Biology subjects

Stansfeld, P. J.

Publications and source records attributed to Stansfeld, P. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Native mass spectrometry of membrane protein lipid and ligand complexes using de novo designed WRAP domains

Membrane proteins engage in dynamic transient interactions with surrounding lipids along with their substrates. Capturing these concomitant contacts often remains a challenging problem. Here, we demonstrate that generative deep learning-designed WRAP domains can preserve weak interactions of membrane protein complexes in native mass spectrometry. Using WRAP-fused GlpG, AqpM, and OmpA as model systems, we show that lipid interactions can be retained and characterized without detergent micelles. We use the approach to show that WRAP-OmpA selectively binds phosphatidylethanolamine lipids within cavities formed at the protein-WRAP interface, while simultaneously accommodating weak chitobiose ligand binding. These findings establish WRAPs as versatile vehicles to probe ligand and lipid interactions of membrane proteins.

biophysics↗

bioRxiv2026

Cardiolipin modulation of MmpL3 in mycobacteria

The complex cell envelope of mycobacteria is characterized by the presence of a unique outer membrane (OM) rich in mycolic acids (MAs). These long-chain, branched fatty acids confer extreme hydrophobicity to the OM, in part rendering the mycobacterial envelope impermeable to antibiotics and host defences. How MAs are transported from the inner membrane (IM) to the OM is largely unknown. The integral membrane protein MmpL3 plays an essential role in this process, but the mechanism(s) by which it exploits the proton motive force to flip and/or release MAs at the IM, in the form of trehalose monomycolates (TMMs), remains elusive. Here, we reconstitute and quantify the proton translocation activity of MmpL3 in artificial lipid bilayers, and discover a novel role for the phospholipid species, cardiolipin (CL), in regulating MmpL3 function. We find that mutations in conserved residues, or binding of known inhibitors in the central channel of MmpL3 do not diminish proton translocation activity. Instead, the specific presence of CL abolishes proton translocation by MmpL3 in vitro. Furthermore, we establish that an MmpL3 variant containing substitutions in a CL-binding site predicted in silico is no longer modulated by CL in vitro, and is unable to support growth of Mycobacterium smegmatis. Our work provides previously unappreciated insights into lipid regulation of MmpL3 activity in mycobacteria, and expands the guiding principles for the development of anti-mycobacterial inhibitors targeting this essential transporter. SignificanceMycobacterial species, including the human pathogen Mycobacterium tuberculosis, are surrounded by a double-membrane cell envelope that makes them intrinsically resistant to many antibiotics. Specifically, the outer membrane (OM) contains unique lipids called mycolic acids (MAs), whose transport pathway across the envelope is poorly understood. In this study, we characterized the biochemical activity of the essential MA transporter MmpL3, and uncovered a novel mechanism for lipid-mediated functional regulation. Our work highlights the importance of protein-lipid interactions in defining transporter activity, provides insights into MA transport and OM assembly in mycobacteria, and sets the stage for the development of anti-mycobacterial strategies.

biochemistry↗

bioRxiv2025

Structure and substrate recognition by the Twin-arginine translocation (Tat) pathway core complex

The twin-arginine translocation (Tat) system is a mechanistically unique protein transport pathway moving folded proteins across membranes (1-4). It is found in all domains of life and is essential for plant photosynthesis and bacterial virulence(2, 5-7). A core complex composed of membrane proteins TatA, TatB, and TatC binds substrate proteins and then recruits additional TatA protomers to form the transport site(8-10). Here we present structures of resting and substrate-bound states of the Tat core complex together with biochemical and functional analysis. Substrate proteins associate with the core complex solely through their N-terminal signal peptides with their Tat targeting sequences making specific contacts with TatC and the peptide body clamped by TatB. The core complex contains highly tilted transmembrane helices that drive extreme local membrane thinning. Based on our results we propose a model for the early steps in Tat transport.

biochemistry↗

bioRxiv2025

Lipid interactions are important for the Tol-Pal complex in maintaining outer membrane lipid homeostasis

Gram-negative bacteria are intrinsically resistant to many antibiotics in part due to the asymmetric architecture and barrier function of their outer membrane (OM). To establish proper lipid asymmetry, cells need to ensure an intricate balance of constituent OM components, especially lipids. In this regard, the conserved, trans-envelope Tol-Pal complex plays a primary role in maintaining OM lipid homeostasis, thus OM integrity, possibly via retrograde phospholipid transport. However, mechanistic details for this process are unknown, owing to the lack of evidence for direct lipid binding. In this study, we discover that the periplasmic protein TolB, a key component of the Tol-Pal system, associates directly with membranes in vitro, via specific interactions with cardiolipin (CL). Using coarse-grained molecular dynamics simulations, we identify a CL-binding site on TolB; a single amino acid mutation at this site abolishes in vitro membrane interaction, consequently impairing cellular Tol-Pal function in maintaining OM homeostasis in Escherichia coli. Curiously, we find that the functional requirement for TolB-CL interactions can be partially bypassed in cells lacking CL, suggesting compensatory effects through other lipids only when CL is absent. Our findings reveal a previously unappreciated lipid-binding role for TolB, and provide novel insights into how the Tol-Pal complex may facilitate phospholipid transport across the cell envelope. Our work will inform future strategies towards developing new antibiotics against Gram-negative bacteria.

biochemistry↗

bioRxiv2025

Insights from aquaporin structures into drug-resistant sleeping sickness

Trypanosoma brucei is the causal agent of African trypanosomiasis in humans and animals, the latter resulting in significant negative economic impacts in afflicted areas of the world. Resistance has arisen to the trypanocidal drugs pentamidine and melarsoprol through mutations in the aquaglyceroporin TbAQP2 that prevent their uptake. Here we use cryogenic electron microscopy to determine the structure of TbAQP2 from Trypanosoma brucei, bound to either the substrate glycerol or to the sleeping sickness drugs, pentamidine or melarsoprol. The drugs bind within the AQP2 channel at a site completely overlapping that of glycerol. Mutations leading to a drug-resistant phenotype were found in the channel lining. Molecular dynamics simulations showed the channel can be traversed by pentamidine, with a low energy binding site at the centre of the channel, flanked by regions of high energy association at the extracellular and intracellular ends. Drug-resistant TbAQP2 mutants are still predicted to bind pentamidine, but the much weaker binding in the centre of the channel observed in the MD simulations would be insufficient to compensate for the high energy processes of ingress and egress, hence impairing transport at pharmacologically relevant concentrations. The structures of drug-bound TbAQP2 represent a novel paradigm for drug-transporter interactions that could provide new mechanisms for targeting drugs into pathogens and human cells.

molecular biology↗

bioRxiv2025

Structure of YbbAP-TesA: a Type VII ABC transporter lipid-hydrolase complex

Type VII ABC transporters are ATP-powered membrane protein complexes that drive key biological processes in the bacterial cell envelope. In E. coli, three of the four Type VII ABC systems have been extensively characterised including: the FtsEX-EnvC cell division complex, the LolCDE-LolA lipoprotein trafficking machinery and the MacAB-TolC efflux pump. Here we describe a fourth E. coli Type VII ABC system, YbbAP-TesA, which combines a Type VII ABC transporter with a multifunctional hydrolytic enzyme. Structures of the complete YbbAP-TesA complex, and of YbbAP with and without bound ATP analogues, capture the long-range transmembrane conformational changes that are the hallmark of this ABC superfamilys mechanotransmission mechanism. We further show that YbbAP-TesA can hydrolyse a variety of ester and thioester substrates and experimentally confirm a constellation of active site residues in TesA. Our data suggests YbbAP has a role in extracting hydrophobic molecules from the inner membrane and presenting these to TesA for hydrolysis. The work extends collective knowledge of the remarkable diversity of the ABC superfamily and establishes a new function for Type VII ABC transporters in bacterial cells. Significance StatementType VII ABC transporters are unique to bacteria and play important roles in bacterial physiology including cell division, antibiotic resistance, siderophore secretion, antibiotic sensing, toxin secretion, biofilm regulation and lipoprotein trafficking. Here we identify a complex that combines an atypical Type VII ABC transporter (YbbAP) with a periplasmic lipid hydrolase (TesA). The YbbAP-TesA complex is structurally and functionally distinct from all known Type VII ABC transporter systems including FtsEX-EnvC, LolCDE-LolA, BceAB-BceS and MacAB-TolC. Structures suggest that YbbAP-TesA uses ATP-driven mechanotransmission to extract substrates from the inner membrane and hydrolyse them in the periplasmic space. The discovery and characterisation of YbbAP-TesA highlights the structural and functional diversity of Type VII ABC transporter complexes and suggests a new function for these proteins in the bacterial cell envelope.

microbiology↗

bioRxiv2025

Computational modelling of toroidal membranes at division and fission sites

The formation of specialised membrane architectures is fundamental to biological processes. Computational modelling of membrane structures offers a means to unravel complex molecular mechanisms that remain inaccessible through in vitro or in vivo approaches. Among these architectures, toroidal membranes, otherwise known as fusion pores or fission pores, occur during the final stages of cell division, exocytosis, endocytosis, and membrane fission; including during the phagocytic-like engulfment of bacterial endospores. Here we have designed MemTorMD, a computational simulation pipeline based on the TS2CG methodology, to enable coarse-grained modelling of free toroidal membranes with biologically-relevant dimensions. This approach allowed us to determine the lipid properties required for stable toroidal membrane formation, providing a biologically accurate simulation surface. Furthermore, we demonstrate the applicability of this approach to investigate the function of proteins that localise at toroidal membranes, and drive molecular events such as membrane fission by a prokaryotic fission protein. Collectively, our work provides insight into membrane and protein behaviour at toroidal membranes and provides a platform for the development of more complex simulations involving additional molecular components at membrane toroids present in a range of processes across life.

microbiology↗

bioRxiv2025

Structural Basis of Lipopolysaccharide Assembly by the Outer Membrane Translocon Holo-Complex

Lipopolysaccharide (LPS) assembly at the surfaces-exposed leaflet of the bacterial outer membrane (OM) is mediated by the OM LPS translocon. An essential transmembrane {beta}-barrel protein, LptD, and a cognate lipoprotein, LptE, translocate LPS selectively into the OM external leaflet via a poorly understood mechanism. Here, we characterize two additional translocon subunits, the lipoproteins LptM and LptY (formerly YedD). We use single-particle cryo-EM analysis, functional assays and molecular dynamics simulations to visualize the roles of LptM and LptY at the translocon holo-complex LptDEMY, uncovering their impact on LptD conformational dynamics. Whereas LptY binds and stabilizes the periplasmic LptD {beta}-taco domain that functions as LPS receptor, LptM intercalates the lateral gate of the {beta}-barrel domain, promoting its opening and access by LPS. Remarkably, we demonstrate a conformational switch of the LptD {beta}-taco/{beta}-barrel interface alternating between contracted and extended states. The LptD {beta}-strand 1, which defines the mobile side of the lateral gate, binds LPS and performs a stroke movement toward the external leaflet during the contracted-to-extended state transition. Our findings establish a detailed mechanistic framework explaining the selective transport of LPS to the membrane external leaflet.

microbiology↗

bioRxiv2025

Structural basis of undecaprenyl phosphate glycosylation leading to polymyxin resistance in Gram-negative bacteria

In Gram-negative bacteria, the enzymatic modification of Lipid A with aminoarabinose (L-Ara4N) leads to resistance against polymyxin antibiotics and cationic antimicrobial peptides. ArnC, an integral membrane glycosyltransferase, attaches a formylated form of aminoarabinose to the lipid undecaprenyl phosphate, enabling its association with the bacterial inner membrane. Here, we present cryo-electron microscopy structures of ArnC from S. enterica in apo and nucleotide-bound conformations. These structures reveal a conformational transition that takes place upon binding of the partial donor substrate. Using coarse-grained and atomistic simulations, we provide insights into substrate coordination before and during catalysis, and we propose a catalytic mechanism that may operate on all similar metal-dependent polyprenyl phosphate glycosyltransferases. The reported structures provide a new target for drug design aiming to combat polymyxin resistance.

biochemistry↗

bioRxiv2025

Conformational Dynamics and Membrane Insertion Mechanism of B4GALNT1 in Ganglioside Synthesis

Glycosphingolipids (GSLs) are crucial membrane components involved in essential cellular pathways. Complex GSLs, known as gangliosides, are synthesised in the ER/Golgi by a series of glycosyltransferase enzymes. Imbalances in GSL metabolism can lead to severe diseases, often affecting the nervous system. B4GALNT1 is a key enzyme in the ganglioside synthetic pathway, synthesising complex gangliosides including GM2 and GD2 from GM3 and GD3. These products are precursors to the major brain gangliosides. Loss of B4GALNT1 function causes hereditary spastic paraplegia 26 (HSP26), while its overexpression is linked to cancers including childhood neuroblastoma. Here, we present crystal structures of the homodimeric B4GALNT1 enzyme demonstrating conformational changes upon binding of donor substrate ligands and product. These structures support a catalytic mechanism that involves dynamic remodelling of the substrate binding site during catalysis. We also demonstrate that processing of lipid substrates by B4GALNT1 is severely compromised when surface loops flanking the active site are mutated from hydrophobic residues to polar. Molecular dynamics simulations support that these loops can insert into the lipid bilayer explaining how B4GALNT1 accesses and processes lipid substrates. By combining structure prediction and molecular simulations we propose that this mechanism of dynamic membrane insertion is exploited by other, structurally distinct GSL synthesising enzymes.

biochemistry↗

bioRxiv2024

Structural insights into terminal arabinosylation biosynthesis of the mycobacterial cell wall arabinan

The emergence of drug-resistant strains exacerbates the global challenge of tuberculosis caused by Mycobacterium tuberculosis (Mtb). Central to the pathogenicity of Mtb is its complex cell envelope, which serves as a barrier against both immune system and pharmacological attacks. Two key components of this envelope, arabinogalactan (AG) and lipoarabinomannan (LAM) are complex polysaccharides that contain integral arabinan domains important for cell wall structural and functional integrity. The arabinofuranosyltransferase AftB terminates the synthesis of these arabinan domains by catalyzing the addition of {beta}-(1[->]2)-linked terminal arabinofuranose residues. Here, we present the cryo-EM structures of Mycobacterium chubuense AftB in its apo and donor substrate analog-bound form, determined to 2.9 [A] and 3.4 [A] resolution, respectively. Our structures reveal that AftB has a GT-C fold transmembrane (TM) domain comprised of eleven TM helices and a periplasmic cap domain. AftB has an irregular tube-shaped cavity that bridges the two proposed substrate binding sites. By integrating structural analysis, biochemical assays, and molecular dynamics simulations, we elucidate the molecular basis of the reaction mechanism of AftB and propose a model for catalysis.

biochemistry↗

bioRxiv2024

Mechanistic studies of mycobacterial glycolipid biosynthesis by the mannosyltransferase PimE

Tuberculosis (TB), exceeded in mortality only by COVID-19 among global infectious diseases, is caused by Mycobacterium tuberculosis (Mtb). The pathogenicity of Mtb is largely attributed to its complex cell envelope, which includes a class of glycolipids called phosphatidyl-myo-inositol mannosides (PIMs), found uniquely in mycobacteria and its related corynebacterineae. These glycolipids maintain the integrity of the mycobacterial cell envelope, regulate its permeability, and mediate host-pathogen interactions. PIMs consist of a phosphatidyl-myo-inositol core decorated with one to six mannose residues and up to four acyl chains. The mannosyltransferase PimE catalyzes the transfer of the fifth PIM mannose residue from a polyprenyl phosphate-mannose (PPM) donor. This step in the biosynthesis of higher-order PIMs contributes to the proper assembly and function of the mycobacterial cell envelope; however, the structural basis for substrate recognition and the catalytic mechanism of PimE remain poorly understood. Here, we present the cryo-electron microscopy (cryo-EM) structures of PimE from Mycobacterium abscessus captured in its apo form and in a product-bound complex with the reaction product Ac1PIM5 and the by-product polyprenyl phosphate (PP), determined at 3.0 [A] and 3.5 [A], respectively. The structures reveal the active site within a distinctive binding cavity that accommodates both donor and acceptor substrates/products. Within the cavity, we identified residues involved in substrate coordination and catalysis, which we confirmed through in vitro enzymatic assays and further validated by in vivo complementation experiments. Molecular dynamics simulations were applied to identify the access pathways and the dynamics involved in substrate binding. Integrating structural, biochemical, genetic, and computational experiments, our study provides comprehensive insights into how PimE functions, opening potential avenues for development of novel anti-TB therapeutics.

biochemistry↗

bioRxiv2024

The partitioning of fatty acids between membrane and storage lipids controls ER membrane expansion.

The biogenesis of membrane-bound organelles involves the synthesis, remodelling and degradation of their constituent phospholipids. How these pathways regulate organelle size, remains still poorly understood. Here we demonstrate that a lipid degradation pathway inhibits the expansion of the endoplasmic reticulum (ER) membrane. Phospholipid diacylglycerol acyltransferases (PDATs) use endogenous phospholipids as fatty acyl donors to generate triglyceride stored in lipid droplets. The significance of this non-canonical triglyceride biosynthetic pathway has remained elusive. We find that the activity of the yeast PDAT Lro1 is regulated by a membrane- proximal domain facing the luminal side of the ER bilayer. To reveal the biological roles of PDATs, we engineered an Lro1 variant with derepressed activity. We show that active Lro1 mediates the retraction of ER membrane expansion driven by phospholipid synthesis. Furthermore, the subcellular distribution and membrane turnover activity of Lro1 are controlled by diacylglycerol, produced by the activity of Pah1, a conserved member of the lipin family. Collectively, our findings reveal a lipid metabolic network that regulates endoplasmic reticulum biogenesis by converting phospholipids into storage lipids.

cell biology↗

bioRxiv2024

Engineering cardiolipin binding to an artificial membrane protein

Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the E. coli rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.

biochemistry↗

bioRxiv2023

A feedback control mechanism governs the synthesis of lipid-linked precursors of the bacterial cell wall

Many bacterial surface glycans such as the peptidoglycan (PG) cell wall, O-antigens, and capsules are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting lipid carrier is effectively distributed among competing pathways has remained unclear for some time. Here, we describe the isolation and characterization of hyperactive variants of Pseudomonas aeruginosa MraY, the essential and conserved enzyme catalyzing the formation of the first lipid-linked PG precursor called lipid I. These variants result in the elevated production of the final PG precursor lipid II in cells and are hyperactive in a purified system. Amino acid substitutions within the activated MraY variants unexpectedly map to a cavity on the extracellular side of the dimer interface, far from the active site. Our structural evidence and molecular dynamics simulations suggest that the cavity is a binding site for lipid II molecules that have been transported to the outer leaflet of the membrane. Overall, our results support a model in which excess externalized lipid II allosterically inhibits MraY, providing a feedback mechanism to prevent the sequestration of lipid carrier in the PG biogenesis pathway. MraY belongs to the broadly distributed polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase (PNPT) superfamily of enzymes. We therefore propose that similar feedback mechanisms may be widely employed to coordinate precursor supply with demand by polymerases, thereby optimizing the partitioning of lipid carriers between competing glycan biogenesis pathways.

microbiology↗

bioRxiv2023

A one-track model for spatiotemporal coordination of Bacillus subtilis septal cell wall synthesis

Bacterial cell division requires synthesis of a septal peptidoglycan (sPG) wall across the middle of the cell. This is accomplished by the divisome synthesis complex in coordination with numerous other division proteins--such as the essential tubulin homolog FtsZ--but the molecular mechanism of its spatiotemporal regulation remains unclear. Here, we investigate the dynamics of sPG synthesis in the model Gram-positive bacterium Bacillus subtilis using live-cell single-molecule imaging of the divisome transpeptidase PBP2B. In contrast to previous models for division, we show that there is a single population of processively-moving PBP2B molecules whose motion is driven by peptidoglycan synthesis and is not associated with FtsZ treadmilling. However, although the motions of PBP2B and FtsZ are asynchronous, we demonstrate that processive PBP2B motion is partially dependent on FtsZ treadmilling. Additionally, we provide evidence that the divisome synthesis complex is multimeric. Our results support a new model for division in B. subtilis where a multimeric synthesis complex follows a single track dependent on sPG synthesis whose activity and dynamics are asynchronous with FtsZ treadmilling.

microbiology↗

bioRxiv2023

Cyclic di-AMP traps proton-coupled K+ transporters of the KUP 1 family in an inward-occluded conformation

Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K+/H+ symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating K+ ions uptake. However, at elevated intracellular K+ concentrations, further K+ accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K+ binding site and therefore traps KimA in an inward-occluded conformation.

biophysics↗

bioRxiv2023

LptM promotes oxidative maturation of the lipopolysaccharide translocon by substrate binding mimicry.

Insertion of lipopolysaccharide (LPS) into the outer membrane (OM) of Gram-negative bacteria is mediated by a druggable OM translocon consisting of a {beta}-barrel membrane protein, LptD, and a lipoprotein, LptE. The {beta}-barrel assembly machinery (BAM) assembles LptD together with LptE to form a plug-and-barrel structure. In the enterobacterium Escherichia coli, formation of two native disulfide bonds in LptD controls LPS translocon activation. Here we report the discovery of LptM (formerly YifL), a conserved lipoprotein that assembles together with LptD and LptE at the BAM complex. We demonstrate that LptM stabilizes a conformation of LptD that can efficiently acquire native disulfide bonds and be released as mature LPS translocon by the BAM complex. Inactivation of LptM causes the accumulation of non-natively oxidized LptD, making disulfide bond isomerization by DsbC become essential for viability. Our structural prediction and biochemical analyses indicate that LptM binds to sites in both LptD and LptE that are proposed to coordinate LPS insertion into the OM. These results suggest that LptM facilitates oxidative maturation of LptD by mimicking LPS binding, thereby activating the LPS translocon.

microbiology↗