Membrane Protein Structure Research Articles

Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein-Lipid Assemblies.

Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand the structural dynamics of these proteins within lipid environments. Lipids, however, have proven problematic for HDX-MS analysis of membrane-embedded proteins due to their presence of impairing proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral overlap. Herein, we describe the integration of a chromatographic phospholipid trap column into the HDX-MS apparatus to enable online sample delipidation prior to protease digestion of deuterium-labeled protein-lipid assemblies. We demonstrate the utility of this method on membrane scaffold protein-lipid nanodisc─both empty and loaded with the ∼115 kDa transmembrane protein AcrB─proving efficient and automated phospholipid capture with minimal D-to-H back-exchange, peptide carry-over, and protein loss. Our results provide insights into the efficiency of phospholipid capture by ZrO2-coated and TiO2 beads and describe how solution conditions can be optimized to maximize not only the performance of our online but also the existing offline, delipidation workflows for HDX-MS. We envision that this HDX-MS method will significantly ease membrane protein analysis, allowing to better interrogate their dynamics in artificial lipid bilayers or even native cell membranes.

Supramolecular organization and dynamics of mannosylated phosphatidylinositol lipids in the mycobacterial plasma membrane

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. Therefore, development of new anti-TB drugs remains one of the highest public health priorities. Mtb has evolved a complex cell envelope that represents a formidable barrier to antibiotics. The Mtb cell envelop consists of four distinct layers enriched for Mtb specific lipids and glycans. Although the outer membrane, comprised of mycolic acid esters, has been extensively studied, less is known about the plasma membrane, which also plays a critical role in impacting antibiotic efficacy. The Mtb plasma membrane has a unique lipid composition, with mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs) comprising more than 50% of the lipids. However, the role of PIMs in the structure and function of the membrane remains elusive. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function.

Synthetic transmembrane channel molecules formed by acyclic cucurbiturils and pillararene: Tuning cation selectivity and generating membrane potential

Biomimetic transmembrane channel molecules developed through chemical synthesis strategies were used for treat genetic diseases which due to the alterations in the channel structure of native membrane proteins and ion transport is deregulated. In this paper, we designed and constructed a novel synthetic transmembrane channel molecules (STCMs), which based on acyclic cucurbiturils (ACBs) and pillar[5]arene (PA[5]). STCMs could imitate the function of natural channel protein molecules and reveal the possible existence structure, there is a hope for new treatment options for genetic diseases. The structure of STCMs have been identified by NMR and FT-IR, the results of fluorescence experiments showed that it could effectively insert into the phospholipid bilayer, specifically recognize and transmit K+. Whole-cell patch-clamp experiments showed that STCMs could insert into the cell membrane of HEK293T cells, and could form channels on the cell membrane to generate current. These works will help to understand the relationship between the structures and properties of STCMs and the selectivity and efficiency of ions or polar molecules. This study presents the molecular structure and function of STCMs which may form a basis for explore the scientific laws of transmembrane transporter.

Melamine-cored glucosides for membrane protein solubilization and stabilization: importance of water-mediated intermolecular hydrogen bonding in detergent performance.

Membrane proteins play essential roles in a number of biological processes, and their structures are important in elucidating such processes at the molecular level and also for rational drug design and development. Membrane protein structure determination is notoriously challenging compared to that of soluble proteins, due largely to the inherent instability of their structures in non-lipid environments. Micelles formed by conventional detergents have been widely used for membrane protein manipulation, but they are suboptimal for long-term stability of membrane proteins, making downstream characterization difficult. Hence, there is an unmet need for the development of new amphipathic agents with enhanced efficacy for membrane protein stabilization. In this study, we designed and synthesized a set of glucoside amphiphiles with a melamine core, denoted melamine-cored glucosides (MGs). When evaluated with four membrane proteins (two transporters and two G protein-coupled receptors), MG-C11 conferred notably enhanced stability compared to the commonly used detergents, DDM and LMNG. These promising findings are mainly attributed to a unique feature of the MGs, i.e., the ability to form dynamic water-mediated hydrogen-bond networks between detergent molecules, as supported by molecular dynamics simulations. Thus, MG-C11 is the first example of a non-peptide amphiphile capable of forming intermolecular hydrogen bonds within a protein-detergent complex environment. Detergent micelles formed via a hydrogen-bond network could represent the next generation of highly effective membrane-mimetic systems useful for membrane protein structural studies.

Combining native mass spectrometry and lipidomics to uncover specific membrane protein-lipid interactions from natural lipid sources.

While it is known that lipids play an essential role in regulating membrane protein structure and function, it remains challenging to identify specific protein-lipid interactions. Here, we present an innovative approach that combines native mass spectrometry (MS) and lipidomics to identify lipids retained by membrane proteins from natural lipid extracts. Our results reveal that the bacterial ammonia channel (AmtB) enriches specific cardiolipin (CDL) and phosphatidylethanolamine (PE) from natural headgroup extracts. When the two extracts are mixed, AmtB retains more species, wherein selectivity is tuned to bias headgroup selection. Using a series of natural headgroup extracts, we show TRAAK, a two-pore domain K+ channel (K2P), retains specific acyl chains that is independent of the headgroup. A brain polar lipid extract was then combined with the K2Ps, TRAAK and TREK2, to understand lipid specificity. More than a hundred lipids demonstrated affinity for each protein, and both channels were found to retain specific fatty acids and lysophospholipids known to stimulate channel activity, even after several column washes. Natural lipid extracts provide the unique opportunity to not only present natural lipid diversity to purified membrane proteins but also identify lipids that may be important for membrane protein structure and function.

Pioneering Understanding of Cell Membrane Components

Cell membranes are critical for cellular life. The effective extraction of proteins and lipids from cell membranes is a necessity for research, but traditional methods may damage the membrane components and limit the accuracy of data. Dr Youzhong Guo at Virginia Commonwealth University has recently developed a revolutionary method for the extraction of membrane components in the format of native cell membrane nanoparticles to allow in-depth structural studies of membrane proteins whilst preserving functionality and limiting damage to vital mechanisms. This exciting work is driving forward the understanding of the structure, function and protein-lipid interactions of membrane protein.

Effect of polyunsaturated free fatty acids on the membrane fusion mechanism.

Membrane fusion is one of the important processes for the survival of eukaryotic cells and the entry of enveloped viruses into the host cells. Lipid composition plays a crucial role by modulating the organization and dynamics of the membrane, as well as the structure and conformation of membrane proteins. The diversity of the lipid acyl chain in its length and degree of unsaturation originates from the variation in free fatty acids (FFAs). We have studied the effect of linoleic (LA) and alpha-linolenic (ALA) acids on the depth-dependent organization, dynamics, and fusion of DOPC/DOPE (70/30 mol%) membranes utilizing steady-state and time-resolved fluorescence spectroscopic methods. Our results suggest that membranes with 5 mol% LA stabilize the stalk-intermediate and promote lipid mixing at the early stage of the process, i.e., the fusion follows the classical stalk model. Conversely, the extents of lipid and content mixing at the stalk intermediate are similar in the presence of 5 mol% of ALA, indicating the fusion mechanism as a nonclassical one like in the DOPC/DOPE (70/30 mol%) membranes. Our results provide an in-depth insight into the effect of the increasing degree of fatty acid tail unsaturation on membrane organization and dynamics and their impact on the membrane fusion mechanism.

Objective Criteria for Estimation of Initial Parameters for the Modeling of Micelle and Liposome Structures from Small-Angle X-ray Scattering Data

The structures of hydrophobic membrane proteins are studied using matrices, which serve as models of cell membranes and are formed by the appropriate amphiphilic molecules, e.g., by surfactant or lipid molecules. To study the structure of a protein incorporated into an artificial membrane, first of all it is necessary to determine the structure of the membrane. The ELLLIP and ELLMIC algorithms were previously developed to address this issue by small-angle X-ray scattering. These algorithms allow the construction of models of ellipsoidal vesicles based on the atomic structure of a lipid or surfactant monomer. However, the results of modeling depend, to a large extent, on the subjective assessment of the initial values of the structural parameters of the matrices and may be wrong due to the ambiguity in the solution of such problems. Here, we present an independent approach to the determination of the initial sizes of model membranes for their subsequent structural modeling, which is based on the analysis of the pair-distance distribution functions derived directly from the small-angle X-ray scattering curve.

PH-tunable membrane-active polymers, NCMNP2a-x, and their potential membrane protein applications.

Accurate 3D structures of membrane proteins are essential for comprehending their mechanisms of action and designing specific ligands to modulate their activities. However, these structures are still uncommon due to the involvement of detergents in the sample preparation. Recently, membrane-active polymers have emerged as an alternative to detergents, but their incompatibility with low pH and divalent cations has hindered their efficacy. Herein, we describe the design, synthesis, characterization, and application of a new class of pH-tunable membrane-active polymers, NCMNP2a-x. The results demonstrated that NCMNP2a-x could be used for high-resolution single-particle cryo-EM structural analysis of AcrB in various pH conditions and can effectively solubilize BcTSPO with the function preserved. Molecular dynamic simulation is consistent with experimental data that shed great insights into the working mechanism of this class of polymers. These results demonstrated that NCMNP2a-x might have broad applications in membrane protein research.

Solid-state NMR chemical shift analysis for determining the conformation of ATP bound to Na,K-ATPase in its native membrane.

Structures of membrane proteins determined by X-ray crystallography and, increasingly, by cryo-electron microscopy often fail to resolve the structural details of unstable or reactive small molecular ligands in their physiological sites. This work demonstrates that 13C chemical shifts measured by magic-angle spinning (MAS) solid-state NMR (SSNMR) provide unique information on the conformation of a labile ligand in the physiological site of a functional protein in its native membrane, by exploiting freeze-trapping to stabilise the complex. We examine the ribose conformation of ATP in a high affinity complex with Na,K-ATPase (NKA), an enzyme that rapidly hydrolyses ATP to ADP and inorganic phosphate under physiological conditions. The 13C SSNMR spectrum of the frozen complex exhibits peaks from all ATP ribose carbon sites and some adenine base carbons. Comparison of experimental chemical shifts with density functional theory (DFT) calculations of ATP in different conformations and protein environments reveals that the ATP ribose ring adopts an C3'-endo (N) conformation when bound with high affinity to NKA in the E1Na state, in contrast to the C2'-endo (S) ribose conformations of ATP bound to the E2P state and AMPPCP in the E1 complex. Additional dipolar coupling-mediated measurements of H-C-C-H torsional angles are used to eliminate possible relative orientations of the ribose and adenine rings. The utilization of chemical shifts to determine membrane protein ligand conformations has been underexploited to date and here we demonstrate this approach to be a powerful tool for resolving the fine details of ligand-protein interactions.

Charge neutralization of the active site glutamates does not limit substrate binding and transport by small multidrug resistance transporter EmrE

EmrE, a small multidrug resistance (SMR) transporter from E. coli, confers broad-spectrum resistance to polyaromatic cations and quaternary ammonium compounds. Previous transport assays demonstrate that EmrE transports a +1 and a +2 substrate with the same stoichiometry of 2 protons:1 cationic substrate. This suggests that EmrE substrate binding capacity is limited to neutralization of the two essential glutamates, E14A and E14B (one from each subunit in the antiparallel homodimer), in the primary binding site. Here we explicitly test this hypothesis, since EmrE has repeatedly broken expectations for membrane protein structure and transport mechanism. We previously showed that EmrE can bind a +1 cationic substrate and proton simultaneously, with cationic substrate strongly associated with one E14 residue while the other remains accessible to bind and transport a proton. Here we demonstrate that EmrE can bind a +2 cation substrate and a proton simultaneously using NMR pH titrations of EmrE saturated with divalent substrates, for a net +1 charge in the transport pore. Further, we find that EmrE can alternate access and transport a +2 substrate and proton at the same time. Together, these results lead us to conclude that E14 charge neutralization does not limit the binding and transport capacity of EmrE.

Structure determination of inactive-state GPCRs with a universal nanobody.

Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. Despite representing one of the largest classes of therapeutic targets, most inactive-state G protein-coupled receptors (GPCRs) have remained inaccessible for cryo-EM because their small size and membrane-embedded nature impedes projection alignment for high-resolution map reconstructions. Here we demonstrate that the same single-chain camelid antibody (nanobody) recognizing a grafted intracellular loop can be used to obtain cryo-EM structures of inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained structures of neurotensin 1 receptor bound to antagonist SR48692, μ-opioid receptor bound to alvimopan, apo somatostatin receptor 2 and histamine receptor 2 bound to famotidine. We expect this rapid, straightforward approach to facilitate the broad exploration of GPCR inactive states without the need for extensive engineering and crystallization.

Study of antimicrobial activity and mechanism of vapor-phase cinnamaldehyde for killing Escherichia coli based on fumigation method.

The vapor-phase antibacterial activity of essential oils makes them suitable for applications in air disinfection and other fields. At present, vapor-phase antibacterial activity of plant-based essential oils has rarely been reported. Herein, we report a new approach to investigate the antimicrobial activity and mechanism of vapor-phase cinnamaldehyde using Escherichia coli (E. coli) and three other pathogenic bacteria (Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus) as model bacteria. Plate fumigation and agar block transfer techniques were used to determine the antimicrobial activities of vapor-phase cinnamaldehyde fumigation on the four types of bacteria, and the mechanism of action was determined by electrical conductivity (EC), OD260nm measurement, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and fluorescence spectroscopy. Cinnamaldehyde had good vapor-phase antibacterial activity against the four types of bacteria. The TEM, EC, and OD260nm measurements showed that after fumigation with cinnamaldehyde, the ultrastructures of the cells were damaged, and plasmolysis, cell collapse, and leakage of intracellular substances were observed. The FTIR and fluorescence spectroscopy analyses showed that the secondary and tertiary structures of bacterial membrane proteins were altered. These findings indicate that the cell membrane is an important target for plant-based essential oils to exert their vapor-phase antimicrobial effects. The results showed that plant-based essential oils can be developed as volatile broad-spectrum disinfection products and vapor-phase antiseptics.

Entamoeba histolytica: Membrane and Non-Membrane Protein Structure, Function, Immune Response Interaction, and Vaccine Development.

Entamoeba histolytica is a protozoan parasite that is the causative agent of amoebiasis. This parasite has caused widespread infection in India, Africa, Mexico, and Central and South America, and results in 100,000 deaths yearly. An immune response is a body's mechanism for eradicating and fighting against substances it sees as harmful or foreign. E. histolytica biological membranes are considered foreign and immunogenic to the human body, thereby initiating the body's immune responses. Understanding immune response and antigen interaction are essential for vaccine development. Thus, this review aims to identify and understand the protein structure, function, and interaction of the biological membrane with the immune response, which could contribute to vaccine development. Furthermore, the current trend of vaccine development studies to combat amoebiasis is also reviewed.

The Epigenetic Dimension of Protein Structure Is an Intrinsic Weakness of the AlphaFold Program.

One of the most important lessons we have learned from sequencing the human genome is that not all proteins have a 3D structure. In fact, a large part of the human proteome is made up of intrinsically disordered proteins (IDPs) which can adopt multiple structures, and therefore, multiple functions, depending on the ligands with which they interact. Under these conditions, one can wonder about the value of algorithms developed for predicting the structure of proteins, in particular AlphaFold, an AI which claims to have solved the problem of protein structure. In a recent study, we highlighted a particular weakness of AlphaFold for membrane proteins. Based on this observation, we have proposed a paradigm, referred to as “Epigenetic Dimension of Protein Structure” (EDPS), which takes into account all environmental parameters that control the structure of a protein beyond the amino acid sequence (hence “epigenetic”). In this new study, we compare the reliability of the AlphaFold and Robetta algorithms’ predictions for a new set of membrane proteins involved in human pathologies. We found that Robetta was generally more accurate than AlphaFold for ascribing a membrane-compatible topology. Raft lipids (e.g., gangliosides), which control the structural dynamics of membrane protein structure through chaperone effects, were identified as major actors of the EDPS paradigm. We conclude that the epigenetic dimension of a protein structure is an intrinsic weakness of AI-based protein structure prediction, especially AlphaFold, which warrants further development.

Transition-Linker Containing Detergents for Membrane Protein Studies.

It is a pressing need, but still challenging to explore the structure and function of membrane proteins (MPs). One of the main obstacles is the limited availability of matched detergents for the handling of specific MPs. We describe herein the design of new detergents by incorporation of a transition linker between the hydrophilic head and the hydrophobic tail. This design allows a gradual change of hydrophobicity between the outside and inside of micelles, in contrast to the abrupt switch in conventional detergents. Notably, many of these detergents assembled into micelles in while retaining low critical micelle concentrations. Meanwhile, thermal stabilizing evaluation identified superior detergents for representative MPs, including G protein-coupled receptors and a transporter protein. Among them, further improved the NMR study of MPs. We anticipate these that results will encourage future detergent expansion through new remodeling on the traditional detergent scaffold.

Refinement of SARS-CoV-2 envelope protein structure in a native-like environment by molecular dynamics simulations.

COVID-19 has become an unprecedented threat to human health. The SARS-CoV-2 envelope (E) protein plays a critical role in the viral maturation process and pathogenesis. Despite intensive investigation, its structure in physiological conditions remains mysterious: no high-resolution full-length structure is available and only an NMR structure of the transmembrane (TM) region has been determined. Here, we present a refined E protein structure, using molecular dynamics (MD) simulations to investigate its structure and dynamics in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer system. Our initial homology model based upon the SARS-CoV E protein structure is shown to be unstable in the lipid bilayer, and the H3 helices tend to move away from the membrane center to the membrane-water interface. A more stable model was developed by replacing all H3 helices with the fully equilibrated H3 structure sampled in the MD simulations. This refined model exhibited more favorable contacts with lipids and water than the original homology model and induced local membrane curvature, decreasing local lipid order. Interestingly, the pore radius profiles showed that the channel in both homology and refined models remained in a closed state throughout the simulations. We also demonstrated the utility of this structure to develop anti-SARS-CoV-2 drugs by docking a library of FDA-approved, investigational, and experimental drugs to the refined E protein structure, identifying 20 potential channel blockers. This highlights the power of MD simulations to refine low-resolution structures of membrane proteins in a native-like membrane environment, shedding light on the structural features of the E protein and providing a platform for the development of novel antiviral treatments.

Lipid-membrane protein interaction visualised by cryo-EM: A review

Membrane proteins reside at interfaces between aqueous and lipid media and solving their molecular structure relies most of the time on removing them from the membrane using detergent. Luckily, this solubilization process does not strip them from all the associated lipids and single-particle cryo-transmission electron microscopy (SP-TEM) has proved a very good tool to visualise both protein high-resolution structure and, often, many of its associated lipids. In this review, we observe membrane protein structures from the Protein DataBank and their associated maps in the Electron Microscopy DataBase and determine how the SP-TEM maps allow lipid visualization, the type of binding sites, the influence of symmetry, resolution and other factors. We illustrate lipid visualization around and inside the protein core, show that some lipid bilayers in the core can be shifted with respect to the membrane and how some proteins can actively bend the lipid bilayer that binds to them. We conclude that resolution improvement in SP-TEM will likely enable many more discoveries regarding the role of lipids bound to proteins.

Yeast as a tool for membrane protein production and structure determination.

Membrane proteins are challenging targets to functionally and structurally characterize. An enduring bottleneck in their study is the reliable production of sufficient yields of stable protein. Here, we evaluate all eukaryotic membrane protein production experiments that have supported the deposition of a high-resolution structure. We focused on the most common yeast host systems, Saccharomyces cerevisiae and Pichia pastoris. The first high-resolution structure of a membrane protein produced in yeast was described in 1999 and today there are 186 structures of α-helical membrane proteins, representing 101 unique proteins from 37 families. Homologous and heterologous production are equally common in S. cerevisiae, while heterologous production dominates in P. pastoris, especially of human proteins, which represent about one-third of the total. Investigating protein engineering approaches (78 proteins from seven families) demonstrated that the majority contained a polyhistidine tag for purification, typically at the C-terminus of the protein. Codon optimization and truncation of hydrophilic extensions were also common approaches to improve yields. We conclude that yeast remains a useful production host for the study of α-helical membrane proteins.

Implications of High-Density Cholesterol Metabolism for Oocyte Biology and Female Fertility.

Cholesterol is an essential component of animal cells. Different regulatory mechanisms converge to maintain adequate levels of this lipid because both its deficiency and excess are unfavorable. Low cell cholesterol content promotes its synthesis and uptake from circulating lipoproteins. In contrast, its excess induces the efflux to high-density lipoproteins (HDL) and their transport to the liver for excretion, a process known as reverse cholesterol transport. Different studies suggest that an abnormal HDL metabolism hinders female fertility. HDL are the only lipoproteins detected in substantial amounts in follicular fluid (FF), and their size and composition correlate with embryo quality. Oocytes obtain cholesterol from cumulus cells via gap junctions because they cannot synthesize cholesterol de novo and lack HDL receptors. Recent evidence has supported the possibility that FF HDL play a major role in taking up excess unesterified cholesterol (UC) from the oocyte. Indeed, genetically modified mouse models with disruptions in reverse cholesterol transport, some of which show excessive circulating UC levels, exhibit female infertility. Cholesterol accumulation can affect the egg´s viability, as reported in other cell types, and activate the plasma membrane structure and activity of membrane proteins. Indeed, in mice deficient for the HDL receptor Scavenger Class B Type I (SR-B1), excess circulating HDL cholesterol and UC accumulation in oocytes impairs meiosis arrest and hinders the developmental capacity of the egg. In other cells, the addition of cholesterol activates calcium channels and dysregulates cell death/survival signaling pathways, suggesting that these mechanisms may link altered HDL cholesterol metabolism and infertility. Although cholesterol, and lipids in general, are usually not evaluated in infertile patients, one study reported high circulating UC levels in women showing longer time to pregnancy as an outcome of fertility. Based on the evidence described above, we propose the existence of a well-regulated and largely unexplored system of cholesterol homeostasis controlling traffic between FF HDL and oocytes, with significant implications for female fertility.