Biology subjects

Edmonds, K. A.

Publications and source records attributed to Edmonds, K. A..

6 recordsLinked to original sources

bioRxiv2026

''Self-gapping'' by a C-terminal domain arginine finger regulates GTP hydrolysis in bacterial zinc metallochaperones

The cellular response to transition metal scarcity is multifaceted and complex. Members of the Cluster of Orthologous Groups 0523 (COG0523) superfamily are proposed to chaperone a bound metal to activate an apoenzyme client and are thus candidate metallochaperones. COG0523 enzymes are GTPases that harbor a conserved Ras-like GTP-binding and hydrolysis domain (G-domain) and a C-terminal domain (CTD) of unknown function connected by a flexible linker. AlphaFold3 modeling posits an "open" GTPase-inactive and "closed" GTPase-active conformation where the GTP and switch 1 (G2) loop are buried at the interface of the two domains. We show here that the CTD functions as a GTP-hydrolysis activation protein (GAP) domain that stimulates GTP hydrolysis by the tethered G-domain. This "self-gapping" activity requires an invariant RxKG sequence in the {beta}2-strand of the CTD in two distantly related bacterial COG0523s from Acinetobacter baumannii, ZigA and MigC. Thermodynamic and kinetic studies reveal that the Arg is analogous to the "arginine finger" motif of a Ras-cognate GAP, while the Lys residue appears to play a catalytic role in GTP hydrolysis. Cognate CTD added in trans to full-length RxK mutant ZigA or MigC rescues Zn(II)-activated GTPase activity whereas the non-cognate CTD shows no rescue. The linker in AbZigA appears to gate Zn(II)-stimulated GTP hydrolysis. Solution NMR studies of RxK AbMigC reveal that the two domains tumble independently of one another in the absence of bound ligands, with cognate CTD added in trans forming a tight complex. The extent to which conformational switching characterizes eukaryotic COG0523s is discussed. Significance StatementThe cellular response to nutrient transition metal limitation is evolutionarily conserved in all kingdoms, providing protection from the loss of these essential inorganic cofactors that power much of metabolism. An important part of this response is the increased cell abundance of members of the enigmatic and ubiquitous Cluster of Orthologous Groups 0523 (COG0523) superfamily. In bacterial pathogens, these enzymes are often associated with the low-zinc adaptive response to host-mediated nutritional immunity, where the host deploys transition metal chelation as an innate immune response to infections. In this work, we provide new mechanistic insights into COG0523 function with the discovery of "self-gapping" by the C-terminal domain of a two-domain G-protein architecture, placed into the context of a metallochaperone model.

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↗

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↗

bioRxiv2018

Structure of the large extracellular loop of FtsX and its interaction with the essential peptidoglycan hydrolase PcsB in Streptococcus pneumoniae

Streptococcus pneumoniae is a leading killer of infants and immunocompromised adults and has become increasingly resistant to major antibiotics. Therefore, the development of new antibiotic strategies is desperately needed. Targeting bacterial cell division is one such strategy, specifically targeting essential proteins for the synthesis and breakdown of peptidoglycan. One complex important to this process is FtsEX. FtsEX comprises an integral membrane protein (FtsX) and cytoplasmic ATPase (FtsE) that resembles an ATP-binding cassette (ABC) transporter. Here, we present NMR solution structural and crystallographic models of the large extracellular domain of FtsX, denoted ECL1. The structure of ECL1 reveals an upper extended {beta}-hairpin and a lower -helical lobe, each extending from a mixed -{beta} core. The helical lobe mediates a physical interaction with the peptidoglycan hydrolase PcsB, via the coiled-coil domain of PcsB (PcsB-CC). Characterization of S. pneumoniae D39 derived strains harboring mutations in the -helical lobe shows that this subdomain is essential for cell viability and required for proper cell division of S. pneumoniae.\n\nIMPORTANCEFtsX is a ubiquitous bacterial integral membrane protein involved in cell division that regulates the activity of peptidoglycan (PG) hydrolases. FtsX is representative of a large group of ABC3 superfamily proteins that function as \"mechanotransmitters,\" proteins that relay signals from inside to the outside of the cell. Here we present a structural characterization of the large extracellular loop (ECL1) of FtsX from the human opportunistic pathogen Streptococcus pneumoniae. We show a direct interaction between the peptidoglycan hydrolase PcsB and FtsX, and demonstrate that this interaction is essential for cell viability. As such, FtsX represents an attractive, conserved target for the development of new classes of antibiotics.

microbiology↗

bioRxiv2018

Tuning site-specific dynamics to drive allosteric activation in a pneumococcal zinc uptake regulator

MarR (multiple antibiotic resistance repressor) family proteins are bacterial repressors that regulate transcription in response to a wide range of chemical signals. Although specific features of MarR family function have been described, the role of atomic motions in MarRs remains unexplored thus limiting insights into the evolution of allostery in this ubiquitous family of repressors. Here, we provide the first experimental evidence that internal dynamics play a crucial functional role in MarR proteins. Streptococcus pneumoniae AdcR (adhesin-competence repressor) regulates ZnII homeostasis and ZnII functions as an allosteric activator of DNA binding. ZnII coordination triggers a transition from independent domains to a more compact structure. We identify residues that impact allosteric activation on the basis of ZnII-induced perturbations of atomic motions over a wide range of timescales. These findings reconcile the distinct allosteric mechanisms proposed for other MarRs and highlight the importance of conformational dynamics in biological regulation.

biochemistry↗