Biology subjects

Corey, R. A.

Publications and source records attributed to Corey, R. A..

35 records · Page 2Linked to original sources

bioRxiv2021

Relative Affinities of Protein-Cholesterol Interactions from Equilibrium Molecular Dynamics Simulations

Specific interactions of lipids with membrane proteins contribute to protein stability and function. Multiple lipid interactions surrounding a membrane protein are often identified in molecular dynamics (MD) simulations and are, increasingly, resolved in cryo-EM densities. Determining the relative importance of specific interaction sites is aided by determination of lipid binding affinities by experimental or simulation methods. Here, we develop a method for determining protein-lipid binding affinities from equilibrium coarse-grained MD simulations using binding saturation curves, designed to mimic experimental protocols. We apply this method to directly obtain affinities for cholesterol binding to multiple sites on a range of membrane proteins and compare our results with free energies obtained from density-based equilibrium methods and with potential of mean force calculations, getting good agreement with respect to the ranking of affinities for different sites. Thus, our binding saturation method provides a robust, high-throughput alternative for determining the relative consequence of individual sites seen in e.g. cryo-EM derived membrane protein structures surrounded by a plethora of ancillary lipid densities.

biophysics↗

bioRxiv2021

Deciphering ion transport and ATPase coupling in the intersubunit tunnel of KdpFABC

KdpFABC, a high-affinity K+ pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K+ limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD simulations to illuminate the mechanisms underlying transport and the coupling to ATP hydrolysis. We unambiguously show that ions are transported via an intersubunit tunnel through KdpA and KdpB. At the subunit interface, the tunnel is constricted by a phenylalanine, which, by polarized cation-{pi} stacking, controls K+ entry into the canonical substrate binding site (CBS) of KdpB. Within the CBS, ATPase coupling is mediated by the charge distribution between an aspartate and a lysine. Interestingly, individual elements of the ion translocation mechanism of KdpFABC identified here are conserved among a wide variety of P-type ATPases from different families. This leads us to the hypothesis that KdpB might represent an early descendant of a common ancestor of cation pumps.

biophysics↗

bioRxiv2021

Structure of the hexameric fungal plasma membrane proton pump in its auto-inhibited state

The fungal plasma membrane H+-ATPase Pma1 is a vital enzyme, generating a proton-motive force that drives the import of essential nutrients. Auto-inhibited Pma1 hexamers in starving fungi are activated by glucose signalling resulting in phosphorylation of the auto-inhibitory domain. As related P-type ATPases are not known to oligomerise, the physiological relevance of Pma1 hexamers remains unknown. We have determined the structure of hexameric Pma1 from Neurospora crassa by cryo-EM at 3.3 [A] resolution, elucidating the molecular basis for hexamer formation and auto-inhibition, and providing a basis for structure-based drug development. Coarse-grained molecular dynamics simulations in a lipid bilayer suggest lipid-mediated contacts between monomers and a substantial protein-induced membrane deformation that could act as a proton-attracting funnel.

biochemistry↗

bioRxiv2021

Identification and assessment of cardiolipin interactions with E. coli inner membrane proteins

Integral membrane proteins are localised and/or regulated by lipids present in the surrounding bilayer. Whilst bacteria such as E. coli have relatively simple membranes when compared to eukaryotic cells, there is ample evidence that many bacterial proteins bind to specific lipids, especially the anionic lipid cardiolipin. Here, we apply molecular dynamics simulations to assess lipid binding to 42 different E. coli inner membrane proteins. Our data reveals a strong asymmetry between the membrane leaflets, with a marked increase of anionic lipid binding to the inner leaflet regions of membrane proteins, particularly for cardiolipin. From our simulations we identify over 700 independent cardiolipin binding sites, allowing us to identify the molecular basis of a prototypical cardiolipin binding site, which we validate against structures of bacterial proteins bound to cardiolipin. This allows us to construct a set of metrics for defining a high affinity cardiolipin binding site on (bacterial) membrane proteins, paving the way for a heuristic approach to defining more complex protein-lipid interactions.

biochemistry↗

bioRxiv2021

Fentanyl binds to the mu-opioid receptor via the lipid membrane and transmembrane helices

Overdose deaths from synthetic opioids, such as fentanyl, have reached epidemic proportions in the USA and are increasing worldwide. Fentanyl is a potent opioid agonist, that is less well reversed by naloxone than morphine. Due to fentanyls high lipophilicity and elongated structure we hypothesised that its unusual pharmacology may be explained by a novel binding mode to the -opioid receptor (MOPr). By employing coarse-grained molecular dynamics simulations and free energy calculations, we determined the routes by which fentanyl and morphine access the orthosteric pocket of MOPr. Morphine accesses MOPr via the aqueous pathway; first binding to an extracellular vestibule, then diffusing into the orthosteric pocket. In contrast, fentanyl takes a novel route; first partitioning into the membrane, before accessing the orthosteric site by diffusing through a ligand-induced gap between the transmembrane helices. This novel lipophilic route may explain the high potency and lower susceptibility of fentanyl to reversal by naloxone.

pharmacology and toxicology↗

bioRxiv2021

Maintenance of Complex I and respiratory super-complexes by NDUF-11 is essential for respiratory function, mitochondrial structure and health in C. elegans

Mitochondrial super-complexes form around a conserved core of monomeric complex I and dimeric complex III; wherein subunit NDUFA11, of the former, is conspicuously situated at the interface. We identified B0491.5 (NDUF-11) as the C. elegans homologue, of which animals homozygous for a CRISPR-Cas9 generated knockout allele arrested at the L2 development stage. Reducing expression by RNAi allowed development to the adult stage, enabling characterisation of the consequences: destabilisation of complex I and its super-complexes, and perturbation of respiratory function. The loss of NADH-dehydrogenase activity is compensated by enhanced complex II activity, resulting in excessive detrimental ROS production. Meanwhile, electron cryo-tomography highlight aberrant cristae morphology and widening of the inter-membrane space and cristae junctions. The requirement of NDUF-11 for balanced respiration, mitochondrial morphology and development highlights the importance of complex I/ super-complex maintenance. Their perturbation by this, or other means, is likely to be the cause of metabolic stress and disease.

molecular biology↗

bioRxiv2020

Evaluating Inositol phospholipid interactions with Inward Rectifier Potassium Channels and characterising their role in Disease

Membrane proteins are frequently modulated by specific protein-lipid interactions. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised. Here, we apply a coarse-grained molecular dynamics free-energy perturbation (CG-FEP) protocol to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step approach, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid. Furthermore, by perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K increases PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. We show good agreement with electrophysiological data where E179K exhibits a reduction in neomycin sensitivity, implying that PIP2 binds more tightly E179K channels. This illustrates the application of CG-FEP to compare affinities between lipid species, and for annotating amino acid residues.

biochemistry↗

bioRxiv2020

The SERCA residue Glu340 mediates inter-domain communication that guides Ca2+ transport

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+ from the cytosol into the SR/ER lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+ binding sites and of the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of five out of SERCAs ten transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+ and ATP analogue reveals a rotated headpiece, altered connectivity between the cytosolic domains and altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in inter-domain communication between the headpiece and the Ca2+-binding transmembrane region.

molecular biology↗

bioRxiv2019

Energy landscape steering in SecYEG mediates dynamic coupling in ATP driven protein translocation

The Sec translocon is a highly conserved membrane complex for transport of polypeptides across, or into, lipid bilayers. In bacteria, the core protein-channel SecYEG resides in the inner-membrane, through which secretion is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the dynamic state of SecYEG throughout the hydrolytic cycle of SecA. We show that the SecYEG channel fluctuates between open and closed states faster ([~]20-fold during transport) than ATP turnover; while the nucleotide status of SecA modulates the rates of opening and closure. Interestingly, a SecY variant (PrlA4), exhibiting faster protein transport, but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore; thereby improving the efficiency of translocation. Thus, contrary to prevailing structure-based models, SecYEG plays an integral part in the translocation mechanism through dynamic allosteric coupling in which SecA steers the energy landscape of the protein-channel.

biophysics↗

bioRxiv2019

Insights into Membrane Protein-Lipid Interactions from Free Energy Calculations

Integral membrane proteins are regulated by specific interactions with lipids from the surrounding bilayer. The structures of protein-lipid complexes can be determined through a combination of experimental and computational approaches, but the energetic basis of these interactions is difficult to resolve. Molecular dynamic simulations provide the primary computational technique to estimate the free energies of these interactions. We demonstrate that the energetics of protein-lipid interactions may be reliably and reproducibly calculated using three simulation-based approaches: potential of mean force calculations. alchemical free energy perturbation, and well-tempered metadynamics. We employ these techniques within the framework of a coarse-grained force field, and apply them to both bacterial and mammalian membrane protein-lipid systems. We demonstrate good agreement between the different techniques, providing a robust framework for their automated implementation within a pipeline for annotation of newly determined membrane protein structures.

biochemistry↗

bioRxiv2019

HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

The bacterial Sec translocon is a multi-component protein complex responsible for translocating diverse proteins across the plasma membrane. For post-translational protein translocation, the Sec-channel - SecYEG - associates with the motor protein SecA to mediate the ATP-dependent transport of unfolded pre-proteins across the membrane. Based on the structure of the machinery, combined with ensemble and single molecule analysis, a diffusional based Brownian ratchet mechanism for protein secretion has been proposed [Allen et al. eLife 2016;5:e15598]. However, the conformational dynamics required to facilitate this mechanism have not yet been fully resolved. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, also ATP-dependent, constriction of SecA around the mature regions of the pre-protein. Thus, ATP binding causes SecY to open and SecA to close, while ATP hydrolysis has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery, and possibly in translocation systems elsewhere. The results highlight the power of HDX-MS for interrogating the dynamic mechanisms of diverse membrane proteins; including their interactions with small molecules such as nucleotides (ATPases and GTPases) and inhibitors (e.g. antibiotics).

biophysics↗

bioRxiv2019

A ‘proton ratchet’ for coupling the membrane potential to protein transport

Transport of proteins across and into membranes is a fundamental biological process with the vast majority being conducted by the ubiquitous Sec machinery. In bacteria, this is usually achieved when the SecY-complex engages the cytosolic ATPase SecA (secretion) or translating ribosomes (insertion). Great strides have been made towards understanding the mechanism of protein translocation. Yet, important questions remain - notably, the nature of the individual steps that constitute transport, and how the proton-motive force (PMF) across the plasma membrane contributes. Here, we apply a recently developed high-resolution protein transport assay to explore these questions. We find that pre-protein transport is limited primarily by the diffusion of arginine residues across the membrane, particularly in the context of bulky hydrophobic sequences. This specific effect of arginine, caused by its positive charge, is mitigated for lysine which can be deprotonated and transported across the membrane in its neutral form. These observations have interesting implications for the mechanism of protein secretion, suggesting a simple mechanism by which PMF can aid transport, and enabling a proton ratchet, wherein re-protonation of exiting lysine residues prevents channel re-entry, biasing transport in the outward direction.

biochemistry↗

bioRxiv2018

Structure and dynamics of the central lipid pool and protein components of the bacterial holo-translocon

The bacterial Sec translocon, SecYEG, associates with accessory proteins YidC and the SecDF-YajC subcomplex to form the bacterial holo-translocon (HTL). The HTL is a dynamic and flexible protein transport machine capable of coordinating protein secretion across the membrane, and efficient lateral insertion of nascent membrane proteins. It has been hypothesized that a central lipid core facilitates the controlled passage of membrane proteins into the bilayer, ensuring efficient formation of their native state. By performing small-angle neutron scattering (SANS) on protein solubilized in match-out deuterated detergent, we have been able to interrogate a naked HTL complex, with the scattering contribution of the surrounding detergent micelle rendered invisible. Such an approach has allowed the confirmation of a lipid core within the HTL, which accommodates between 8 and 29 lipids. Coarse-grained molecular dynamics simulations of the HTL also demonstrate a dynamic, central pool of lipids. An opening at this lipid rich region between YidC and the SecY lateral gate may provide an exit gateway for newly synthesized, correctly oriented, membrane protein helices to emerge from the HTL.

biophysics↗

bioRxiv2018

The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase and ion channel activity, or specific chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident calcium-regulated lipid scramblase. Our crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional structures solved by cryo-EM reveal extensive conformational changes extending from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity. Our results suggest mechanisms by which missense variants of TMEM16K could cause SCAR10 ataxia, providing new hypotheses to explore for therapy.

biochemistry↗

bioRxiv2018

Dynamic action of the Sec machinery during initiation, protein translocation and termination revealed by single molecule fluorescence

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. This is mediated, for the majority of proteins, by the highly conserved Sec machinery. The bacterial translocon - SecYMKEG - resides in the plasma membrane, where translocation is driven through rounds of ATP hydrolysis by the cytoplasmic SecA ATPase, and the proton motive force (PMF). We have used single molecule Forster resonance energy transfer (FRET) alongside a combination of confocal and total internal reflection microscopy to gain access to SecY pore dynamics and translocation kinetics on timescales spanning milliseconds to minutes. This allows us to dissect and characterise the translocation process in unprecedented detail. We show that SecA, signal sequence, pre-protein and ATP hydrolysis each have important and specific roles in unlocking and opening the Sec channel, priming it for transport. After channel opening, translocation proceeds in two phases: an initiation phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~ 40 amino acids per second for the model pre-protein substrate proOmpA. The initiation and translocation phases are both coupled to ATP hydrolysis while termination is ATP-independent. Distributions of translocation rates reflect the stochastic nature of the translocation process and are consistent with the recently proposed Brownian ratchet model [Allen et al. doi: 10.7554/eLife.15598]. The results allow us unparalleled access to the kinetics of the complex reaction and provide a framework for understanding the molecular mechanism of protein secretion.

biophysics↗

bioRxiv2017

ATP-induced asymmetric pre-protein folding: a potential driver of protein translocation?

The transport of proteins across membranes is a fundamental and essential process, achieved in every cell by the Sec translocon. In prokaryotes, SecYEG associates with the motor protein SecA to carry out ATP-driven pre-protein secretion - a vital step in the biogenesis of most periplasmic, outer membrane and secreted proteins. Structural data of the SecA-SecYEG complex has provided considerable insight into underlying mechanism of this process. Previously, we have proposed a Brownian ratchet model for protein translocation, whereby the free energy of ATP binding and hydrolysis favours the progression of pre-protein across the membrane from the cytosol toward the outside [Allen, Corey et al. eLife 2016]. Here, we use atomistic molecular dynamics simulation of a SecA-SecYEG complex engaged with preprotein to further address the mechanism underlying this process. The data describe pre-protein secondary structure formation within the channel, which exhibits a nucleotide-dependent asymmetry between the cytoplasmic and exterior cavities. The results suggest ATP-dependent pre-protein transport is partly driven by pre-protein secondary structure formation. The model previously described, and refined here, could easily be adapted for the transport of proteins across various other membranes, such as the endoplasmic reticular and mitochondrial inner membranes.

biophysics↗

bioRxiv2017

Identification and functional analyses of cardiolipin binding sites on the bacterial Sec translocase

The transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of pre-proteins across the plasma membrane, powered by ATP hydrolysis and the trans-membrane proton-motive-force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly by cardiolipin, a specialised phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific cardiolipin binding sites on the Thermotoga maritima SecA-SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites using in vitro mutagenesis, native mass spectrometry and biochemical analysis of Escherichia coli SecYEG. The results show that the two sites account for the preponderance of functional cardiolipin binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for cardiolipin in the conferral of PMF-stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery and thereby stimulate protein transport, by an as yet unknown mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, towards investigation of both the nature and functional implications of protein-lipid interactions.\n\nSignificance StatementMany proteins are located in lipid membranes surrounding cells and cellular organelles. The membrane can impart important structural and functional effects on the protein, making understanding of this interaction critical. Here, we apply computational simulation to the identification of conserved lipid binding sites on an important highly conserved bacterial membrane protein, the Sec translocase (SecA-SecYEG), which uses ATP and the proton motive force (PMF) to secrete proteins across the bacterial plasma membrane. We experimentally validate and reveal the conserved nature of these binding sites, and use functional analyses to investigate the biological significance of this interaction. We demonstrate that these interactions are specific, transient, and critical for both ATP- and PMF- driven protein secretion.

biophysics↗