-sulfonation ensures rapid clearance of diverse reactive sulfur species under conditions where these species accumulate, permitting S. aureus to harness their cytoprotective effects while avoiding cellular toxicity.

biochemistry↗

bioRxiv2025

CH···S hydrogen bonds drive molecular recognition of ergothioneine by the microbial transporter

Many bacteria harbor an ATP-binding cassette (ABC) transporter named EgtU specific for the human dietary antioxidant and 2-thioimidazole-containing low molecular weight thiol ergothioneine (ET). How the solute binding domain, EgtUC, discriminates among ET and other similar molecules is unknown. Here, we use a "chimeric" mutagenesis strategy and two distantly related EgtUCs from Streptococcus pneumoniae and Helicobacter pylori to show that a suite of EgtUC alkyl CH***S hydrogen bonds to the ET thione S atom are central determinants of molecular recognition. Small perturbations in CH***S distance and angle give rise to sharply attenuated transport-competent ET-bound "closed" state lifetimes and increased motional disorder in the binding pocket, not around the S atom itself, but distally in weakening NH***O hydrogen bonds. This is, to our knowledge, the first work to describe the impact of alkyl CH***S H-bonding in a biological protein-ligand complex in water.

biochemistry↗

bioRxiv2024

A narrow ratio of nucleic acid to SARS-CoV-2 N-protein enables phase separation

SARS-CoV-2 Nucleocapsid protein (N) is a viral structural protein that packages the 30kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation (LLPS). N, in both soluble and condensed forms, has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation and its reversibility. The conditions that stabilize and destabilize N condensates and the role of N-N interactions are poorly understood. We have investigated LLPS formation and dissolution in a minimalist system comprised of N protein and an ssDNA oligomer just long enough to support assembly. The short oligo allows us to focus on the role of N-N interaction. We have developed a sensitive FRET assay to interrogate LLPS assembly reactions from the perspective of the oligonucleotide. We find that N alone can form oligomers but that oligonucleotide enables their assembly into a three-dimensional phase. At a [~]1:1 ratio of N to oligonucleotide LLPS formation is maximal. We find that a modest excess of N or of nucleic acid causes the LLPS to break down catastrophically. Under the conditions examined here assembly has a critical concentration of about 1 {micro}M. The responsiveness of N condensates to their environment may have biological consequences. A better understanding of how nucleic acid modulates N-N association will shed light on condensate activity and could inform antiviral strategies targeting LLPS.

biochemistry↗

bioRxiv2022

Discovery and structure of a widespread bacterial ABC transporter specific for ergothioneine

Ergothioneine (ET) is the 2-thiourea derivative of trimethylhistidine that is biosynthesized only by select fungi and bacteria, notably Mycobacterium tuberculosis, and functions as a potent scavenger of reactive oxygen species. Although ET is obtained in the diet and accumulates in vertebrate cells via an ET-specific transporter, the extent to which ET broadly functions in bacterial cells unable to synthesize it is unknown. Here we show that spd_1642-1643 in Streptococcus pneumoniae D39, a Gram-positive respiratory pathogen, encodes a novel ergothioneine uptake ATP-binding cassette (ABC) transporter, which we designate EgtUV. EgtU is a permease-solute binding domain (SBD) fusion protein, and the SBD binds ET with high affinity and exquisite specificity in the cleft between the two subdomains, with cation-{pi} interactions engaging the betaine moiety and a water-mediated hydrogen bonding network surrounding the C2-sulfur-containing imidazole ring. Bioinformatics studies reveal that EgtUV is uniquely strongly conserved among known quaternary amine-specific transporters and widely distributed in firmicutes, including the human pathogens Listeria monocytogenes, as BilEB, Enterococcus faecalis and Staphylococcus aureus. This discovery significantly diversifies the LMW thiol pool in Gram-positive human pathogens that may contribute to antioxidant defenses in the infected host.

biochemistry↗

bioRxiv2021

Functional asymmetry and chemical reactivity of CsoR family persulfide sensors

CstR is a persulfide-sensing member of the functionally diverse copper-sensitive operon repressor (CsoR) superfamily that regulates the bacterial response to hydrogen sulfide (H2S) and more oxidized reactive sulfur species (RSS) in Gram-positive pathogens. A cysteine thiol pair on CstR reacts with RSS to form a mixture of interprotomer di-, tri- and tetrasulfide crosslinks, which drives transcriptional derepression of CstR-regulated genes. In some bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA), CstR and CsoR, a Cu(I)-sensor, exhibit no regulatory crosstalk in cells, despite maintaining an identical pair of cysteines. We report a sequence similarity network (SSN) analysis of the entire CsoR superfamily, together with the first crystallographic structure of a CstR protein and mass spectrometry-based kinetic profiling experiments to obtain new insights into the molecular basis of RSS specificity in CstRs. The more N-terminal cysteine is the attacking Cys in CstR and is far more nucleophilic than in a CsoR. This cysteine, C30 in SpCstR, is separated from the resolving thiol, C59, by an Asn55 wedge. Chemical reactivity experiments reveal a striking asymmetry of reactivity, preserved in all CstRs and with all oxidants tested; however, the distribution of crosslinked products varies markedly among CstRs. Substitution of N55 with Ala in SpCstR significantly impacts the distribution of species, despite adopting the same structure as the parent repressor. We show that CstRs react with hydrogen peroxide, a finding that contrasts sharply with other structurally distinct persulfide sensors from Gram-negative bacteria. This suggests that other factors may enhance the specificity and repressor activity of CstRs in cells.

biochemistry↗

bioRxiv2020

The DNA binding domain of the Vibrio vulnificus SmcR transcription factor is flexible and recognizes diverse DNA sequences

The quorum-sensing regulon in vibrios is controlled by the LuxR/HapR family of transcriptional regulators. In Vibrio vulnificus, this regulator is called SmcR, and it controls expression of numerous virulence behaviors, including biofilm formation and elastase production. The consensus binding site of Vibrio LuxR/HapR/SmcR proteins is palindromic, as is common for regulators that bind as dimers with helix-turn-helix N-terminal DNA binding domains. However, the LuxR/HapR/SmcR consensus site is highly degenerate and asymmetric with variations in sequence at each promoter. To determine the mechanism of DNA site recognition, we generated separation-of-function mutants of SmcR that either repress or activate transcription but not both. The SmcR N55I protein is defective at transcription activation due to loss of binding to most DNA binding sites in activated promoters but retains interaction with RNA polymerase (RNAP) alpha. SmcR S76A, L139R, and N142D are defective for interaction with RNAP alpha but retain functional DNA binding activity. Using X-ray crystallography, we show that the wild-type SmcR dimer and the three RNAP-interaction mutants exhibit two conformations of the helix-turn-helix DNA binding domain. Conversely, the SmcR N55I X-ray crystal structure is limited to only one conformation and is restricted in recognition of single base-pair variations in DNA binding site sequences. These data support a model in which two mechanisms drive SmcR transcriptional activation: interaction with RNA polymerase and a multi-conformational DNA binding domain that permits recognition of variable DNA sites. Thus, the LuxR/HapR/SmcR proteins balance specificity for quorum-sensing targets and diversity to accommodate binding at hundreds of sites genome-wide. SignificanceThe cell-cell communication system called quorum sensing controls expression of genes required for virulence in Vibrio bacteria species, including the potent human pathogen Vibrio vulnificus. The master transcriptional regulator of quorum-sensing genes in vibrios belongs to the LuxR/HapR/SmcR family. These regulators directly activate and repress transcription of >100 genes via binding to degenerate sites in promoter regions. We used X-ray crystallography to determine the structure of mutant SmcR proteins. Our experiments reveal that SmcR recognizes diverse sequences via a DNA binding domain that samples multiple conformations to accommodate variations in palindromic DNA sequences. Significantly, the DNA binding domain of SmcR is completely conserved in LuxR/HapR/SmcR family proteins, suggesting that this mechanism is representative of quorum-sensing regulation in other vibrios.

biochemistry↗

bioRxiv2020

Structural determinants of persulfide-sensing specificity in a dithiol-based transcriptional regulator

Cysteine thiol-based transcriptional regulators orchestrate coordinated regulation of redox homeostasis and other cellular processes by "sensing" or detecting a specific redox-active molecule, which in turn activates the transcription of a specific detoxification pathway. The extent to which these sensors are truly specific in cells for a singular class of reactive small molecule stressors, e.g., reactive oxygen or sulfur species, is largely unknown. Here we report novel structural and mechanistic insights into a thiol-based transcriptional repressor SqrR, that reacts exclusively with organic and inorganic oxidized sulfur species, e.g., persulfides, to yield a unique tetrasulfide bridge that allosterically inhibits DNA operator-promoter binding. Evaluation of five crystallographic structures of SqrR in various derivatized states, coupled with the results of a mass spectrometry-based kinetic profiling strategy, suggest that persulfide selectivity is determined by structural frustration of the disulfide form. This energetic roadblock effectively decreases the reactivity toward major oxidants to kinetically favor formation of the tetrasulfide product. These findings lead to the identification of an uncharacterized repressor from the increasingly antibiotic-resistant bacterial pathogen, Acinetobacter baumannii, as a persulfide sensor, illustrating the predictive power of this work and potential applications to bacterial infectious disease.

biochemistry↗

bioRxiv2019

Structural Differences Between the Woodchuck Hepatitis Virus Core Protein in Dimer and Capsid States Indicate Entropic and Conformational Regulation of Assembly

Hepadnaviruses are hepatotropic enveloped DNA viruses with an icosahedral capsid. Hepatitis B virus (HBV) causes chronic infection in an estimated 240 million people; Woodchuck Hepatitis virus (WHV), an HBV homologue, has been an important model system for drug development. The dimeric capsid protein (Cp) plays multiple functions during the viral life cycle and thus has become an important target for a new generation of antivirals. Purified HBV and WHV Cp spontaneously assemble into 120-dimer capsids. Though they have 65% identity, WHV Cp has error-prone assembly with stronger protein-protein association. We have taken advantage of the differences in assembly to investigate the basis of assembly regulation. We have determined the structures of the WHV capsid to 4.5 [A] resolution by cryo-EM and the WHV Cp dimer to 2.9 [A] resolution by crystallography and examined the biophysical properties of the dimer. We found, in dimer, the subdomain that makes protein-protein interactions is partially disordered and rotated 21{degrees} from its position in capsid. This subdomain is susceptible to proteolysis, consistent with local disorder. These data show there is an entropic cost for assembly that is compensated for by the energetic gain of burying hydrophobic interprotein contacts. We propose a series of stages in assembly that incorporate disorder-to-order transition and structural shifts. WHV assembly shows similar susceptibility to HBV antiviral molecules, suggesting that HBV assembly follows similar transitions and indicating WHVs importance as a model system for drug development. We suggest that a cascade of structural changes may be a common mechanism for regulating high fidelity capsid assembly in HBV and other viruses.\n\nAuthor summaryCapsid assembly is a key step in the Hepatitis B virus (HBV) life cycle; it requires sophisticated regulation to ensure the production of capsids and viruses with high-fidelity. HBV capsids are constructed from 120 self-assembling capsid protein dimers (Cp). It was hypothesized that changes in Cp structure regulated the assembly reaction. Here we determined structures of dimer and capsid for Woodchuck Hepatitis virus (WHV), an HBV homologue. We observed that the component of the dimer involved in subunit-subunit interactions undergoes a disorder-order transition and changes structure concomitant with assembly. Meanwhile WHV Cp displays similar susceptibility to HBV antiviral. We propose that this structural transition entropically regulates assembly and may be a common theme in other viruses.

biophysics↗