Biology subjects

Coupland, C. E.

Publications and source records attributed to Coupland, C. E..

7 recordsLinked to original sources

bioRxiv2024

Cryo-EM of native membranes reveals an intimate connection between the Krebs cycle and respiration in mycobacteria

Imaging of endogenous protein complexes in their native membranes can reveal protein-protein interactions that are lost upon detergent solubilization. To investigate interactions in the mycobacterial oxidative phosphorylation machinery, we prepared inverted membrane vesicles from Mycobacterium smegmatis and enriched for vesicles containing complexes of interest by affinity chromatography. Electron cryomicroscopy (cryo-EM) of these vesicles revealed that malate-quinone oxidoreductase (Mqo), an enzyme from the Krebs cycle, physically associates with the electron transport chain Complex III2IV2 (CIII2CIV2) supercomplex. Analysis of the Mqo:CIII2CIV2 interaction shows that CIII2CIV2 is necessary for malate-driven, but not NADH- driven, electron transport chain activity and oxygen consumption. Further, the association of Mqo with CIII2CIV2 enables electron transfer from malate to CIII2CIV2 with millisecond kinetics. Together, these findings indicate a connection between the Krebs cycle and respiration that directs electrons along a single branch of the mycobacterial electron transport chain.

biochemistry↗

bioRxiv2024

Vesicle Picker: A tool for efficient identification of membrane protein complexes in vesicles

Electron cryomicroscopy (cryo-EM) has recently allowed determination of near-atomic resolution structures of membrane proteins and protein complexes embedded in lipid vesicles. However, particle selection from electron micrographs of these vesicles can be challenging due to the strong signal contributed from the lipid bilayer. This challenge often requires iterative and laborious particle selection workflows to generate a dataset of high-quality particle images for subsequent analysis. Here we present Vesicle Picker, an open-source program built on the Segment Anything model. Vesicle Picker enables automatic identification of vesicles in cryo-EM micrographs with high recall and precision. It then exhaustively selects all potential particle locations, either at the perimeter or uniformly over the surface of the projection of the vesicle. The program is designed to interface with cryoSPARC, which performs both upstream micrograph processing and downstream single particle image analysis. We demonstrate Vesicle Pickers utility by determining a high-resolution map of the vacuolar-type ATPase from micrographs of native synaptic vesicles (SVs) and identifying an additional protein or protein complex in the SV membrane.

biophysics↗

bioRxiv2024

High resolution cryo-EM of V-ATPase in native synaptic vesicles

Neurotransmitter release into the synaptic cleft between neurons enables the intercellular communication central to the function of the brain. In the presynaptic neuron, the proton pumping vesicular- or vacuolar-type ATPase (V-ATPase) powers neurotransmitter loading into synaptic vesicles (SVs), with the V1 complex dissociating from the membrane region of the enzyme before exocytosis. We isolated SVs from rat brain using SidK, a V-ATPase-binding bacterial effector protein. Single particle electron cryomicroscopy of the vesicles allowed high-resolution structure determination of V-ATPase within the native SV membrane. In the structure, regularly spaced cholesterol molecules decorate the enzymes rotor and the abundant SV protein synaptophysin binds the complex stoichiometrically. Conditions where ATP hydrolysis drives glutamate loading result in loss of V1 from the SV membrane, suggesting that SV loading is sufficient to induce V-ATPase dissociation.

biochemistry↗

bioRxiv2023

Mapping Structural and Dynamic Divergence Across the MBOAT family

Membrane Bound O-acyltransferases (MBOATs) are membrane embedded enzymes which catalyse acyl chain transfer to a diverse group of substrates including lipids, small-molecules and proteins. Recent MBOAT structures reveal a conserved structural core, despite wide-ranging functional specificity across both prokaryotes and eukaryotes. The structural basis of catalytic specificity, regulation and interactions with the surrounding environment remain uncertain, hindering effective therapeutic targeting. Here, we combine comparative molecular dynamics (MD) simulations with bioinformatics to assess molecular and interactional divergence across the family. In simulations, MBOATs differentially distort the bilayer depending on their substrate type. Additionally, we identify specific lipid binding sites surrounding MBOAT reactant gates into the surrounding membrane. We use bioinformatics to reveal a conserved role for re-entrant loop-2 in stabilisation of the MBOAT fold and identify a key hydrogen bond involved in DGAT1 dimerisation. Finally, we predict differences in MBOAT core solvation and water gating properties across the family. These data are pertinent to the design of MBOAT specific inhibitors that encompass dynamic information within cellular mimetic environments.

biochemistry↗

bioRxiv2022

LipIDens: Simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins.

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. The LipIDens code is open-source and embedded within a notebook format to assist automation and usability.

biophysics↗

bioRxiv2021

Structure and Mechanism of Hedgehog Acyl Transferase

The iconic Sonic Hedgehog (SHH) morphogen pathway is a fundamental orchestrator of embryonic development and stem cell maintenance, and is implicated in cancers in various organs. A key step in signalling is transfer of a palmitate group to the N-terminal cysteine residue of SHH, catalysed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT) resident in the endoplasmic reticulum (ER). Here, we present the high-resolution cryo-EM structure of HHAT bound to substrate analogue palmityl-coenzyme A and a SHH mimetic megabody. Surprisingly, we identified a heme group bound to an HHAT cysteine residue and show that this modification is essential for HHAT structure and function. A structure of HHAT bound to potent small molecule inhibitor IMP-1575 revealed conformational changes in the active site which occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the novel mechanism by which HHAT adapts the membrane environment to transfer a long chain fatty acid across the ER membrane from cytosolic acyl-CoA to a luminal protein substrate. This structure of a member of the protein-substrate membrane-bound O-acyltransferase (MBOAT) superfamily provides a blueprint for other protein substrate MBOATs, such as WNT morphogen acyltransferase Porcupine and ghrelin O-acyltransferase GOAT, and a template for future drug discovery.

biophysics↗

bioRxiv2020

Photochemical probe identification of the small-molecule binding site in a mammalian membrane-bound O-acyltransferase

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports development of a photochemical probe to interrogate the small-molecule binding site in the human MBOAT Hedgehog acyltransferase (HHAT) based on HHAT inhibitor RUSKI-201. Structure-activity relationship investigation identified the improved enantiomeric inhibitor IMP-1575, which is the most potent HHAT inhibitor reported to-date, and guided rational design of a photocrosslinking probe that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed via kinetic analysis. Our results provide an optimal HHAT inhibitor IMP-1575 (Ki = 38 nM) and a strategy for mapping of interaction sites in MBOATs.

biochemistry↗