Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer.

Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components and properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP-lipid complexes directly from intact and customizable lipid membranes. Both the lipid composition and membrane properties (such as curvature, tension, and fluidity) of these bilayers can be precisely customized to a target membrane. Subsequent direct nMS analysis of these intact proteolipid vesicles can yield the oligomeric states of the embedded IMPs, identify bound lipids, and determine the membrane properties that can regulate the observed IMP-lipid organization. Applying this method, we show how lipid binding regulates neurotransmitter release and how membrane composition regulates the functional oligomeric state of a transporter.

Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery.

Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca2+. Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca2+-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca2+-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of docked, release-ready vesicles. Dynamic single-molecule imaging of Complexin binding to release-ready vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by chaperones, Munc13 and Munc18. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 "template" complex is a functional intermediate in the production of primed, release-ready vesicles, which requires the coordinated action of Munc13 and Munc18.

The role of interfacial lipids in stabilizing membrane protein oligomers.

Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways but is often difficult to define or predict. Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT, one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na+/H+ antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors.

Deciphering the molecular organization of GET pathway chaperones through native mass spectrometry.

Get3/4/5 chaperone complex is responsible for targeting C-terminal tail-anchored membrane proteins to the endoplasmic reticulum. Despite the availability of several crystal structures of independent proteins and partial structures of subcomplexes, different models of oligomeric states and structural organization have been proposed for the protein complexes involved. Here, using native mass spectrometry (Native-MS), coupled with intact dissociation, we show that Get4/5 exclusively forms a tetramer using both Get5/5 and a novel Get4/4 dimerization interface. Addition of Get3 to this leads to a hexameric (Get3)2-(Get4)2-(Get5)2 complex with closed-ring cyclic architecture. We further validate our claims through molecular modeling and mutational abrogation of the proposed interfaces. Native-MS has become a principal tool to determine the state of oligomeric organization of proteins. The work demonstrates that for multiprotein complexes, native-MS, coupled with molecular modeling and mutational perturbation, can provide an alternative route to render a detailed view of both the oligomeric states as well as the molecular interfaces involved. This is especially useful for large multiprotein complexes with large unstructured domains that make it recalcitrant to conventional structure determination approaches.

High-resolution mass spectrometry of small molecules bound to membrane proteins.

Small molecules are known to stabilize membrane proteins and to modulate their function and oligomeric state, but such interactions are often hard to precisely define. Here we develop and apply a high-resolution, Orbitrap mass spectrometry-based method for analyzing intact membrane protein-ligand complexes. Using this platform, we resolve the complexity of multiple binding events, quantify small molecule binding and reveal selectivity for endogenous lipids that differ only in acyl chain length.

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces.

Mass spectrometry (MS) applications for intact protein complexes typically require electrospray (ES) ionization and have not been achieved via direct desorption from surfaces. Desorption ES ionization (DESI) MS has however transformed the study of tissue surfaces through release and characterisation of small molecules. Motivated by the desire to screen for ligand binding to intact protein complexes we report the development of a native DESI platform. By establishing conditions that preserve non-covalent interactions we exploit the surface to capture a rapid turnover enzyme-substrate complex and to optimise detergents for membrane protein study. We demonstrate binding of lipids and drugs to membrane proteins deposited on surfaces and selectivity from a mix of related agonists for specific binding to a GPCR. Overall therefore we introduce this native DESI platform with the potential for high-throughput ligand screening of some of the most challenging drug targets including GPCRs.

Mass spectrometric analysis of dimer-disrupting mutations in Plasmodium triosephosphate isomerase.

Electrospray ionization mass spectrometry (ESI MS) under nanospray conditions has been used to examine the effects of mutation at two key dimer interface residues, Gln (Q) 64 and Thr (T) 75, in Plasmodium falciparum triosephosphate isomerase. Both residues participate in an intricate network of intra- and intersubunit hydrogen bonds. The gas phase distributions of dimeric and monomeric protein species have been examined for the wild type enzyme (TWT) and three mutants, Q64N, Q64E, and T75S, under a wide range of collision energies (40-160 eV). The results established the order of dimer stability as TWT > T75S > Q64E âˆ¼ Q64N. The mutational effects on dimer stability are in good agreement with the previously reported estimates, based on the concentration dependence of enzyme activity. Additional experiments in solution, using inhibition of activity by a synthetic dimer interface peptide, further support the broad agreement between gas phase and solution studies.

Cyclic AMP-dependent protein lysine acylation in mycobacteria regulates fatty acid and propionate metabolism.

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guérin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

Oligomeric organization of membrane proteins from native membranes at nanoscale spatial and single-molecule resolution.

The oligomeric organization of membrane proteins in native cell membranes is a critical regulator of their function. High-resolution quantitative measurements of oligomeric assemblies and how they change under different conditions are indispensable to understanding membrane protein biology. We report Native-nanoBleach, a total internal reflection fluorescence microscopy-based single-molecule photobleaching step analysis technique to determine the oligomeric distribution of membrane proteins directly from native membranes at an effective spatial resolution of ~10 nm. We achieved this by capturing target membrane proteins in native nanodiscs with their proximal native membrane environment using amphipathic copolymers. We applied Native-nanoBleach to quantify the oligomerization status of structurally and functionally diverse membrane proteins, including a receptor tyrosine kinase (TrkA) and a small GTPase (KRas) under growth-factor binding and oncogenic mutations, respectively. Our data suggest that Native-nanoBleach provides a sensitive, single-molecule platform to quantify membrane protein oligomeric distributions in native membranes under physiologically and clinically relevant conditions.

Structure and function of the human apoptotic scramblase Xkr4.

Phosphatidylserine externalization on the surface of dying cells is a key signal for their recognition and clearance by macrophages and is mediated by members of the X-Kell related (Xkr) protein family. Defective Xkr-mediated scrambling impairs clearance, leading to inflammation. It was proposed that activation of the Xkr4 apoptotic scramblase requires caspase cleavage, followed by dimerization and ligand binding. Here, using a combination of biochemical approaches we show that purified monomeric, full-length human Xkr4 (hXkr4) scrambles lipids. CryoEM imaging shows that hXkr4 adopts a novel conformation, where three conserved acidic residues create an electronegative surface embedded in the membrane. Molecular dynamics simulations show this conformation induces membrane thinning, which could promote scrambling. Thinning is ablated or reduced in conditions where scrambling is abolished or reduced. Our work provides insights into the molecular mechanisms of hXkr4 scrambling and suggests the ability to thin membranes might be a general property of active scramblases.

A proteome-wide quantitative platform for nanoscale spatially resolved extraction of membrane proteins into native nanodiscs.

The intricate molecular environment of the native membrane profoundly influences every aspect of membrane protein (MP) biology. Despite this, the most prevalent method of studying MPs uses detergent-like molecules that disrupt and remove this vital local membrane context. This severely impedes our ability to quantitatively decipher the local molecular context and comprehend its regulatory role in the structure, function, and biogenesis of MPs. Using a library of membrane-active polymers we have developed a platform for the high-throughput analysis of the membrane proteome. The platform enables near-complete spatially resolved extraction of target MPs directly from their endogenous membranes into native nanodiscs that maintain the local membrane context. We accompany this advancement with an open-access database that quantifies the polymer-specific extraction variability for 2065 unique mammalian MPs and provides the most optimized condition for each of them. Our method enables rapid and near-complete extraction and purification of target MPs directly from their endogenous organellar membranes at physiological expression levels while maintaining the nanoscale local membrane environment. Going beyond the plasma membrane proteome, our platform enables extraction from any target organellar membrane including the endoplasmic reticulum, mitochondria, lysosome, Golgi, and even transient organelles such as the autophagosome. To further validate this platform, we took several independent MPs and demonstrated how our resource can enable rapid extraction and purification of target MPs from different organellar membranes with high efficiency and purity. Further, taking two synaptic vesicle MPs, we show how the database can be extended to capture multiprotein complexes between overexpressed MPs. We expect these publicly available resources to empower researchers across disciplines to efficiently capture membrane 'nano-scoops' containing a target MP and interface with structural, functional, and other bioanalytical approaches. We demonstrate an example of this by combining our extraction platform with single-molecule TIRF imaging to demonstrate how it can enable rapid determination of homo-oligomeric states of target MPs in native cell membranes.

Studying Membrane Protein-Lipid Specificity through Direct Native Mass Spectrometric Analysis from Tunable Proteoliposomes.

Native mass spectrometry (nMS) has emerged as a key analytical tool to study the organizational states of proteins and their complexes with both endogenous and exogenous ligands. Specifically, for membrane proteins, it provides a key analytical dimension to determine the identity of bound lipids and to decipher their effects on the observed structural assembly. We recently developed an approach to study membrane proteins directly from intact and tunable lipid membranes where both the biophysical properties of the membrane and its lipid compositions can be customized. Extending this, we use our liposome-nMS platform to decipher the lipid specificity of membrane proteins through their multiorganelle trafficking pathways. To demonstrate this, we used VAMP2 and reconstituted it in the endoplasmic reticulum (ER), Golgi, synaptic vesicle (SV), and plasma membrane (PM) mimicking liposomes. By directly studying VAMP2 from these customized liposomes, we show how the same transmembrane protein can bind to different sets of lipids in different organellar-mimicking membranes. Considering that the cellular trafficking pathway of most eukaryotic integral membrane proteins involves residence in multiple organellar membranes, this study highlights how the lipid-specificity of the same integral membrane protein may change depending on the membrane context. Further, leveraging the capability of the platform to study membrane proteins from liposomes with curated biophysical properties, we show how we can disentangle chemical versus biophysical properties, of individual lipids in regulating membrane protein assembly.

Isolation and Lipidomic Profiling of Neuronal Lipid Droplets: Unveiling the Lipid Landscape for insights into Neurodegenerative Disorders.

Recent advances have expanded the role of lipid droplets (LDs) beyond passive lipid storage, implicating their involvement in various metabolic processes across mammalian tissues. Neuronal LDs, long debated in existence, have been identified in several neural structures, raising questions about their contribution to neurodegenerative disorders. Elucidating the specific chemical makeup of these organelles within neurons is critical for understanding their implication in neural pathologies. This study outlines an improved methodology to stimulate and isolate mature LDs from cultured primary neurons, offering insights into their unique lipid-protein composition. Integrating this method with high-throughput techniques may unveil disease-specific alterations in lipid metabolism, providing avenues for potential therapeutic interventions.

Determination of oligomeric organization of membrane proteins from native membranes at nanoscale-spatial and single-molecule resolution.

The oligomeric organization of membrane proteins in native cell membranes is a critical regulator of their function. High-resolution quantitative measurements of oligomeric assemblies and how they change under different conditions are indispensable to the understanding of membrane protein biology. We report a single-molecule imaging technique (Native-nanoBleach) to determine the oligomeric distribution of membrane proteins directly from native membranes at an effective spatial resolution of ∼10 nm. We achieved this by capturing target membrane proteins in "native nanodiscs" with their proximal native membrane environment using amphipathic copolymers. We established this method using structurally and functionally diverse membrane proteins with well-established stoichiometries. We then applied Native-nanoBleach to quantify the oligomerization status of a receptor tyrosine kinase (TrkA) and a small GTPase (KRas) under conditions of growth-factor binding or oncogenic mutations, respectively. Native-nanoBleach provides a sensitive, single-molecule platform to quantify membrane protein oligomeric distributions in native membranes at an unprecedented spatial resolution.

Structure of Geobacter cytochrome OmcZ identifies mechanism of nanowire assembly and conductivity.

OmcZ nanowires produced by Geobacter species have high electron conductivity (>30 S cm-1). Of 111 cytochromes present in G. sulfurreducens, OmcZ is the only known nanowire-forming cytochrome essential for the formation of high-current-density biofilms that require long-distance (>10 µm) extracellular electron transport. However, the mechanisms underlying OmcZ nanowire assembly and high conductivity are unknown. Here we report a 3.5-Å-resolution cryogenic electron microscopy structure for OmcZ nanowires. Our structure reveals linear and closely stacked haems that may account for conductivity. Surface-exposed haems and charge interactions explain how OmcZ nanowires bind to diverse extracellular electron acceptors and how organization of nanowire network re-arranges in different biochemical environments. In vitro studies explain how G. sulfurreducens employ a serine protease to control the assembly of OmcZ monomers into nanowires. We find that both OmcZ and serine protease are widespread in environmentally important bacteria and archaea, thus establishing a prevalence of nanowire biogenesis across diverse species and environments.

ATG9 vesicles comprise the seed membrane of mammalian autophagosomes.

As the autophagosome forms, its membrane surface area expands rapidly, while its volume is kept low. Protein-mediated transfer of lipids from another organelle to the autophagosome likely drives this expansion, but as these lipids are only introduced into the cytoplasmic-facing leaflet of the organelle, full membrane growth also requires lipid scramblase activity. ATG9 harbors scramblase activity and is essential to autophagosome formation; however, whether ATG9 is integrated into mammalian autophagosomes remains unclear. Here we show that in the absence of lipid transport, ATG9 vesicles are already competent to collect proteins found on mature autophagosomes, including LC3-II. Further, we use styrene-maleic acid lipid particles to reveal the nanoscale organization of protein on LC3-II membranes; ATG9 and LC3-II are each fully integrated into expanding autophagosomes. The ratios of these two proteins at different stages of maturation demonstrate that ATG9 proteins are not continuously integrated, but rather are present on the seed vesicles only and become diluted in the expanding autophagosome membrane.

Novel Roles for Diacylglycerol in Synaptic Vesicle Priming and Release Revealed by Complete Reconstitution of Core Protein Machinery.

Here we introduce the full functional reconstitution of genetically-validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca 2+ . Using this novel setup, we discover new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca 2+- triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca 2+ -dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of ready-release vesicles. Dynamic single-molecule imaging of Complexin binding to ready-release vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by Munc13 and Munc18 chaperones. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 'template' complex is a functional intermediate in the production of primed, ready-release vesicles, which requires the coordinated action of Munc13 and Munc18. SIGNIFICANCE STATEMENT: Munc13 and Munc18 are SNARE-associated chaperones that act as "priming" factors, facilitating the formation of a pool of docked, release-ready vesicles and regulating Ca 2+ -evoked neurotransmitter release. Although important insights into Munc18/Munc13 function have been gained, how they assemble and operate together remains enigmatic. To address this, we developed a novel biochemically-defined fusion assay which enabled us to investigate the cooperative action of Munc13 and Munc18 in molecular terms. We find that Munc18 nucleates the SNARE complex, while Munc13 promotes and accelerates the SNARE assembly in a DAG-dependent manner. The concerted action of Munc13 and Munc18 stages the SNARE assembly process to ensure efficient 'clamping' and formation of stably docked vesicles, which can be triggered to fuse rapidly (∼10 msec) upon Ca 2+ influx.

Distinct disulfide isomers of µ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.

In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of µ-conotoxin KIIIA, which was predicted originally to have a [C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely related µ-conotoxins. The two major isomers of synthetic µ-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric collision-induced dissociation fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a [C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of Na(V)1.2 (K(d) values of 5 and 230 nM, respectively). The solution structure for µ-KIIIA based on nuclear magnetic resonance data was recalculated with the [C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the µ-KIIIA structure calculated with the incorrect [C1-C9,C2-C15,C4-C16] disulfide pattern, with an α-helix spanning residues 7-12. In addition, the major folding isomers of µ-KIIIB, an N-terminally extended isoform of µ-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as µ-KIIIA, and both blocked Na(V)1.2 (K(d) values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic µ-KIIIA and µ-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of µ-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means of further expanding the snail's repertoire of active peptides.

Combined electron transfer dissociation-collision-induced dissociation fragmentation in the mass spectrometric distinction of leucine, isoleucine, and hydroxyproline residues in Peptide natural products.

Distinctions between isobaric residues have been a major challenge in mass spectrometric peptide sequencing. Here, we propose a methodology for distinction among isobaric leucine, isoleucine, and hydroxyproline, a commonly found post-translationally modified amino acid with a nominal mass of 113 Da, through a combined electron transfer dissociation-collision-induced dissociation approach. While the absence of c and z(•) ions, corresponding to the Yyy-Xxx (Xxx = Leu, Ile, or Hyp) segment, is indicative of the presence of hydroxyproline, loss of isopropyl (Δm = 43 Da) or ethyl radicals (Δm = 29 Da), through collisional activation of z radical ions, are characteristic of leucine or isoleucine, respectively. Radical migration processes permit distinctions even in cases where the specific z(•) ions, corresponding to the Yyy-Leu or -Ile segments, are absent or of low intensity. This tandem mass spectrometric (MS(n)) method has been successfully implemented in a liquid chromatography-MS(n) platform to determine the identity of 23 different isobaric residues from a mixture of five different peptides. The approach is convenient for distinction of isobaric residues from any crude peptide mixture, typically encountered in natural peptide libraries or proteomic analysis.