Biology subjects

Khalid, S.

Publications and source records attributed to Khalid, S..

At least 19 recordsLinked to original sources

bioRxiv2026

YhdP forms an envelope-spanning phospholipid-binding bridge in complex with DedD

The outer membrane of Gram-negative bacteria is built from phospholipids synthesised at the inner membrane, but how they cross the periplasm remains unresolved. YhdP is the leading candidate for anterograde phospholipid transfer and is predicted to form an envelope-spanning bridge. Here we purify YhdP at endogenous levels and show by cryo-electron microscopy that it contains a continuous cavity, alongside an additional density that proteomics identifies as the cell wall-binding protein DedD. Liposome co-sedimentation shows that the C-terminal helices of YhdP bind membranes directly, and single-molecule tracking reveals that endogenous YhdP is as immobile as an outer membrane protein, indicating an envelope-spanning architecture in vivo. Native mass spectrometry demonstrates that the YhdP groove accommodates all major classes of E. coli phospholipid without headgroup preference. DedD engages YhdP through its disordered region and increases lipid density at the groove entrance, suggesting a potential role in lipid loading.

microbiology↗

bioRxiv2026

A bacterial Rhesus transporter retunes a structurally conserved ammonium pore into a reversible nitrogen valve

How conserved proteins acquire new physiological functions is a central question in molecular evolution. Rather than inventing new architectures, evolution often repurposes existing scaffolds, preserving core structural features while retuning the molecular logic that connects mechanism to physiology. Membrane transporters offer a powerful test of this principle because substrate selectivity, directionality, flux and energetic cost must be coordinated at the interface between the cell and its environment. The Amt/Mep/Rh superfamily controls one of the most fundamental requirements of cellular life: the movement of reduced nitrogen across biological membranes. Despite sharing a highly conserved ammonium-conducting pore, these proteins support distinct physiological roles, including nitrogen acquisition, sensing and homeostatic control. Here, we combined targeted mutagenesis, electrophysiology, yeast complementation and molecular dynamics simulations to define the transport logic of NeRh50, a bacterial Rhesus protein from the ammonia-oxidising bacterium Nitrosomonas europaea. We show that NeRh50 is not simply an AmtB-like ammonium importer. Instead, it uses the conserved Amt/Mep/Rh pore as a branched transport system in which inward uptake and export-linked transport can be separated genetically and mechanistically. Two conserved pore residues define this division of labour. A residue at the external entrance couples ammonium recruitment to productive inward uptake, whereas a second residue deeper in the pore enables a distinct transport mode required for substrate release when intracellular nitrogen accumulates. Thus, conserved Amt/Mep/Rh pore landmarks do not impose a single mechanism. Their local chemistry can be reassigned to generate different transport outputs, allowing NeRh50 to function as a reversible nitrogen valve. These findings reveal how minimal retuning within an ancient membrane-protein scaffold can rewire transport directionality and adapt nitrogen handling to ecological and physiological demand.

biochemistry↗

bioRxiv2026

Lipid-associated architecture and divergent ammonium transport distinguish a bacterial Rhesus protein

Nitrification is a major process in the global nitrogen cycle, initiated by the oxidation of ammonia to nitrite. The ammonia-oxidising bacterium Nitrosomonas europaea catalyses this first and rate-limiting step and depends on ammonium both as an energy substrate and as a nitrogen source, making ammonium acquisition central to its physiology. Cellular ammonium transport is mediated by the Amt/Mep/Rh superfamily, whose members share a conserved fold despite divergent physiological roles. Remarkably, N. europaea lacks a canonical prokaryotic Amt transporter and instead expresses NeRh50, a Rh-family protein proposed to have been acquired from a eukaryotic lineage. Here, we determine a cryo-EM structure of NeRh50 that reveals tightly associated lipids at the monomer-monomer interfaces, including density consistent with an unusual inverted lipid orientation. Native mass spectrometry and molecular dynamics simulations further support persistent lipid association at these interfaces. NeRh50 also differs functionally from canonical AmtB, selectively mediating electrogenic ammonium transport with ~70-fold lower apparent affinity. Transport is enhanced under alkaline conditions but not by an imposed proton gradient, suggesting membrane-potential dependence. Together, these findings reveal divergence from canonical Amt proteins in both membrane-associated architecture and transport properties, providing insight into the functional and evolutionary diversification of the Amt/Mep/Rh superfamily.

biochemistry↗

bioRxiv2026

Cooling fast and slow: Characterising the effects of vitrification in cryo-EM and the subsequent recovery of equilibrium populations

Single-particle cryogenic electron microscopy (cryo-EM) has enabled near-atomic resolution structure determination of diverse biomolecules. Because the high vacuum required for electron microscopy prevents the imaging of liquid-phase samples, cryo-EM samples are prepared by plunging the sample into a cryogen, rapidly cooling the sample and suspending the ensemble of biomolecules in a matrix of water glass. However, the effects of this vitrification on the biomolecular ensemble are unknown, complicating efforts to use cryo-EM to derive conformational ensembles of biomolecules. To study these effects, we carried out extensive molecular dynamics simulations (over 50 milliseconds) of the Trp-cage miniprotein at equilibrium and undergoing rapid cooling. We simulated seven cooling rates spanning three orders of magnitude, with the slowest coolings matching experimental rates. By inspecting molecular mobility and density-temperature equations of state for water with and without protein, we found that water vitrification is unaltered by the protein. To track protein conformation changes, and to relate them to conformational kinetics, we made a Markov State Model (MSM) of Trp-cage from 5.4 milliseconds of equilibrium sampling at 277 K. We observed that MSM states with a characteristic time longer than the duration of the non-equilibrium cooling, tend to be more robust to artefacts induced by such cooling. Critically, although we observe that some states vanish in the equilibrium ensemble at 230 K, none do in our nonequilibrium cooled ensembles. However, to account for perturbations induced by nonequilibrium cooling for more labile states, we developed a thermodynamic inference framework for recovering equilibrium populations from the measured vitrified ensembles. These results indicate that cryo-EM has the capacity to be a reliable and accurate biophysical technique for the study of biomolecular ensembles. SignificanceCryogenic electron microscopy images biomolecules trapped in vitreous ice. To vitrify the sample, it must be cooled over the course of 22 microseconds. However, the degree to which this cooling causes the ensemble of the molecules to be perturbed from equilibrium is unknown. Here we present extensive molecular dynamics simulations to quantify the equilibrium dynamics of the Trp-cage miniprotein and the effects of cooling on its conformational ensemble. By simulating cooling at seven different rates, including the slowest experimental rates that still result in vitrification, we connect the kinetic properties of a proteins conformational state to the change in state population from cooling. We show that cooling-induced population shifts are small but observable. We further introduce a thermodynamic-inference method to recover equilibrium populations from the cooled ensembles.

biophysics↗

bioRxiv2025

Assembly of two functionally-distinct protein import complexes in the outer membrane of plant chloroplasts

The TOC translocon delivers thousands of nucleus-encoded proteins to chloroplasts and related non-photosynthetic plastids. It comprises the {beta}-barrel channel, Toc75, and multiple isoforms of receptor GTPases, Toc33 and Toc159. However, exactly how TOC complexes are assembled in different plastid types is unknown. Here, we present detailed characterization of two distinct TOC complexes, TOC-P and TOC-N, from photosynthetic chloroplasts and non-photosynthetic plastids, respectively. The assembled complexes are distinguished by having different sets of receptors, but both possess Toc75 which we identify as a central hub in TOC biogenesis: assembly is driven by TOC75 expression, with the Toc33 and Toc159 being added sequentially thereafter. Integrative structural analysis revealed a modular architecture for TOC-P comprising a cytosolic GTPase receptor module linked flexibly to a membrane {beta}-barrel channel module. TOC-N has a similar overall architecture, albeit with some clear differences that likely account for observed functional differences related to client specificity.

cell biology↗

bioRxiv2025

Interactions between tick-borne encephalitis virus non-structural protein 1 and blood-brain barrier tight junction proteins: potential clues to strain-specific neuropathogenicity

Tick-borne encephalitis virus (TBEV) invades the central nervous system (CNS) through strain-specific mechanisms that remain poorly understood. In mosquito-borne orthoflaviviruses such as dengue and yellow fever viruses, the non-structural protein 1 (NS1) has been shown to disrupt endothelial barrier integrity by targeting tight junction proteins (TJPs), thereby facilitating viral neuroinvasion. However, comparable mechanisms in TBEV remain largely unexplored. Here, we investigate the potential interaction of NS1 from high (Hypr)- and low (Vs)-pathogenicity TBEV strains to different blood-brain barrier (BBB) TJPs, using AlphaFold3 (AF3) multimer modelling and in vitro binding assays. We find that NS1 from the highly pathogenic strain exhibits higher predicted interactions with multiple TJPs, including two junctional adhesion molecules (JAM-A, JAM-B) and Claudin-10, which are critical component of the paracellular barrier. In contrast, low pathogenic strain Vs interaction was limited to JAM-A and Claudin-5. Experimental validation using recombinant NS1 proteins revealed strain-specific binding profiles: Hypr NS1 displayed high-affinity, saturable direct binding to immobilized JAM-A (KD = 0.271 {micro}g/mL), whereas Vs NS1 showed negligible interaction (KD = 0.000023 {micro}g/mL). No binding to ZO-1, a barrier scaffold lacking an extracellular domain, was observed for either strain. This differential interaction may be modulated by 22 specific amino acid substitutions localized to the Wing and {beta}-ladder domains, which distinguish the highly neurovirulent Hypr strain from the avirulent Vs strain. Notably, despite its weak JAM-A interaction, the Vs strain is associated with slow-progressing infections that can culminate in chronic neurological disease, highlighting the need for further investigation into noncanonical pathways of neuroinvasion. GraphicsO_ST_ABSA graphical illustration of low-and high-pathogenic TBEV mediated compromised blood-brain barrier (BBB)C_ST_ABSThe left panel illustrates the cellular architecture of the BBB, including pericytes, endothelial cells, astrocytes, and blood vessels. The central panel depicts an intact BBB with tight junction proteins (JAM-A, ZO-1, Claudin) maintaining barrier integrity. The right panel shows strain-specific pathogenesis, highlighting a direct interaction between NS1 from the highly pathogenic Hypr strain and JAM-A, which may contribute to differential neuroinvasive outcomes. The schematic was created using BioRender.com. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/669603v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@3e03c2org.highwire.dtl.DTLVardef@168e8f8org.highwire.dtl.DTLVardef@9703f1org.highwire.dtl.DTLVardef@83e24a_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Small siphophage binding to an open state of the LptDE outer membrane lipopolysaccharide translocon

Bacteriophages are bacterial viruses that provide alternatives to small-molecule drugs to combat infections by antibiotic-resistant bacteria. To infect a bacterial host, a phage needs to bind to the bacterial surface via receptor binding proteins (RBPs), which are critical for determining host specificity. For functionally important receptors, the RBP-receptor interaction could be exploited via phage steering, where emerging bacterial resistance due to receptor modification could make bacteria less fit or virulent. Despite this, relatively little is known about RBP-receptor interactions. Here we build on the recent discovery of coliphages that have the outer membrane (OM) lipopolysaccharide translocon LptDE as their terminal receptor and show via cryogenic electron microscopy (cryoEM) that, surprisingly, the RBP of the small siphophage Oekolampad binds to a hitherto unobserved, open state of LptDE. The open lateral gate of LptD is occupied by a {beta}-strand peptide originating from the degraded N-terminal jellyroll domain of LptD, suggesting the possibility of LptD inhibition via peptidomimetics. A structure of LptDE in complex with the superinfection exclusion (SE) protein Rtp45 of the Oekolampad-related phage Rtp shows a mechanism of SE where Rtp45-induced conformational changes in LptD resulting from steric clashes preclude RBP binding. Finally, analysis of spontaneous Oekolampad-resistant E. coli mutants identifies mutations in LptD that abolish the LptDE-RBP interaction in vitro. SDS-EDTA sensitivity assays of the mutants show no major defects in LptDE function, suggesting that phage steering via LptDE might be challenging. SignificanceThe outer membrane (OM) of Gram-negative bacteria is a protective barrier generated by lipopolysaccharide (LPS), a complex glycolipid that makes up the outer leaflet of the OM. Following its synthesis and transport from the inner membrane, LPS is inserted into the OM by LptDE, an OM protein complex essential for most Gram-negative bacteria. LPS insertion requires a hitherto unobserved, open state of LptDE. Here we report the unexpected finding that the receptor binding protein (RBP) of a bacteriophage binds to an open state of LptDE, allowing isolation and visualisation via cryogenic electron microscopy. The lateral gate of the LptDE-RBP complex is occupied by a {beta}-strand peptide, suggesting that the LptDE of pathogenic bacteria could be inhibited by peptidomimetics.

microbiology↗

bioRxiv2025

Allocation of resources among multiple daughter cells

Cell division commonly produces two daughter cells, but there are many exceptions where large cells produce multiple daughters. Multiple fission of some green algae and bacteria, cellularization during embryogenesis of plants and insects, and growth of Ichthyosporeans, Chytrids, and Apicomplexans all provide variations on this theme. In some yeast species, a large multi-nucleate mother cell grows multiple buds (daughters) simultaneously. Here we address how mothers partition growth equally among their buds in the multi-budding yeast Aureobasidium pullulans. Bud growth is directed by actin cable networks that appear to be optimized for even partitioning despite complex cell geometries. Even partitioning does not rely on compensatory mechanisms to adjust bud volumes, but rather stems directly from effective equalization of polarity sites. These results reveal how conserved cell polarity and cytoskeletal networks are adapted to build complex morphologies in fungi.

cell biology↗

bioRxiv2025

Atomistic modelling of lysophospholipids from the Campylobacter jejuni lipidome

Lysophospholipids are an important class of lipids in both prokaryotic and eukaryotic organisms. These lipids typically constitute a very small proportion (<1%) of the bacterial lipidome, but can constitute 20-45% of the Campylobacter jejuni lipidome under stress conditions. It is thus of importance to include these lipids in model C. jejuni membrane simulations for an accurate representation of the lipidic complexity of these systems. Here, we present atomistic models for four lysophospholipids from the C. jejuni lipidome, each derived from existing phospholipid models. Herein we use molecular dynamics simulations to evaluate the ability of these models to reproduce the expected micellar, hexagonal, and lamellar phases at varying levels of hydration. Mixtures of phospholipids and lysophospholipids emulating the C. jejuni lipidome under ideal growth conditions were found to self-assemble into bilayers in solution. The properties of these mixed bilayers were compared to those containing only phospholipids: the presence of the selected lysophospholipids causes a subtle thinning of the bilayer and a reduction in area per lipid, but no significant change in lipid diffusion. We further test the mixed bilayer model running simulations in which a native inner membrane protein is embedded within the bilayer. Finally, we show that lysophospholipids facilitate the formation of pores in the membrane, with lysophospholipid-containing bilayers more susceptible to electroporation than those containing only phospholipids.

biochemistry↗

bioRxiv2024

Macrophage WDFY3, a protector against autoimmunity

Efficient efferocytosis is essential for maintaining homeostasis. Excessive apoptotic cell (AC) death and impaired macrophage efferocytosis lead to autoantigen release and autoantibody production, immune activation, and organ damage. It remains unclear whether these immunogenic autoantigens are the sole cause of increased autoimmunity or if efferocytosis of ACs directly influences macrophage function, impacting their ability to activate T cells and potentially amplifying autoimmune responses. Additionally, it has not been established if enhancing macrophage efferocytosis or modulating macrophage responses to AC engulfment can be protective in autoimmune-like disorders. Our previous work showed WDFY3 is crucial for efficient macrophage efferocytosis. This study reveals that myeloid knockout of Wdfy3 exacerbates autoimmunity in young mice with increased AC burden by systemic injections of ACs and in middle-aged mice developing spontaneous autoimmunity, whereas ectopic overexpression of WDFY3 suppresses autoimmunity in these models. Macrophages, as efferocytes, can activate T cells and the inflammasome upon engulfing ACs, which are suppressed by overexpressing WDFY3. This work uncovered the role of WDFY3 as a protector against autoimmunity by promoting macrophage efferocytosis thus limiting autoantigen production, as well as mitigating T cell activation and inflammasome activation.

immunology↗

bioRxiv2024

Two structurally mobile regions control the conformation and function of metamorphic meiotic HORMAD proteins

Metamorphic HORMA domain proteins (HORMADs) nucleate protein complex formation by refolding their mobile safety belt region to bind short motifs on interactors. Meiotic HORMADs (mHORMADs) bind proteinaceous axial elements to orchestrate complex chromosomal events that underpin fertility, including pairing and recombination between homologous chromosomes. However, the mechanisms supporting the diverse roles of mHORMADs remain unclear. Here, we show that mHORMADs have a second structurally mobile region, the {beta}5-C loop, which controls mHORMAD conformation and function. Molecular dynamics and in vivo approaches show that functional specialisation of C. elegans paralogs HTP-1 and HTP-2 depends on the interplay between their {beta}5-C loop and safety belt. The {beta}5-C loop can interact with the same HORMA core surface as the safety belt, forming a "loop engaged" conformation. The {beta}5-C loop HORMA core interaction is essential for axis loading of HTP-1 and its paralog HTP-3, and is also present in yeast, plant, and mammalian mHORMADs, suggesting that it represents a conserved functional feature of mHORMADs. Our study reveals that mHORMADs have expanded the bimodal folding landscape first identified in Mad2, paving the way to elucidate how non-canonical HORMAD conformations control meiotic chromosome function to ensure fertility.

molecular biology↗

bioRxiv2024

Faster but not sweeter: A model of E. coli Re-level lipopolysaccharide for Martini 3 and a Martini 2 version with accelerated kinetics

Lipopolysaccharide (LPS) is a complex glycolipid molecule that is the main lipidic component of the outer leaflet of the outer membrane of Gram-negative bacteria. It has very limited lateral motion compared to phospholipids, which are more ubiquitous in biological membranes, including in the inner leaflet of the outer membrane of Gram-negative bacteria. The slow-moving nature of LPS can present a hurdle for molecular dynamics simulations given the (pragmatically) accessible timescales to simulations are currently limited to microseconds, during which LPS displays some conformational dynamics, but hardly any lateral diffusion. Thus, it is not feasible to observe phenomena such as insertion of molecules, including antibiotics/antimicrobials directly into the outer membrane from the extracellular side nor to observe LPS dissociating from proteins via molecular dynamics using currently available models at the atomistic and more coarse-grained levels of granularity. Here we present a model of deep rough LPS compatible with the Martini 2 coarse-grained force field with scaled down non-bonded interactions to enable faster diffusion. We show that the faster-diffusing LPS model is able to reproduce the salient biophysical properties of the standard models, but due to its faster lateral motion, molecules are able to penetrate deeper into membranes containing the faster model. We show that faster LPS model is able to reproduce experimentally determined patterns of interaction with outer membrane proteins, while also allowing for LPS to associate/dissociate with proteins within microsecond timescales. We also complete the Martini 3 LPS toolkit for E. coli by presenting a (standard) model of deep rough LPS for this forcefield.

bioinformatics↗

bioRxiv2023

Phospholipid transport to the bacterial outer membrane through an envelope-spanning bridge

The outer membrane of Gram-negative bacteria provides a formidable barrier, essential for both pathogenesis and antimicrobial resistance. Biogenesis of the outer membrane requires the transport of phospholipids across the cell envelope. Recently, YhdP was implicated as a major protagonist in the transport of phospholipids from the inner membrane to the outer membrane however the molecular mechanism of YhdP mediated transport remains elusive. Here, utilising AlphaFold, we observe YhdP to form an elongated assembly of 60 {beta} strands that curve to form a continuous hydrophobic groove. This architecture is consistent with our negative stain electron microscopy data which reveals YhdP to be approximately 250 [A] in length and thus sufficient to span the bacterial cell envelope. Furthermore, molecular dynamics simulations and in vivo bacterial growth assays indicate essential helical regions at the N- and C-termini of YhdP, that may embed into the inner and outer membranes respectively, reinforcing its envelope spanning nature. Our in vivo crosslinking data reveal phosphate-containing substrates captured along the length of the YhdP groove, providing direct evidence that YhdP transports phospholipids. This finding is congruent with our molecular dynamics simulations which demonstrate the propensity for inner membrane lipids to spontaneously enter the groove of YhdP. Collectively, our results support a model in which YhdP bridges the cell envelope, providing a hydrophobic environment for the transport of phospholipids to the outer membrane.

microbiology↗

bioRxiv2023

An ancestral dual function of OmpM as outer membrane tether and nutrient uptake channel in diderm Firmicutes

The outer membrane (OM) in diderm, or Gram-negative, bacteria must be tethered to peptidoglycan (PG) for mechanical stability and to maintain cell morphology. Most diderm phyla from the Terrabacteria group lack well-characterised OM attachment systems such as Brauns lipoprotein, Pal and OmpA, but instead have the OmpM protein. OmpM has a periplasmic region containing an S-layer homology domain and a {beta}-barrel region that is embedded in the OM. Here, we have determined the structure of the most abundant OmpM protein from the diderm firmicute Veillonella parvula by single particle cryogenic electron microscopy (cryo-EM). We reveal an OM portion similar to well-characterised trimeric general porins and a mobile periplasmic PG-binding region. Combining the cryo-EM data with crystal structures, structure predictions and molecular dynamics simulations, we show that the periplasmic region of OmpM likely adopts multiple conformations. Single-channel electrophysiology demonstrates ion conductance properties similar to E. coli OmpF. Finally, we demonstrate via functional assays that the four OmpM paralogues are likely to be the only general porins in V. parvula. Together, our results show that OM tethering and nutrient acquisition are genetically linked in V. parvula (and other diderm Firmicutes), suggesting that concurrent loss of two key OM-related functions, tethering and nutrient uptake, promoted the multiple OM loss events that have been inferred in the Terrabacteria.

biochemistry↗

bioRxiv2023

Activation and substrate specificity of the human P4-ATPase ATP8B1

Asymmetric distribution of phospholipids in eukaryotic membranes is essential for maintaining cell integrity, signaling pathways, and vesicular trafficking. P4-ATPases, also known as flippases, participate in creating and maintaining this asymmetry through active transport of phospholipids from the exoplasmic to the cytosolic leaflet. In this study, we present a total of nine cryo-electron microscopy structures at a resolution ranging from 2.4 to 3.1 [A], along with functional and computational studies of the human flippase ATP8B1-CDC50A complex, describing the autophosphorylation steps from ATP, substrate recognition and occlusion, as well as its regulation by phosphoinositides. Our findings show that the P4-ATPase transport site is filled with water upon phosphorylation from ATP. Additionally, we identify two different autoinhibited states, a closed and an outward-open conformation. Furthermore, we identified and characterized the PI(3,4,5)P3 binding site of ATP8B1 in an electropositive pocket between transmembrane segments 5, 7, 8, and 10. Our study also highlights the structural basis of ATP8B1 broad specificity for lipids and identifies a new transport substrate for P4-ATPases, phosphatidylinositol (PI). We report the critical role of the sn-2 ester bound of glycerophospholipids in substrate recognition by ATP8B1. These findings provide fundamental insights into ATP8B1 regulation, the catalytic cycle, and substrate recognition in P4-ATPases.

biochemistry↗

bioRxiv2022

Energy extraction from air: structural basis of atmospheric hydrogen oxidation

Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival. This recently discovered yet globally significant process regulates the composition of the atmosphere, enhances soil biodiversity, and drives primary production in certain extreme environments. Atmospheric H2 oxidation has been attributed to still uncharacterised members of the [NiFe]-hydrogenase superfamily. However, it is unresolved how these enzymes overcome the extraordinary catalytic challenge of selectively oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2, and how the derived electrons are transferred to the respiratory chain. Here we determined the 1.52 [A] resolution CryoEM structure of the mycobacterial hydrogenase Huc and investigated its mechanism by integrating kinetics, electrochemistry, spectroscopy, mass spectrometry, and molecular dynamics simulations. Purified Huc is an oxygen-insensitive enzyme that couples the oxidation of atmospheric H2 at its large subunit to the hydrogenation of the respiratory electron carrier menaquinone at its small subunit. The enzyme uses a narrow hydrophobic gas channel to selectively bind atmospheric H2 at the expense of O2, while three [3Fe-4S] clusters and their unusual ligation by a D-histidine modulate the electrochemical properties of the enzyme such that atmospheric H2 oxidation is energetically feasible. Huc forms an 833 kDa complex composed of an octamer of catalytic subunits around a membrane-associated central stalk, which extracts and transports menaquinone a remarkable 94 [A] from the membrane, enabling its reduction. These findings provide a mechanistic basis for the biogeochemically and ecologically critical process of atmospheric H2 oxidation. Through the first characterisation of a group 2 [NiFe]-hydrogenase, we also uncover a novel mode of energy coupling dependent on long-range quinone transport and pave way for the development of biocatalysts that oxidize H2 in ambient air.

biochemistry↗

bioRxiv2022

Conformational dynamics and putative substrate extrusion pathways of the N-glycosylated outer membrane factor CmeC from Campylobacter jejuni

The outer membrane factor CmeC of the efflux machinery CmeABC plays an important role in conferring antibiotic and bile resistance to Campylobacter jejuni. Curiously, the protein is N-glycosylated, with the glycans playing a key role in the effective function of this system. In this work we have employed atomistic equilibrium molecular dynamics simulations of CmeC in a representative model of the C. jejuni outer membrane to characterise the dynamics of the protein and its associated glycans. We show that the glycans are more conformationally labile than had previously been thought. The extracellular loops of CmeC visit the open and closed states freely suggesting the absence of a gating mechanism on this side, while the narrow periplasmic entrance remains tightly closed, regulated via coordination to solvated cations. We identify several cation binding sites on the interior surface of the protein. Additionally, we used steered molecular dynamics simulations to elucidate translocation pathways for a bile acid and a macrolide antibiotic. These, and additional equilibrium simulations suggest that the anionic bile acid utilises multivalent cations to climb the ladder of acidic residues that line the interior surface of the protein.

biophysics↗

bioRxiv2022

Impact on S. aureus and E. coli membranes of treatment with chlorhexidine and alcohol solutions: insights from molecular simulations and nuclear magnetic resonance.

Membranes form the first line of defence of bacteria against potentially harmful molecules in the surrounding environment. Understanding the protective properties of these membranes represents an important step towards development of targeted anti-bacterial agents such as sanitizers. Use of propanol, isopropanol and chlorhexidine can significantly decrease the threat imposed by bacteria in the face of growing anti-bacterial resistance via mechanisms that include membrane disruption. Here we have employed molecular dynamics simulations and nuclear magnetic resonance to explore the impact of chlorhexidine and alcohol on the S. aureus cell membrane, as well as the E. coli inner and outer membranes. We identify how sanitizer components partition into these bacterial membranes, and show that chlorhexidine is instrumental in this process.

biophysics↗