Biology subjects

Stewart, A. G.

Publications and source records attributed to Stewart, A. G..

16 recordsLinked to original sources

bioRxiv2026

The Lower γ Region Ensures Unidirectional Rotation and Torque Generation in the Latter Half of the 80° Substep of F1-ATPase

F1-ATPase achieves unidirectional rotation of its {gamma} shaft through coordinated conformational cycling of the 3{beta}3 ring, yet how the shaft itself enforces directionality remains unclear. Here we analyzed an axle-less TF1 lacking the lower half of the rotor shaft by combining single-molecule rotation assays with cryo-EM structural analysis under catalysis conditions. Under ATP-saturated conditions, wild-type TF1 exhibited only three pauses per turn corresponding to the catalytic dwells, whereas axle-less TF1 exhibited six pauses per turn, indicating the presence of an additional intermediate during rotation. This previously unreported intermediate dwell was observed at 40{degrees} between the binding and catalytic dwells. High-speed recordings revealed frequent backsteps confined to the 40{degrees} transition between the intermediate and catalytic dwells. Analysis of the dwell-time distributions indicated an approximately zero free energy bias between these two states, consistent with thermally driven interconversion. Cryo-EM resolved three corresponding intermediates--binding, intermediate, and catalytic dwells--showing a major {beta} conformational change from 0{degrees} to 40{degrees}, but minimal {beta} rearrangement between 40{degrees} and 80{degrees}, suggesting that the {beta} power stroke is not operative in this interval. Analysis of the {gamma} rotational scheme indicates that the driving force changes across the 0-80{degrees} step: the 0-40{degrees} advance proceeds via a power stroke to an intermediate dwell, whereas the subsequent 40{degrees} transition to the catalytic dwell lacks a power stroke and is dominated by thermal fluctuations. Taken together, these findings suggest that the lower {gamma} region contributes to unidirectional rotation and torque generation specifically in the latter half of the 0-80{degrees} step, while the initial half can proceed even without contributions from the lower {gamma} region.

biophysics↗

bioRxiv2026

Mechanism of phospholipid transport to the bacterial outer membrane by TAM.

Gram-negative bacteria transport phospholipids from the inner membrane (IM) to the outer membrane (OM) via poorly understood processes. These processes are essential for cell growth and the establishment of an antibiotic-resistant barrier. Here, we conducted single-particle cryo-electron microscopy, in vivo functional assays, and lipidomics to investigate the role of the "translocation and assembly module" (TAM) in lipid transport. We found that the OM-embedded subunit TamA anchors the IM-embedded bridge-like subunit TamB to the OM by forming a functional stable hybrid-barrel structure with the highly conserved C-terminal domain of unknown function 490 (DUF490). Using in vivo disulfide-tethering experiments we found that a highly conserved amphipathic helix within TamB DUF490 is important for TAM to function in OM maintenance. We also found that TamB DUF490 forms a {beta}-taco channel containing lipid-like densities and that the lipophilic property of the channel is important for TAM to maintain the levels of cardiolipin in the OM. Not only do our data support a novel model in which TAM acts to direct specific lipid classes into the OM, but it also supports the notion that TamB is a bacterial evolutionary prototype of a structurally homologous superfamily of eukaryotic bridge-like lipid transfer proteins.

biochemistry↗

bioRxiv2025

The structure and composition of native human nucleosomes

Since the first high-resolution structures of recombinantly assembled nucleosomes, efforts have shifted towards understanding chromatin structure in a native context. Most of these efforts have focused on native-like, yet still recombinantly assembled, nucleosomes that contain native DNA sequences. To date, no high-resolution structures of native human nucleosomes have been reported. Here we report the high-resolution cryo-EM structure of native human nucleosomes isolated from HEK293 cells. The HEK293-NCP structure reveals that native human nucleosomes store 145 bp of DNA. Despite the DNA sequence diversity of native nucleosomes, we observe conserved nucleotides that support the idea of a nucleosome positioning code. In addition to these striking features of nucleosomal DNA, we note alternate conformations of several DNA contacting histone residues that hint at dynamics in the HEK293-NCP. To complement the HEK293-NCP structure, we provide a mass spectrometry analysis of histone modifications and variants present in the sample, which demonstrates that a typical HEK293-NCP is composed of canonical histones with N-terminal tails that are methylated at K9, K27 and/or K36 of histone H3. Altogether these findings have implications for biological processes such as chromatin remodelling and transcription and improve our understanding of nucleosome and chromatin structure in a native context.

biochemistry↗

bioRxiv2025

Metagenomic resolution of spotted-fever group Rickettsia tasmanensis and novel DNA viruses in Australian wildlife ticks, with spatial modelling of Rickettsia exposure zones.

Australia's spotted fever group (SFG) rickettsiae cause significant illness, yet microbial diversity in wildlife ticks remains incompletely understood. Metagenomic sequencing of nine tick pools representing three Ixodidae species, Ixodes tasmani, I. holocyclus, and Haemaphysalis bancrofti, from Australian wildlife revealed a genome of a novel SFG Rickettsia species from I. tasmani ticks collected from koalas in New South Wales. Phylogenomic analysis confirmed this as a distinct species closely related to Rickettsia tasmaniensis fragments previously reported from Tasmanian devils. Additionally, we discovered four novel DNA Anellovirus species forming a new genus 'Sintorquevirus' and one Circovirus from likely vertebrate blood meals, revealing wildlife viral diversity. Species distribution modelling of I. tasmani for assessing Rickettsia risk revealed suitable habitat along Australia's eastern coastlines, with overlap between vector distribution, marsupial hosts, and population centres. This identified potential Candidatus R. tasmanensis exposure zones in coastal regions where positive samples originated, highlighting areas for surveillance.

microbiology↗

bioRxiv2025

Functional and Structural Characterization of F1-ATPase with common ancestral core domains in stator ring

Extant F1-ATPases exhibit diverse rotational stepping behaviors--3-, 6-, or 9-step cycles--yet the evolutionary origin of these patterns remains unclear. Here, we used ancestral sequence reconstruction to infer the catalytic {beta} and non-catalytic subunits of a putative ancestral F1-ATPase. We then fused their functionally critical domains into the thermostable F1 from Bacillus PS3, yielding a stable chimeric enzyme. Cryo-EM revealed two distinct conformational states--binding and catalytic dwell states--separated by a [~]34{degrees} rotation of the {gamma} subunit, suggesting a fundamental six-step mechanism akin to that of extant 6-stepping F1-ATPases. Single-molecule rotation assays with ATP and the slowly hydrolyzed ATP analog ATP{gamma}S demonstrated that the chimeric motor is intrinsically a 6-stepper, pausing at binding and catalytic dwell positions separated by 32.1{degrees}, although the binding dwell is significantly prolonged by an unknown mechanism. These findings indicate that F1-ATPase was originally a 6-stepper and diversified into 3-, 6- and 9-step forms in evolutional adaptation. Based on these results, we discuss plausible features of the entire FoF1 complex, along with potential physiological contexts in last universal common ancestor and related lineages.

biophysics↗

bioRxiv2024

The molecular structure of an axle-less F1-ATPase

F1Fo ATP synthase is a molecular rotary motor that can generate ATP using a transmembrane proton motive force. Isolated F1-ATPase catalytic cores can hydrolyse ATP, passing through a series of conformational states involving rotation of the central {gamma} rotor subunit and the opening and closing of the catalytic {beta} subunits. Cooperativity in F1-ATPase has long thought to be conferred through the {gamma} subunit, with three key interaction sites between the {gamma} and {beta} subunits being identified. Single molecule studies have demonstrated that the F1 complexes lacking the {gamma} axle still "rotate" and hydrolyse ATP, but with less efficiency. We solved the cryogenic electron microscopy structure of an axle-less Bacillus sp. PS3 F1-ATPase. The unexpected binding-dwell conformation of the structure in combination with the observed lack of interactions between the axle-less {gamma} and the open {beta} subunit suggests that the complete {gamma} subunit is important for coordinating efficient ATP binding of F1-ATPase.

biochemistry↗

bioRxiv2023

Potassium dependent structural changes in the selectivity filter of HERG potassium channels

The fine tuning of biological electrical signaling is mediated by variations in the rates of opening and closing of gates that control ion flux through different ion channels. Human ether-a-go-go related gene (HERG) potassium channels have uniquely rapid inactivation kinetics which are critical to the role they play in regulating cardiac electrical activity. Here, we have exploited the K+ sensitivity of HERG inactivation to determine structures of both a conductive and non-conductive selectivity filter structure of HERG. We propose that inactivation is the result of a high propensity for flipping of the selectivity filter valine carbonyl oxygens. Molecular dynamics simulations point to a low energy barrier, and hence rapid kinetics, for flipping of the valine 625 carbonyl oxygens facilitated by a previously unrecognized interaction between S620 and Y616 that stabilizes the transition state between conducting and non-conducting structures. Our model represents a new mechanism by which ion channels fine tune their activity that explains the uniquely rapid inactivation kinetics of HERG. HighlightsStructures of a conductive and non-conductive HERG selectivity filter have been determined. Reduced potassium causes flipping of selectivity filter valine carbonyl oxygens. The sidechain of S620 on the pore helix coordinates distinct sets of interactions between conductive, non-conductive, and transition states.

biophysics↗

bioRxiv2022

Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients

Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells (PBMCs) of convalescent patients using SARS-CoV-2 receptor binding domains (RBDs) carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and class 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (mAb S309) by orders of magnitude. They also provide potent prophylactic and therapeutic in vivo protection of hACE2 mice against viral challenge. Our results indicate that exposure to Wuhan SARS-CoV-2 induces antibodies that maintain potent and broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.

immunology↗

bioRxiv2021

Molecular basis for GIGYF-TNRC6 complex assembly in miRNA-mediated translational repression

The GIGYF proteins associate with 4EHP and RNA-associated proteins to elicit transcript-specific translational repression. However, the mechanism by which the GIGYF1/2-4EHP complex is recruited to its target transcripts remain unclear. Here we report the crystal structures of the GYF domains from GIGYF1 and GIGYF2 in complex with proline-rich sequences from miRISC-binding proteins TNRC6C and TNRC6A, respectively. The TNRC6 proline-rich motifs bind to a conserved array of aromatic residues on the surface of the GIGYF1/2 GYF domain, bridging 4EHP to Argonaute-miRNA mRNA targets. Our structures also reveal a phenylalanine residue conserved from yeast to human GYF domains that contributes to GIGYF2 thermostability. The molecular details we outline here are likely to be conserved between GIGYF1/2 and other RNA-binding proteins to elicit 4EHP-mediated repression in different biological contexts.

biochemistry↗

bioRxiv2020

The six steps of the F1-ATPase rotary catalytic cycle

F1Fo ATP synthase interchanges phosphate transfer energy and proton motive force via a rotary catalysis mechanism. When isolated, its F1-ATPase catalytic core can hydrolyze ATP, rotating its {gamma} rotor subunit. Although previous structural studies have contributed greatly to understanding rotary catalysis in F1, the structure of one major conformational state detected in single-molecule studies, termed the binding dwell state, has not yet been determined. Here, by exploiting a temperature-sensitive F1-ATPase mutant from Bacillus PS3, the structure of this binding dwell state was established together with that of the catalytic dwell state. Each state showed three catalytic {beta} subunits in different conformations, providing the complete set of six {beta} subunit conformational states taken up during catalysis cycle. These structures provide molecular details for the power-stroke conformational change that occurs upon ATP binding and induces a ~80{degrees} {gamma} subunit rotation, as well as a second torque-generating conformational change, triggered by hydrolysis and product release, that produces a ~40{degrees} rotation. This study also identifies a putative phosphate-releasing tunnel that indicates how ADP and phosphate releasing steps are coordinated. Overall these findings provide a structural basis for the entire F1-ATPase rotary catalysis cycle.

biophysics↗

bioRxiv2020

Potent SARS-CoV-2 binding and neutralization through maturation of iconic SARS-CoV-1 antibodies

Antibodies against coronavirus spike protein potently protect against infection and disease, however it remains unclear if such protection can be extended to variant coronaviruses. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here we explore antibody maturation strategies to change and broaden their specificity, enabling potent binding and neutralization of SARS-CoV-2. Using targeted mutagenesis as well as light chain shuffling on phage, we identified variants with considerably increased affinity and neutralization potential. The most potent antibody, derived from the NIH-developed mAb m396, neutralized live SARS-CoV-2 virus with a half-maximal inhibitory concentration (IC50) of 160 ng/ml. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of antibodies can give rise to variants targeting diverse epitopes. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.

immunology↗

bioRxiv2020

Subunit epsilon of E. coli F1Fo ATP synthase attenuates enzyme activity by modulating central stalk flexibility

F1Fo ATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. Proton flow through the Fo motor generates rotation of the central stalk, inducing conformational changes in the F1 motor that catalyzes ATP production via flexible coupling. Here we present a range of cryo-EM structures of E. coli ATP synthase in different rotational and inhibited states observed following a 45 second incubation with 10 mM MgATP. The structures generated describe multiple changes that occur following addition of MgATP, with the inhibitory C-terminal domain of subunit {varepsilon} ({varepsilon}CTD) disassociating from the central stalk to adopt a condensed "down" conformation. The transition to the {varepsilon}CTD down state increases the torsional flexibility of the central stalk allowing its foot to rotate by [~]50{degrees}, with further flexing in the peripheral stalk enabling the c-ring to rotate by two sub-steps in the Fo motor. Truncation mutants lacking the second helix of the {varepsilon}CTD suggest that central stalk rotational flexibility is important for F1Fo ATP synthase function. Overall this study identifies the potential role played by torsional flexing within the rotor and how this could be influenced by the {varepsilon} subunit.

biophysics↗

bioRxiv2020

Glutamate transporters contain a conserved chloride channel with two hydrophobic gates

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, therefore its precise control is vital for maintaining normal brain function and preventing excitotoxicity1. Removal of extracellular glutamate is achieved by plasma membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism2-5. Glutamate transporters also conduct chloride ions via a channel-like process that is thermodynamically uncoupled from transport6-8. However, the molecular mechanisms that allow these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, revealing an aqueous cavity that is formed during the transport cycle. Using functional studies and molecular dynamics simulations, we show that this cavity is an aqueous-accessible chloride permeation pathway gated by two hydrophobic regions, and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function and add a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family.

biophysics↗

bioRxiv2020

Using whole-genome sequencing and a pentaplex real-time PCR to characterize third-generation cephalosporin-resistant Enterobacteriaceae from Southeast Queensland, Australia

SynopsisThird-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 264 patients presenting at a regional Australian hospital over a 14-month period. Alongside routine mass spectrometry speciation and antibiotic sensitivity testing, isolates were examined using rapid ([~]40 min) real-time PCR assays targeting the most common extended spectrum {beta}-lactamases (ESBLs; CTX-M-1 and CTX-M-9 groups, plus TEM, SHV, and an internal 16S ribosomal DNA control). AmpC CMY {beta}-lactamase prevalence was also examined. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance were identified in 97.0% and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings in Enterobacteriaceae, most (98.3%) isolates harbored at least one {beta}-lactamase gene, with 144 (50%) encoding blaCTX-M-1 group, 92 (31.9%) blaCTX-M-9 group, 48 (16.7%) blaSHV, 133 (46.2%) blaTEM, and 34 (11.8%) blaCMY. A subset of isolates (n=98) were subjected to whole-genome sequencing (WGS) to identify the presence of cryptic resistance determinants, and to verify genotyping accuracy. WGS of {beta}-lactamase negative or carbapenem-resistant isolates identified uncommon ESBLs and carbapenemases, including blaNDM and blaIMP, and confirmed all PCR-positive genotypes. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.

microbiology↗

bioRxiv2020

Structural Analysis of a Filamentous Chaperonin from Sulfolobus solfataricus

Chaperonins are biomolecular complexes that assist protein folding. Thermophilic Factor 55 (TF55) is a group II chaperonin found in the archaeal genus Sulfolobus that has , {beta} and {gamma} subunits. Using cryo-electron microscopy, we have determined the structure of the {beta}-only complex of S. solfataricus TF55 complexes to 3.6-4.2 [A] resolution and a filamentous form to 5.2 [A] resolution. The structures of the TF55{beta} complexes formed in the presence of ADP or ATP highlighted an open state in which nucleotide exchange can occur before progressing in the refolding cycle. The structure of the filamentous state indicates how helical protrusions facilitate end-on-end interactions. SynopsisThe isolated complex and filamentous forms of TF55{beta} chaperonin from the thermophilic archaea Sulfolobus solfataricus are reported. Using cryo-EM, nucleotide-bound complexes of TF55{beta} at 3.6-4.2 [A] resolution reveal an open conformation, while a 5.2 [A] reconstruction of the filamentous chaperonin reveals contacts at the apical domain similar to crystal-packed structures.

biophysics↗

bioRxiv2019

Visualizing movements in E. coli F1Fo ATP synthase indicates how the F1 and Fo motors are coupled

F1Fo ATP synthase functions as a biological rotary generator and makes a major contribution to cellular energy production. It is comprised of two motors that are coupled together by a central \"rotor\" and peripheral \"stator\" stalk. Proton flow through the Fo motor generates rotation of the central stalk that induces conformation changes that catalyze production of ATP in the F1 motor. Here we provide 3-4 [A] resolution cryo-EM structures of E. coli F1Fo ATP synthase in 10 mM MgADP. In addition to generating a comprehensive structural model of E. coli F1Fo ATP synthase to provide a framework to interpret mutagenesis studies, we describe a rotational sub-step of the Fo motor c-ring associated with long-range conformational changes that suggests an elegant mechanism by which the F1 and Fo motors can be coupled with minimal energy loss.

biophysics↗