Membrane Protein Topology Research Articles

Structural Dynamics and Conformational Equilibria of SERCA Regulatory Proteins in Membranes by Solid-State NMR Restrained Simulations

Solid-state NMR spectroscopy is emerging as a powerful approach to determine structure, topology, and conformational dynamics of membrane proteins at the atomic level. Conformational dynamics are often inferred and quantified from the motional averaging of the NMR parameters. However, the nature of these motions is difficult to envision based only on spectroscopic data. Here, we utilized restrained molecular dynamics simulations to probe the structural dynamics, topology and conformational transitions of regulatory membrane proteins of the calcium ATPase SERCA, namely sarcolipin and phospholamban, in explicit lipid bilayers. Specifically, we employed oriented solid-state NMR data, such as dipolar couplings and chemical shift anisotropy measured in lipid bicelles, to refine the conformational ensemble of these proteins in lipid membranes. The samplings accurately reproduced the orientations of transmembrane helices and showed a significant degree of convergence with all of the NMR parameters. Unlike the unrestrained simulations, the resulting sarcolipin structures are in agreement with distances and angles for hydrogen bonds in ideal helices. In the case of phospholamban, the restrained ensemble sampled the conformational interconversion between T (helical) and R (unfolded) states for the cytoplasmic region that could not be observed using standard structural refinements with the same experimental data set. This study underscores the importance of implementing NMR data in molecular dynamics protocols to better describe the conformational landscapes of membrane proteins embedded in realistic lipid membranes.

A Disease-causing Mutation Illuminates the Protein Membrane Topology of the Kidney-expressed Prohibitin Homology (PHB) Domain Protein Podocin

Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocin(P118L)) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2(P134S)). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. Podocin(P118L) and MEC-2(P134S) did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.

Biochemically characterizing the subcellular localization of peroxisomal proteins by fractionation, protease protection, and carbonate extraction.

Traditional biochemical approaches, as well as the complementary methods of living cell fluorescence microscopy and immunofluorescence microscopy, can serve to characterize the subcellular localization of proteins and organelles. This chapter describes methods for isolation of crude organelle fractions from methanol- or oleate-grown Pichia pastoris, followed by protease protection and carbonate extraction assays to dissect the subcellular localization of peroxisomal matrix and membrane proteins. These biochemical tools can be used to analyze the targeting efficiency of proteins to the peroxisome membrane and matrix, as well as the topology of membrane proteins.

Lipid-Mediated Polypeptide Interactions in Membranes: Case Study on the Synergism Between Linear Cationic Antimicrobial Peptides

Biophysical investigations, that aim to explain the antimicrobial activities of cationic linear peptides will be presented (1,2). In particular we aim to understand how some mixtures of these peptides exhibit synergistic activities. Therefore, the structure, topology and dynamics of PGLa and magainin 2 were investigated in oriented phospholipid bilayers using solid-state NMR in the presence or absence of the other peptide and as a function of the membrane lipid composition (2,3). Furthermore, fluorescence spectroscopy was used to investigate how the peptides interact with each other within the lipid bilayer environment.Whereas, magainin 2 exhibits stable in-planar alignments under all conditions investigated PGLa adopts a number of different membrane topologies with considerable tilt angle variations (2,3). Notably, the hydrophobic thickness modulates the alignment of PGLa (1). In equimolar mixtures of PGLa and magainin 2 the former adopts transmembrane orientations in DMPC when at the same time magainin 2 remains associated with the surface (1). In contrast in bilayers, which represents better the natural membrane composition (1-palmitoyl-2-oleoyl-phospholipids), both peptides adopt a surface oriented topology. Therefore, lipid-mediated interactions play a fundamental role in determining the topology of membrane peptides and proteins (3) and thereby potentially also the regulation of their activities. These results have important consequences for the mechanistic models explaining synergistic activities of the peptide mixtures and will be discussed in the context of unpublished data where membrane structure and interactions are correlated with biological activities. Furthermore, recent results obtained with DNP enhanced solid-state NMR spectroscopy of supported lipid bilayer samples using prototype equipment will be presented.

Localization and Topology of Thylakoid Membrane Proteins in Land Plants

Localization and Topology of Thylakoid Membrane Proteins in Land Plants

Characterization of Avirulent TnphoA Mutants in <i>Agrobacterium tumefaciens</i> to Enhance Transformation Efficiency

Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane protein. Agrobacterium strain A6007 was mutagenized with E. coli strain mm294A plasmid pRK609 having TnphoA to obtain mutants defective in virulence. Because alkaline phosphatase activity is only detected when the PhoA gene product from the transposon is secreted out of the protoplasm, the virulence mutants are located in genes that code for transmembrane or periplasmic proteins. Attempts were made to obtain the sequences adjacent to the TnphoA inserts through several different approaches including Inverse PCR, Cloning, and Tail PCR. Transposon-adjacent sequence was obtained from one membrane anchor subunit in Bradyrhizobium japonicum i.e. succinate dehydrogenase which has enhanced transformation efficiency.

Signal peptide discrimination and cleavage site identification using SVM and NN

About 15% of all proteins in a genome contain a signal peptide (SP) sequence, at the N-terminus, that targets the protein to intracellular secretory pathways. Once the protein is targeted correctly in the cell, the SP is cleaved, releasing the mature protein. Accurate prediction of the presence of these short amino-acid SP chains is crucial for modelling the topology of membrane proteins, since SP sequences can be confused with transmembrane domains due to similar composition of hydrophobic amino acids. This paper presents a cascaded Support Vector Machine (SVM)-Neural Network (NN) classification methodology for SP discrimination and cleavage site identification.The proposed method utilises a dual phase classification approach using SVM as a primary classifier to discriminate SP sequences from Non-SP. The methodology further employs NNs to predict the most suitable cleavage site candidates. In phase one, a SVM classification utilises hydrophobic propensities as a primary feature vector extraction using symmetric sliding window amino-acid sequence analysis for discrimination of SP and Non-SP. In phase two, a NN classification uses asymmetric sliding window sequence analysis for prediction of cleavage site identification.The proposed SVM-NN method was tested using Uni-Prot non-redundant datasets of eukaryotic and prokaryotic proteins with SP and Non-SP N-termini. Computer simulation results demonstrate an overall accuracy of 0.90 for SP and Non-SP discrimination based on Matthews Correlation Coefficient (MCC) tests using SVM. For SP cleavage site prediction, the overall accuracy is 91.5% based on cross-validation tests using the novel SVM-NN model.

Sensitivity enhancement and contrasting information provided by free radicals in oriented-sample NMR of bicelle-reconstituted membrane proteins

Elucidating structure and topology of membrane proteins (MPs) is essential for unveiling functionality of these important biological constituents. Oriented-sample solid-state NMR (OS-NMR) is capable of providing such information on MPs under nearly physiological conditions. However, two dimensional OS-NMR experiments can take several days to complete due to long longitudinal relaxation times combined with the large number of scans to achieve sufficient signal sensitivity in biological samples. Here, free radicals 5-DOXYL stearic acid, TEMPOL, and CAT-1 were added to uniformly 15N-labeled Pf1 coat protein reconstituted in DMPC/DHPC bicelles, and their effect on the longitudinal relaxation times (T1Z) was investigated. The dramatically shortened T1Z’s allowed for the signal gain per unit time to be used for either: (i) up to a threefold reduction of the total experimental time at 99% magnetization recovery or (ii) obtaining up to 74% signal enhancement between the control and radical samples during constant experimental time at “optimal” relaxation delays. In addition, through OS-NMR and high-field EPR studies, free radicals were able to provide positional constraints in the bicelle system, which provide a description of the location of each residue in Pf1 coat protein within the bicellar membranes. This information can be useful in the determination of oligomerization states and immersion depths of larger membrane proteins.

Biochemical properties of human dehydrogenase/reductase (SDR family) member 7

Dehydrogenase/reductase (SDR family) member 7 (DHRS7, retSDR4, SDR34C1) is a previously uncharacterized member of the short-chain dehydrogenase/reductase (SDR) superfamily. While human SDR members are known to play an important role in various (patho)biochemical pathways including intermediary metabolism and biotransformation of xenobiotics, only 20% of them are considered to be well characterized. Based on phylogenetic tree and SDR sequence clusters analysis DHRS7 is a close relative to well-known SDR member 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) that participates in metabolism of endogenous and xenobiotic substances with carbonyl group. The aim of present study is to determine the basic biochemical properties of DHRS7 and its possible involvement in metabolism of substrates with carbonyl group. For the first time the computational predictions of this membrane protein and membrane topology were experimentally confirmed. DHRS7 has been demonstrated to be an integral protein facing the lumen of the endoplasmic reticulum with lack of posttranscriptional glycosylation modification. Subsequently, NADP(H) cofactor preference and enzymatic reducing activity of DHRS7 was determined towards endogenous substrates with a steroid structure (cortisone, 4-androstene-3,17-dion) and also toward relevant exogenous substances bearing a carbonyl group harmful to human health (1,2-naphtoquinone, 9,10-phenantrenequinone). In addition to 11β-HSD1, DHRS7 is another enzyme from SDR superfamily that have been proved, at least in vitro, to contribute to the metabolism of xenobiotics with carbonyl group.

Positive Charge Loading at Protein Termini Is Due to Membrane Protein Topology, Not a Translational Ramp

In the great majority of genomes, the use of positive charge increases, on average, approaching protein N-termini. Such charged residues slow ribosomes by interacting with the negatively charged exit tunnel. This has been proposed to be selectively advantageous as it provides an elongation speed ramp at translational starts. Positive charges, however, are known to orientate proteins in membranes by the positive-inside rule whereby excess charge lies on the cytoplasmic side of the membrane. Which of these two models better explains the N-terminal loading of positively charged amino acids? We find strong evidence that the tendency for average positive charge use to increase at termini is exclusively due to membrane protein topology: 1) increasing N-terminal positive charge is not found in cytosolic proteins, but in transmembrane ones with cytosolic N-termini, with signal sequences contributing additional charge; 2) positive charge density at N-termini corresponds to the length of cytoplasmically exposed transmembrane tails, its usage increasing just up until the membrane; 3) membrane-related patterns are repeated at C-termini, where no ramp is expected; and 4) N-terminal positive charge patterns are no different from those seen internally in proteins in membrane-associated domains. The overall apparent increase in positive charge across all N-termini results from membrane proteins using positive charge adjacent to the cytosolic leaflet, combined with a skewed distribution of where N-termini cross the plasma membrane; 5) while Escherichia coli was predicted to have a 5' ribosomal occupancy ramp of at least 31 codons, in contrast to what is seen in yeast, we find in ribosomal footprinting data no evidence for such a ramp. In sum, we find no need to invoke a translational ramp to explain the rising positive charge densities at N-termini. The membrane orientation model makes a full account of the trend.

Fifty Years Fused to Lac

I recount the history of how I became interested in the use of gene fusions for studying biological problems. Initially, selections for mutations that would restore function to an inactivated lac operon unexpectedly yielded fusions in which lac was expressed from the controlling elements of upstream genes. Subsequently, by chance, I generated strains in which the lac operon was transposed from its normal position on the chromosome to a position close to the trp operon, thus facilitating sets of useful fusions of the two operons. The development of a more generalized technique for obtaining fusions by my student Malcolm Casadaban opened up a much broader set of biological problems that could be approached with fusions. Work on these problems included the study of protein translocation across membranes, the analysis of membrane protein topology, and the discovery of the pathway of electron transfer that leads to disulfide bond formation in proteins.

Bioinspired Exosome-Mimetic Nanovesicles for Targeted Delivery of Chemotherapeutics to Malignant Tumors

Exosomes, the endogenous nanocarriers that can deliver biological information between cells, were recently introduced as new kind of drug delivery system. However, mammalian cells release relatively low quantities of exosomes, and purification of exosomes is difficult. Here, we developed bioinspired exosome-mimetic nanovesicles that deliver chemotherapeutics to the tumor tissue after systemic administration. The chemotherapeutics-loaded nanovesicles were produced by the breakdown of monocytes or macrophages using a serial extrusion through filters with diminishing pore sizes (10, 5, and 1 μm). These cell-derived nanovesicles have similar characteristics with the exosomes but have 100-fold higher production yield. Furthermore, the nanovesicles have natural targeting ability of cells by maintaining the topology of plasma membrane proteins. In vitro, chemotherapeutic drug-loaded nanovesicles induced TNF-α-stimulated endothelial cell death in a dose-dependent manner. In vivo, experiments in mice showed that the chemotherapeutic drug-loaded nanovesicles traffic to tumor tissue and reduce tumor growth without the adverse effects observed with equipotent free drug. Furthermore, compared with doxorubicin-loaded exosomes, doxorubicin-loaded nanovesicles showed similar in vivo antitumor activity. However, doxorubicin-loaded liposomes that did not carry targeting proteins were inefficient in reducing tumor growth. Importantly, removal of the plasma membrane proteins by trypsinization eliminated the therapeutic effects of the nanovesicles both in vitro and in vivo. Taken together, these studies suggest that the bioengineered nanovesicles can serve as novel exosome-mimetics to effectively deliver chemotherapeutics to treat malignant tumors.

High-accuracy prediction of transmembrane inter-helix contacts and application to GPCR 3D structure modeling

Residue-residue contacts across the transmembrane helices dictate the three-dimensional topology of alpha-helical membrane proteins. However, contact determination through experiments is difficult because most transmembrane proteins are hard to crystallize. We present a novel method (MemBrain) to derive transmembrane inter-helix contacts from amino acid sequences by combining correlated mutations and multiple machine learning classifiers. Tested on 60 non-redundant polytopic proteins using a strict leave-one-out cross-validation protocol, MemBrain achieves an average accuracy of 62%, which is 12.5% higher than the current best method from the literature. When applied to 13 recently solved G protein-coupled receptors, the MemBrain contact predictions helped increase the TM-score of the I-TASSER models by 37% in the transmembrane region. The number of foldable cases (TM-score >0.5) increased by 100%, where all G protein-coupled receptor templates and homologous templates with sequence identity >30% were excluded. These results demonstrate significant progress in contact prediction and a potential for contact-driven structure modeling of transmembrane proteins. www.csbio.sjtu.edu.cn/bioinf/MemBrain/

Topological Plasticity of Enzymes Involved in Disulfide Bond Formation Allows Catalysis in Either the Periplasm Or the Cytoplasm

The transmembrane enzymes disulfide bond forming enzyme B (DsbB) and vitamin K epoxide reductase (VKOR) are central to oxidative protein folding in the periplasm of prokaryotes. Catalyzed formation of structural disulfide bonds in proteins also occurs in the cytoplasm of some hyperthermophilic prokaryotes through currently, poorly defined mechanisms. We aimed to determine whether DsbB and VKOR can be inverted in the membrane with retention of activity. By rational design of inversion of membrane topology, we engineered DsbB mutants that catalyze disulfide bond formation in the cytoplasm of Escherichia coli. This represents the first engineered inversion of a transmembrane protein with demonstrated conservation of activity and substrate specificity. This successful designed engineering led us to identify two naturally occurring and oppositely oriented VKOR homologues from the hyperthermophile Aeropyrum pernix that promote oxidative protein folding in the periplasm or cytoplasm, respectively, and hence defines the probable route for disulfide bond formation in the cytoplasm of hyperthermophiles. Our findings demonstrate how knowledge on the determinants of membrane protein topology can be used to de novo engineer a metabolic pathway and to unravel an intriguingly simple evolutionary scenario where a new “adaptive” cellular process is constructed by means of membrane protein topology inversion.

Mapping Peptide Thiol Accessibility in Membranes Using a Quaternary Ammonium Isotope-Coded Mass Tag (ICMT)

The plasma membrane contains a diverse array of proteins, including receptors, channels, and signaling complexes, that serve as decision-making centers. Investigation of membrane protein topology is important for understanding the function of these types of protein. Here, we report a method to determine protein topology in the membrane that utilizes labeling of cysteine with isotope-coded mass tags. The mass tags contain a thiol reactive moiety, linker, and a quaternary ammonium group to aid ionization in the mass spectrometer and were synthesized in both light and heavy (deuterated) forms. The probes were found to be membrane impermeable when applied to lipid vesicles. To assess the utility of the probes for mapping peptide thiol topology, we employed a two-step labeling procedure. Vesicles containing α-helical transmembrane peptides were labeled with heavy (or light) probe, solubilized by detergent, and then labeled by an excess of the complementary probe. Peptide for which the cysteine was oriented in the center of the lipid bilayer was not labeled until the lipid vesicles were lysed with detergent, consistent with the membrane impermeability of the probes and reduced ionization of the thiol in the hydrophobic membrane. Peptide for which the cysteine was positioned in the headgroup zone of the lipid bilayer was labeled rapidly. Peptide for which the cysteine was positioned below the headgroup abutting the hydrocarbon region was labeled at a reduced rate compared to the fully accessible cysteine. Moreover, the effect of lipid bilayer structure on the kinetics of peptide and lipid flipping in the bilayer was readily measured with our two-step labeling method. The small sample size required, the ease and rapidity of sample preparation, and the amenability of MALDI-TOF mass spectral analysis to the presence of lipids will enable future facile investigation of membrane proteins in a cellular context.

In vitro reconstitution of lipid-dependent dual topology and postassembly topological switching of a membrane protein

Phospholipids could exert their effect on membrane protein topology either directly by interacting with topogenic signals of newly inserted proteins or indirectly by influencing the protein assembly machinery. In vivo lactose permease (LacY) of Escherichia coli displays a mixture of topological conformations ranging from complete inversion of the N-terminal helical bundle to mixed topology and then to completely native topology as phosphatidylethanolamine (PE) is increased from 0% to 70% of membrane phospholipids. These topological conformers are interconvertible by postassembly synthesis or dilution of PE in vivo. To investigate whether coexistence of multiple topological conformers is dependent solely on the membrane lipid composition, we determined the topological organization of LacY in an in vitro proteoliposome system in which lipid composition can be systematically controlled before (liposomes) and after (fliposomes) reconstitution using a lipid exchange technique. Purified LacY reconstituted into preformed liposomes of increasing PE content displayed inverted topology at low PE and then a mixture of inverted and proper topologies with the latter increasing with increasing PE until all LacY adopted its native topology. Interconversion between topological conformers of LacY was observed in a PE dose-dependent manner by either increasing or decreasing PE levels in proteoliposomes postreconstitution of LacY, clearly demonstrating that membrane protein topology can be changed simply by changing membrane lipid composition independent of other cellular factors. The results provide a thermodynamic-based lipid-dependent model for shifting the equilibrium between different conformational states of a membrane protein.

Solid-State NMR-Based Approaches for Supramolecular Structure Elucidation

Supramolecular chemistry provides structural and conformational information about complexes formed from multiple molecules. While the molecule is held together by strong intramolecular contacts like covalent bonds, supramolecular structures can be further stabilized by weaker or transient intermolecular interactions. These interactions can confer a great diversity and sensitivity to exogenous factors like temperature, pressure, or ionic strength to multimolecular arrangements. Solid-state nuclear magnetic resonance (ssNMR) can provide atomic-scale structural and dynamical information in highly disordered or heterogeneous biological systems, even in complex environments such as cellular membranes or whole cells. In these systems, the molecule of interest no longer exists as a separate unit, but it entangles with its surroundings in a dynamic interplay. Researchers have long accounted for the complexity of these intermolecular arrangements through a rather phenomenological description. But now the focus is shifting toward a detailed understanding of supramolecular structure at atomic resolution, constantly expanding our understanding of the stunning influence of the environment. In this Account, we discuss how ssNMR can help to dissect the remarkable interplay between intra- and intermolecular interactions. We describe biochemical and spectroscopic strategies that tailor ssNMR spectroscopic methods to the challenge of supramolecular structure investigation. In particular, we consider protein-protein interactions or the protein-membrane topology, and we review recent applications of these techniques. Furthermore, we summarize methods for integrating ssNMR information with other experimental techniques or computational methods, and we offer perspectives on how this overall information allows us to target increasingly large and intricate supramolecular structures of biomolecules. Advancements in ssNMR methodology and instrumentation, including the incorporation of signal enhancement methods such as dynamic nuclear polarization will further increase the potential of ssNMR spectroscopy, and together with additional developments in the field of NMR-hybrid strategies, ssNMR may become an ideal tool to study the heterogeneous, dynamic, and often transient nature of molecular interactions in complex biological systems.

Role of membrane‐proximal basic residues in cell surface trafficking of GPR15

Membrane‐proximal basic residues (MPBRs) in the cytoplasmic region are one of the important factors that determine the topology of membrane proteins during biogenesis in the endoplasmic reticulum (ER), known as a positive‐inside rule. However, MPBRs have been also found to be critically involved in the ER export of certain membrane proteins such as Golgi‐resident glycosyltransferases. Here we investigated the role of cytoplasmic MPBRs (Arg310Arg311) in the cell surface trafficking of GPR15, a G protein‐coupled receptor that serves as an HIV co‐receptor. The mutation of Arg310Arg311 to Ala did not alter the topology but abolished the O‐glycosylation and cell surface expression of GPR15. Immunocytochemistry indicated that Ala mutant can exit ER and reach ER‐Golgi intermediate compartment (ERGIC) and cis‐Golgi, but not trans‐Golgi. Our study suggests the possibility that MPBRs play a pivotal role in the anterograde trafficking of a broad range of membrane proteins.

Inmembrane, a bioinformatic workflow for annotation of bacterial cell-surface proteomes

BackgroundThe annotation of surface exposed bacterial membrane proteins is an important step in interpretation and validation of proteomic experiments. In particular, proteins detected by cell surface protease shaving experiments can indicate exposed regions of membrane proteins that may contain antigenic determinants or constitute vaccine targets in pathogenic bacteria.ResultsInmembrane is a tool to predict the membrane proteins with surface-exposed regions of polypeptide in sets of bacterial protein sequences. We have re-implemented a protocol for Gram-positive bacterial proteomes, and developed a new protocol for Gram-negative bacteria, which interface with multiple predictors of subcellular localization and membrane protein topology. Through the use of a modern scripting language, inmembrane provides an accessible code-base and extensible architecture that is amenable to modification for related sequence annotation tasks.ConclusionsInmembrane easily integrates predictions from both local binaries and web-based queries to help gain an overview of likely surface exposed protein in a bacterial proteome. The program is hosted on the Github repository http://github.com/boscoh/inmembrane.

Membrane Protein Structure Determination Through Assembly of Trans-Membrane Helices Guided by Limited Experimental Data

We present BCL::Fold, a new computational algorithm that rapidly predicts the topology of helical membrane proteins through assembly of secondary structure elements. A symmetry folding mode allows for the prediction of homomultimers. Final models are evaluated using a knowledge-based energy potential. In a benchmark test of 40 proteins with up to 1200 residues, the method samples the correct topology in 39 cases. We will demonstrate the combination of this algorithm with limited NMR, EPR, and cryo-EM data for membrane protein structure determination. Although great progress in determining the structure of integral, helical, multi-span membrane proteins was made in recent years, only 10% of the expected 1000 membrane protein folds are represented by at least one example in the protein databank. For the 900 membrane proteins topologies that remain to be determined, billions of topologies are possible with an increasing number of trans-membrane spans. This large structural space is contrasted by experimental datasets that - if available at all - are limited in resolution of density map or number of structural restraints. It is the objective of BCL::Fold to enable and accelerate structure determination for these proteins.