Structure Of Integral Membrane Proteins Research Articles

NDT-C11 as a Viable Novel Detergent for Single Particle Cryo-EM.

Single particle cryo electron microscopy (cryo-EM) is now the major method for the determination of integral membrane protein structure. For the success of a given project the type of membrane mimetic used for extraction from the native cell membrane, purification to homogeneity and finally cryo-grid vitrification is crucial. Although small molecule amphiphiles - detergents - are the most widely used membrane mimetic, specific tailoring of detergent structure for single particle cryo-EM is rare and the demand for effective detergents not satisfied. Here, we compare the popular detergent lauryl maltose-neopentyl glycol (LMNG) with the novel detergent neopentyl glycol-derived triglucoside-C11 (NDT-C11) in its behavior as free detergent and when bound to two types of multisubunit membrane protein complexes - cyanobacterial photosystem I (PSI) and mammalian F-ATP synthase. We conclude that NDT-C11 has high potential to become a very useful detergent for single particle cryo-EM of integral membrane proteins.

Molecular Dynamics of Outer Membrane-Embedded Polysaccharide Secretion Porins Reveals Closed Resting-State Surface Gates Targetable by Virtual Fragment Screening for Drug Hotspot Identification.

Recent advances in iterative neural network analyses (e.g., AlphaFold2 and RoseTTA fold) have been revolutionary for protein 3D structure prediction, especially for difficult-to-manipulate α-helical/β-barrel integral membrane proteins. These model structures are calculated based on the coevolution of amino acids within the protein of interest and similarities to existing protein structures; the local effects of the membrane on folding and stability of the calculated model structures are not considered. We recently reported the discovery, 3D modeling, and characterization of 18-β-stranded outer-membrane (OM) WzpX, WzpS, and WzpB β-barrel secretion porins for the exopolysaccharide (EPS), major spore coat polysaccharide (MASC), and biosurfactant polysaccharide (BPS) pathways (respectively) in the Gram-negative social predatory bacterium Myxococcus xanthus DZ2. However, information was not obtained regarding the dynamic behavior of surface-gating WzpX/S/B loop domains or on potential treatments to inactivate these porins. Herein, we developed a molecular dynamics (MD) protocol to study the core stability and loop dynamism of neural network-based integral membrane protein structure models embedded in an asymmetric OM bilayer, using the M. xanthus WzpX, WzpS, and WzpB proteins as test candidates. This was accomplished through integration of the CHARMM-graphical user interface (GUI) and Molecular Operating Environment (MOE) workflows to allow for a rapid simulation system setup and facilitate data analysis. In addition to serving as a method of model structure validation, our molecular dynamics simulations revealed a minimal movement of extracellular WzpX/S/B loops in the absence of an external stimulus as well as druggable cavities between the loops. Virtual screening of a commercial fragment library against these cavities revealed putative fragment-binding hotspots on the cell-surface face of each β-barrel, along with key interacting residues, and identified promising hits for the design of potential binders capable of plugging the β-barrels and inhibiting polysaccharide secretion.

Membrane protein isolation and structure determination in cell-derived membrane vesicles

Integral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and structure determination of proteins in membrane vesicles derived directly from cells. Structures of the ion channel Slo1 from total cell membranes and from cell plasma membranes were determined at 3.8Å and 2.7Å resolution, respectively. The plasma membrane environment stabilizes Slo1, revealing an alteration of global helical packing, polar lipid, and cholesterol interactions that stabilize previously unresolved regions of the channel and an additional ion binding site in the Ca2+ regulatory domain. The two methods presented enable structural analysis of both internal and plasma membrane proteins without disrupting weakly interacting proteins, lipids, and cofactors that are essential to biological function.

OPRLM: Orientations of proteins in realistic lipid membranes.

A new web tool and a database, OPRLM (Orientations of Proteins in Realistic Lipid Membranes), has been created based on the OPM database and the broadly used GHARMM-GUI toolset. The database provides a complete set of experimental 3D structures of integral and peripheral membrane proteins from the Protein Data Bank that are positioned in planar and curved lipid bilayers using implicit and explicit membrane representations. Membrane systems vary from single-component artificial lipid bilayers to multi-component bilayers with complex lipid composition corresponding to 18 biomembranes with either asymmetric or symmetric distribution of lipid types between two leaflets: 14 “average” eukaryotic cellular and organelle membranes (plasma membranes of mammals, plants, and fungi, endoplasmic reticulum and apparatus Golgi membranes of mammals and fungi, mammalian membranes of endosomes and lysosomes, mitochondrial outer and inner membranes, plant vacuole membranes, thylakoid membranes of plants and cyanobacteria), the outer and the inner membranes of Gram-negative bacteria (E. coli), and “average” cell membranes of Gram-positive bacteria and archaebacteria. All these membrane systems are accessible for public use in CHARMM-GUI Archive (https://www.charmm-gui.org/docs/archive/biomembrane). The web tool allows users of any level of expertise an easy setup of protein-membrane systems for atomic molecular dynamics (MD) simulations in pre-assembled realistic lipid membranes formed by mixtures of explicit lipids. OPRLM is publicly available at https://oprlm.org/.

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.

Cryo-electron Microscopic Analysis of Single-Pass Transmembrane Receptors.

Single-pass transmembrane receptors (SPTMRs) represent a diverse group of integral membrane proteins that are involved in many essential cellular processes, including signal transduction, cell adhesion, and transmembrane transport of materials. Dysregulation of the SPTMRs is linked with many human diseases. Despite extensive efforts in past decades, the mechanisms of action of the SPTMRs remain incompletely understood. One major hurdle is the lack of structures of the full-length SPTMRs in different functional states. Such structural information is difficult to obtain by traditional structural biology methods such as X-ray crystallography and nuclear magnetic resonance (NMR). The recent rapid development of single-particle cryo-electron microscopy (cryo-EM) has led to an exponential surge in the number of high-resolution structures of integral membrane proteins, including SPTMRs. Cryo-EM structures of SPTMRs solved in the past few years have tremendously improved our understanding of how SPTMRs function. In this review, we will highlight these progresses in the structural studies of SPTMRs by single-particle cryo-EM, analyze important structural details of each protein involved, and discuss their implications on the underlying mechanisms. Finally, we also briefly discuss remaining challenges and exciting opportunities in the field.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs).

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Genomics-based strategies toward the identification of a Z-ISO carotenoid biosynthetic enzyme suitable for structural studies.

Over the past 20years, structural genomics efforts have proven enormously successful for the determination of integral membrane protein structures, particularly for those of prokaryotic origin. However, traditional genomic expansion screens have included up to hundreds of targets, necessitating the use of robotics and other automation not available to most laboratories. Moreover, such large-scale screens of eukaryotic targets are not easily performed at such a scale. To have broader appeal, traditional structural genomic approaches need to be modified and improved such that they are feasible for most laboratories and especially so for proteins from eukaryotic organisms. One such refinement, termed "microgenomic expansion," has been recently described. This approach improves the process of target selection by making target screening a two-step process, with a minimal number of targets tested at each step. Microgenomic expansion methods are applied here theoretically to a project that has the objective of acquiring a structure for the plant 15-cis-ζ-carotene isomerase, Z-ISO.

Insights into the Role of Membrane Lipids in the Structure, Function and Regulation of Integral Membrane Proteins

Membrane proteins exist within the highly hydrophobic membranes surrounding cells and organelles, playing key roles in cellular function. It is becoming increasingly clear that the membrane does not just act as an appropriate environment for these proteins, but that the lipids that make up these membranes are essential for membrane protein structure and function. Recent technological advances in cryogenic electron microscopy and in advanced mass spectrometry methods, as well as the development of alternative membrane mimetic systems, have allowed experimental study of membrane protein–lipid complexes. These have been complemented by computational approaches, exploiting the ability of Molecular Dynamics simulations to allow exploration of membrane protein conformational changes in membranes with a defined lipid content. These studies have revealed the importance of lipids in stabilising the oligomeric forms of membrane proteins, mediating protein–protein interactions, maintaining a specific conformational state of a membrane protein and activity. Here we review some of the key recent advances in the field of membrane protein–lipid studies, with major emphasis on respiratory complexes, transporters, channels and G-protein coupled receptors.

CG2AT2: Recovering Atomistic Details from Coarse Grain Simulations

The structure and function of most integral membrane proteins is modulated by the molecular components of the lipid bilayer. While a number of experimental and computational approaches exist to probe the structural basis of these interactions, the energetics between individual components are more difficult to resolve. With Molecular Dynamics the largest constraint is the amount of sampling required to reach convergence. The use of coarse grain simulations ameliorate this at the cost of resolution.

Effects of 2-monoacylglycerol on in meso crystallization and the crystal structures of integral membrane proteins

Monoacylglycerols (MAGs) are the lipids most commonly used for crystallizing membrane proteins by the in meso method. The acyl chain and glycerol components of MAGs are ester-linked at the sn-1 hyd...

Solution structure and oligomeric state of the E. coliglycerol facilitator

Protein dynamics at atomic resolution can provide deep insights into the biological activities of proteins and enzymes but they can also make structure and dynamics studies challenging. Despite their well-known biological and pharmaceutical importance, integral membrane protein structure and dynamics studies lag behind those of water-soluble proteins mainly owing to solubility problems that result upon their removal from the membrane. Escherichia coli glycerol facilitator (GF) is a member of the aquaglyceroporin family that allows for the highly selective passive diffusion of its substrate glycerol across the inner membrane of the bacterium. Previous molecular dynamics simulations and hydrogen-deuterium exchange studies suggested that protein dynamics play an important role in the passage of glycerol through the protein pore. With the aim of studying GF dynamics by solution and solid-state nuclear magnetic resonance (NMR) spectroscopy we optimized the expression of isotope-labelled GF and explored various solubilizing agents including detergents, osmolytes, amphipols, random heteropolymers, lipid nanodiscs, bicelles and other buffer additives to optimize the solubility and polydispersity of the protein. The GF protein is most stable and soluble in lauryl maltose neopentyl glycol (LMNG), where it exists in a tetramer-octamer equilibrium. The solution structures of the GF tetramer and octamer were determined by negative-stain transmission electron microscopy (TEM), size-exclusion chromatography small-angle X-ray scattering (SEC-SAXS) and solid-state magic-angle spinning NMR spectroscopy. Although NMR sample preparation still needs optimization for full structure and dynamics studies, negative stain TEM and SEC-SAXS revealed low-resolution structures of the detergent-solubilized tetramer and octamer particles. The non-native octamer appears to form from the association of the cytoplasmic faces of two tetramers, the interaction apparently mediated by their disordered N- and C-termini. This information may be useful in future studies directed at reducing the heterogeneity and self-association of the protein.

Sample Preparation and Technical Setup for NMR Spectroscopy with Integral Membrane Proteins.

NMR spectroscopy is a method of choice to characterize structure, function, and dynamics of integral membrane proteins at atomic resolution. Here, we describe protocols for sample preparation and characterization by NMR spectroscopy of two integral membrane proteins with different architecture, the α-helical membrane protein MsbA and the β-barrel membrane protein BamA. The protocols describe recombinant expression in E. coli, protein refolding, purification, and reconstitution in suitable membrane mimetics, as well as key setup steps for basic NMR experiments. These include experiments on protein samples in the solid state under magic angle spinning (MAS) conditions and experiments on protein samples in aqueous solution. Since MsbA and BamA are typical examples of their respective architectural classes, the protocols presented here canalso serve asa reference for other integral membrane proteins.

Hydrogen/Deuterium Exchange Mass Spectrometry for the Structural Analysis of Detergent-Solubilized Membrane Proteins.

Integral membrane proteins are involved in numerous biological functions and represent important drug targets. Despite their abundance in the human proteome, the number of integral membrane protein structures is largely underrepresented in the Protein Data Bank. The challenges associated with the biophysical characterization of such biological systems are well known. Most structural approaches, including X-ray crystallography, SAXS, or mass spectrometry (MS), require the complete solubilization of membrane proteins in aqueous solutions. Detergents are frequently used for this task, but may interfere with the analysis, as is the case with MS. The use of "MS-friendly" detergents, such as non-ionic alkyl glycoside detergents, has greatly facilitated the analysis of detergent-solubilized membrane proteins. Here, we describe a protocol, which we have successfully implemented in our laboratory to study the structure and dynamics of detergent-solubilized integral membrane proteins by Hydrogen/Deuterium eXchange and Mass Spectrometry (HDX-MS). The procedure does not require detergent removal prior to MS analysis, instead taking advantage of the ultra-high pressure chromatographic system to separate deuterated peptides from "MS-friendly" detergents.

Regulation of KcsA by Anionic Phospholipids

Membrane lipids are known to modulate the structure and function of integral membrane proteins. This effect can be achieved directly, by specific binding sites on the protein surface, or indirectly, by changing the physical properties of the membrane bilayer. In the former, binding sites at the interface between two protein subunits, known as non-annular sites, have been implicated. Anionic lipids have been shown to modulate various functional properties of the KcsA K+-channel in different manners, and are required for channel function. Here, we present molecular dynamics simulations of KcsA with anionic and zwitterionic lipids bound in non-annular sites, in order to deduce the molecular mechanism of action of membrane lipids in the prototypical K+-channel.

Studying Lipid-Protein Interactions with Electron Paramagnetic Resonance Spectroscopy of Spin-Labeled Lipids.

Spin label electron paramagnetic resonance (EPR) of lipid-protein interactions reveals crucial features of the structure and assembly of integral membrane proteins. Spin-label EPR spectroscopy is the technique of choice to characterize the protein solvating lipid shell in its highly dynamic nature, because the EPR spectra of lipids that are spin-labeled close to the terminal methyl end of their acyl chains display two spectral components, those corresponding to lipids directly contacting the protein and those corresponding to lipids in the bulk fluid bilayer regions of the membrane. In this chapter, typical spin label EPR procedures are presented that allow determination of the stoichiometry of interaction of spin-labeled lipids with the intramembranous region of membrane proteins or polypeptides, as well as the association constant of the spin-labeled lipid with respect to the host lipid. The lipids giving rise to a so-called immobile spectral component in the EPR spectrum of such samples are identified as the motionally restricted first-shell lipids solvating membrane proteins in biomembranes. Stoichiometry and selectivity are directly related to the structure of the intramembranous sections of membrane-associated proteins or polypeptides and can be used to study the state of assembly of such proteins in the membrane. Since these characteristics of lipid-protein interactions are discussed in detail in the literature (see ref. Marsh, Eur Biophys J 39:513-525, 2010 for a recent review), here we focus more on how to spin label model membranes and biomembranes and how to measure and analyze the two-component EPR spectra of spin-labeled lipids in phospholipid bilayers that contain proteins or polypeptides. After a description of how to prepare spin-labeled model and native biological membranes, we present the reader with computational procedures for determining the molar fraction of motionally restricted lipids when both, one or none of the pure isolated-mobile or immobile-spectral components are available. With these topics, this chapter complements a previous methodological paper (Marsh, Methods 46:83-96, 2008). The interpretation of the data is discussed briefly, as well as other relevant and recent spin label EPR techniques for studying lipid-protein interactions, not only from the point of view of lipid chain dynamics.

Computational Studies for Structure-Based Drug Designing Against Transmembrane Receptors: pLGICs and Class A GPCRs

Biological cell is the fundamental building block of every living system. The plasma membrane, a phospholipid bilayer consisting of two asymmetric leaflets, defines its existence by separating the interior from the exterior. This low dielectric barrier selectively prevents the passage of hydrophilic and charged compounds including small ions. Integral transmembrane proteins span the entire bilayer and take part in small-molecule transport and complex signaling pathways while functioning as receptors and/or ion channels. These proteins carry important biological functions and hence are attractive drug targets. Present review considers the members of two important protein superfamilies that provided the major pharmaceutical drug-targets, viz., Cys-loop pentameric ligand gated ion channels (pLGICs) and class A G-protein-coupled receptors (GPCRs). The crystal structures of integral membrane proteins are difficult to obtain. It limited their structural investigation and associated structure-based drug designing (SBDD). However, recent advancement in crystallographic techniques yielded some important crystal structures. While the advancement of computational science guided their study even in the absence of crystal structures through the homology/comparative modelling approaches. These proteins possess multiple ligand binding sites including both orthosteric and allosteric sites. Addressing the multidimensional problem of understanding the structure and dynamics of such big proteins, multisite-protein-ligand complexes is now possible with molecular dynamics simulation approach, enabled with highly enhanced computational power. Overall the discussion highlights the understanding of structure-function relationship that guides SBDD of these interesting and important transmembrane proteins.

Nanopore-Confined Bilayers: A Model of Biomembranes with Defined Curvature and a Tool for Oriented Sample Magnetic Resonance

It is well established that biological function of cellular membranes is determined by both membrane proteins and the membrane biochemical composition. Moreover, the membrane shape/local curvature may also play a role in modulating membrane properties and the proteins’ function. While specific biomembrane compositions are more readily replicated in biophysical bench experiments, formation of bilayers of specific shapes and curvature represents a more challenging task. Macroscopic alignment of such membranes – an important prerequisite of high resolution magnetic resonance and other studies – is even more difficult especially over a broad range of experimental conditions such pH, ionic strength, and temperature. Here we describe methods for forming self-assembled lipid nanotubular bilayers inside cylindrical nanopores composed of anodic aluminum oxide (AAO). Such hybrid nanostructures, named lipid nanotube arrays, represent a new type of substrate-supported and macroscopically-aligned lipid bilayers of defined curvature that have many attractive features for both membrane biophysics and structure-function protein studies by spectroscopic techniques. Optical properties of AAO allow for assessing the integrity of membrane protein complexes by UV-vis while high density of the deposited lipids and proteins enable examination by other biophysical methods, including DSC, QCM, and magnetic resonance. The latter studies have shown that the individual lipids in such nanopore-confined structures maintain fast uniaxial diffusion and a high degree of macroscopic alignment. The macroscopic alignment enables detailed studies of effects of lipid composition on structure of integral membrane proteins by solid state oriented sample NMR, EPR and DEER. Accessibility of either both or mainly inner leaflet of the nanotubular bilayers to water-soluble species provides for studies of protein, peptides, and drug binding. Supported by DE-FG02-02ER15354 to AIS.

The cubicon method for concentrating membrane proteins in the cubic mesophase.

The lipid cubic phase (in meso) method is an important approach for generating crystals and high-resolution X-ray structures of integral membrane proteins. However, as a consequence of instability, it can be impossible-using traditional methods-to concentrate certain membrane proteins and complexes to values suitable for in meso crystallization and structure determination. The cubicon method described here exploits the amphiphilic nature of membrane proteins and their natural tendency to partition preferentially into lipid bilayers from aqueous solution. Using several rounds of reconstitution, the protein concentration in the bilayer of the cubic mesophase can be ramped up stepwise from less than a milligram per milliliter to tens of milligrams per milliliter for crystallogenesis. The general applicability of the method is demonstrated with five integral membrane proteins: the β2-adrenergic G protein-coupled receptor (β2AR), the peptide transporter (PepTSt), diacylglycerol kinase (DgkA), the alginate transporter (AlgE) and the cystic fibrosis transmembrane conductance regulator (CFTR). In the cases of β2AR, PepTSt, DgkA and AlgE, an effective 20- to 45-fold concentration was realized, resulting in a protein-laden mesophase that allowed the formation of crystals using the in meso method and structure determination to resolutions ranging from 2.4 Å to 3.2 Å. In addition to opening up in meso crystallization to a broader range of integral membrane protein targets, the cubicon method should find application in situations that require membrane protein reconstitution in a lipid bilayer at high concentrations. These applications include functional and biophysical characterization studies for ligand screening, drug delivery, antibody production and protein complex formation. A typical cubicon experiment can be completed in 3-5 h.

Comprehensive analysis of the numbers, lengths and amino acid compositions of transmembrane helices in prokaryotic, eukaryotic and viral integral membrane proteins of high-resolution structure

We report a comprehensive analysis of the numbers, lengths and amino acid compositions of transmembrane helices in 235 high-resolution structures of integral membrane proteins. The properties of 1551 transmembrane helices in the structures were compared with those obtained by analysis of the same amino acid sequences using topology prediction tools. Explanations for the 81 (5.2%) missing or additional transmembrane helices in the prediction results were identified. Main reasons for missing transmembrane helices were mis-identification of N-terminal signal peptides, breaks in α-helix conformation or charged residues in the middle of transmembrane helices and transmembrane helices with unusual amino acid composition. The main reason for additional transmembrane helices was mis-identification of amphipathic helices, extramembrane helices or hairpin re-entrant loops. Transmembrane helix length had an overall median of 24 residues and an average of 24.9 ± 7.0 residues and the most common length was 23 residues. The overall content of residues in transmembrane helices as a percentage of the full proteins had a median of 56.8% and an average of 55.7 ± 16.0%. Amino acid composition was analysed for the full proteins, transmembrane helices and extramembrane regions. Individual proteins or types of proteins with transmembrane helices containing extremes in contents of individual amino acids or combinations of amino acids with similar physicochemical properties were identified and linked to structure and/or function. In addition to overall median and average values, all results were analysed for proteins originating from different types of organism (prokaryotic, eukaryotic, viral) and for subgroups of receptors, channels, transporters and others.