Integral Membrane Protein Complex Research Articles

Discovery of Therapeutic Antibodies Targeting Complex Multi-Spanning Membrane Proteins.

Complex integral membrane proteins, which are embedded in the cell surface lipid bilayer by multiple transmembrane spanning polypeptides, encompass families of proteins that are important target classes for drug discovery. These protein families include G protein-coupled receptors, ion channels, transporters, enzymes, and adhesion molecules. The high specificity of monoclonal antibodies and the ability to engineer their properties offers a significant opportunity to selectively bind these target proteins, allowing direct modulation of pharmacology or enabling other mechanisms of action such as cell killing. Isolation of antibodies that bind these types of membrane proteins and exhibit the desired pharmacological function has, however, remained challenging due to technical issues in preparing membrane protein antigens suitable for enabling and driving antibody drug discovery strategies. In this article, we review progress and emerging themes in defining discovery strategies for a generation of antibodies that target these complex membrane protein antigens. We also comment on how this field may develop with the emerging implementation of computational techniques, artificial intelligence, and machine learning.

Distance tuneable integral membrane protein containing floating bilayers via in situ directed self-assembly.

Model membranes allow for structural and biophysical studies on membrane biochemistry at the molecular level, albeit on systems of reduced complexity which can limit biological accuracy. Floating supported bilayers offer a means of producing planar lipid membrane models not adhered to a surface, which allows for improved accuracy compared to other model membranes. Here we communicate the incorporation of an integral membrane protein complex, the multidomain β-barrel assembly machinery (Bam), into our recently developed in situ self-assembled floating supported bilayers. Using neutron reflectometry and quartz crystal microbalance measurements we show this sample system can be fabricated using a two-step self-assembly process. We then demonstrate the complexity of the model membrane and tuneability of the membrane-to-surface distance using changes in the salt concentration of the bulk solution. Results demonstrate an easily fabricated, biologically accurate and tuneable membrane assay system which can be utilized for studies on integral membrane proteins within their native lipid matrix.

Trifunctional Saxitoxin Conjugates for Covalent Labeling of Voltage-Gated Sodium Channels.

Voltage-gated sodium ion channels (NaV s) are integral membrane protein complexes responsible for electrical signal conduction in excitable cells. Methods that enable selective labeling of NaV s hold potential value for understanding how channel regulation and post-translational modification are influenced during development and in response to diseases and disorders of the nervous system. We have developed chemical reagents patterned after (+)-saxitoxin (STX) - a potent and reversible inhibitor of multiple NaV isoforms - and affixed with a reactive electrophile and either a biotin cofactor, fluorophore, or 'click' functional group for labeling wild-type channels. Our studies reveal enigmatic structural effects of the probes on the potency and efficiency of covalent protein modification. Among the compounds analyzed, a STX-maleimide-coumarin derivative is most effective at irreversibly blocking Na+ conductance when applied to recombinant NaV s and endogenous channels expressed in hippocampal neurons. Mechanistic analysis supports the conclusion that high-affinity toxin binding is a prerequisite for covalent protein modification. Results from these studies are guiding the development of next-generation tool compounds for selective modification of NaV s expressed in the plasma membranes of cells.

Pentameric ligand gated ion channels' intracellular domain interaction with RIC3 chaperone.

Pentameric ligand-gated ion channels (pLGICs) are cys-loop receptors important in transduction of electrical signals between neurons in the peripheral and central nervous system. Their significant role in neurotransmission has increased the research in neuropharmacology. Members of this protein family are found in mammals, but also in prokaryotes that can often incorporate additional domains whose roles are largely uncharacterized. pLGICs include nicotinic acetylcholinereceptors, gamma-amino butyric acid A receptors, glycine receptors and type 3 serotonin receptors (5HT3). These receptors are integral membrane protein complexes composed of five subunits that surround the central pore. Each subunit incorporates a large extracellular domain which contains the agonist-binding site, four transmembrane segments (TM1-4) which form the ion pore, a large intracellular domain (ICD) between TM3 and TM4, and a short extracellular C-terminal region. The ionotropic serotonin subtype-3 (5-HT3) receptor is of current interest as a therapeutic target in the treatment of alcohol abuse. Selective pharmacological antagonists reduce alcohol consumption in preclinical and clinical models. To date only two applications have been fully realized in the clinic: the treatment of emesis and irritable-bowel syndrome. Competitive antagonists, called setrons, target 5HT3R, are used in the management of nausea and vomiting associated with radiation and chemotherapies in cancer patients. These drugs interact with off-target subunits of the receptor and consequently lead to many undesired effects including headache and drowsiness. In order to understand and characterize effective control of neurotransmitter-gated pLGICs, we have explored the interaction of 5HT3R with RIC-3 chaperone which mediates functional expression of the channels. In the present study, synthetic pLGIC peptides were used to probe and characterize the interaction with RIC-3 in native sources such as mouse brains and neuronal cell lines.

The conformations and basal conformational dynamics of translocation factor SecDF vary with translocon SecYEG interaction

The general secretory, or Sec, system is a primary protein export pathway from the cytosol of Escherichia coli and all eubacteria. Integral membrane protein complex SecDF is a translocation factor that enhances polypeptide secretion, which is driven by the Sec translocase, consisting of translocon SecYEG and ATPase SecA. SecDF is thought to utilize a proton gradient to effectively pull precursor proteins from the cytoplasm into the periplasm. Working models have been developed to describe the structure and function of SecDF, but important mechanistic questions remain unanswered. Atomic force microscopy (AFM) is a powerful technique for studying the dynamics of single-molecule systems including membrane proteins in near-native conditions. The sharp tip of the AFM provides direct access to membrane-external protein conformations. Here, we acquired AFM images and kymographs (∼100 ms resolution) to visualize SecDF protrusions in near-native supported lipid bilayers and compared the experimental data to simulated AFM images based on static structures. When studied in isolation, SecDF exhibited a stable and compact conformation close to the lipid bilayer surface, indicative of a resting state. Interestingly, upon SecYEG introduction, we observed changes in both SecDF conformation and conformational dynamics. The population of periplasmic protrusions corresponding to an intermediate form of SecDF, which is thought to be active in precursor protein handling, increased more than ninefold. In conjunction, our dynamics measurements revealed an enhancement in the transition rate between distinct SecDF conformations when the translocon was present. Together, this work provides a novel vista of basal-level SecDF conformational dynamics in near-native conditions.

Dynamics of the translocation pore of the human peroxisomal protein import machinery.

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and imported in a posttranslational manner. Intricate protein import machineries have evolved that catalyze the different stages of translocation. In humans, PEX5L was found to be an essential component of the peroxisomal translocon. PEX5L is the main receptor forsubstrate proteins carrying a peroxisomal targeting signal (PTS). Substrates are bound by soluble PEX5L in the cytosol after which the cargo-receptor complex is recruited to peroxisomal membranes. Here, PEX5L interacts with the docking protein PEX14 and becomes part of an integral membrane protein complex that facilitates substrate translocation into the peroxisomal lumen in a still unknown process. In this study, we show that PEX5L containing complexes purified from human peroxisomal membranes constitute water-filled pores when reconstituted into planar-lipid membranes. Channel characteristics were highly dynamic in terms of conductance states, selectivity and voltage- and substrate-sensitivity. Our results show that a PEX5L associated pore exists in human peroxisomes, which can be activated by receptor-cargo complexes.

ChanFAD: A Functional Annotation Database for Ion Channels.

Ion channels are integral membrane protein complexes critical for regulation of membrane potential, cell volume, and other signaling events. As complex molecular assemblies with many interacting partners, ion channels have multiple structural and functional domains. While channel sequence and functional data are readily available across multiple online resources, there is an unmet need for functional annotation directly relating primary sequence information, 2D interactions, and three-dimensional protein structure. To this end, we present ChanFAD (Channel Functional Annotation Database), to provide the research community with a centralized resource for ion channel structure and functional data. ChanFAD provides functional annotation of PDB structures built on the National Center for Biotechnology Information’s iCn3D structure viewing tool while providing additional information such as primary sequence, organism, and relevant links to other databases. Here we provide a brief tour of ChanFAD functionality while showing example use cases involving drug-channel interactions and structural changes based on mutation. ChanFAD is freely available and can be accessed at https://www.chanfad.org/.

Direct visualization of conformations and conformational dynamics of the proton-driven translocation factor SecDF in supported lipid bilayers

The general secretory, or Sec, system is a primary method of exporting proteins from the cytosol of Escherichia coli and all eubacteria. Integral membrane protein complex SecDF is a translocation factor that enhances the polypeptide secretion process that is driven by the Sec translocase, consisting of translocon SecYEG and ATPase SecA. Studies have suggested that SecDF utilizes a proton gradient to undergo cycles of conformational changes to effectively pull polypeptide species into the periplasm, independent of the ATP hydrolysis performed by SecA.

Crystal structure of a photosynthetic LH1-RC in complex with its electron donor HiPIP

Photosynthetic electron transfers occur through multiple components ranging from small soluble proteins to large integral membrane protein complexes. Co-crystallization of a bacterial photosynthetic electron transfer complex that employs weak hydrophobic interactions was achieved by using high-molar-ratio mixtures of a soluble donor protein (high-potential iron-sulfur protein, HiPIP) with a membrane-embedded acceptor protein (reaction center, RC) at acidic pH. The structure of the co-complex offers a snapshot of a transient bioenergetic event and revealed a molecular basis for thermodynamically unfavorable interprotein electron tunneling. HiPIP binds to the surface of the tetraheme cytochrome subunit in the light-harvesting (LH1) complex-associated RC in close proximity to the low-potential heme-1 group. The binding interface between the two proteins is primarily formed by uncharged residues and is characterized by hydrophobic features. This co-crystal structure provides a model for the detailed study of long-range trans-protein electron tunneling pathways in biological systems.

Lipid and protein dynamics of stacked and cation-depletion induced unstacked thylakoid membranes

Chloroplast thylakoid membranes in plants and green algae form 3D architectures of stacked granal membranes interconnected by unstacked stroma lamellae. They undergo dynamic structural changes as a response to changing light conditions that involve grana unstacking and lateral supramolecular reorganization of the integral membrane protein complexes. We assessed the dynamics of thylakoid membrane components and addressed how they are affected by thylakoid unstacking, which has consequences for protein mobility and the diffusion of small electron carriers. By a combined nuclear and electron paramagnetic-resonance approach the dynamics of thylakoid lipids was assessed in stacked and cation-depletion induced unstacked thylakoids of Chlamydomonas (C.) reinhardtii. We could distinguish between structural, bulk and annular lipids and determine membrane fluidity at two membrane depths: close to the lipid headgroups and in the lipid bilayer center. Thylakoid unstacking significantly increased the dynamics of bulk and annular lipids in both areas and increased the dynamics of protein helices. The unstacking process was associated with membrane reorganization and loss of long-range ordered Photosystem II- Light-Harvesting Complex II (PSII-LHCII) complexes. The fluorescence lifetime characteristics associated with membrane unstacking are similar to those associated with state transitions in intact C. reinhardtii cells. Our findings could be relevant for understanding the structural and functional implications of thylakoid unstacking that is suggested to take place during several light-induced processes, such as state transitions, photoacclimation, photoinhibition and PSII repair.

Generating therapeutic monoclonal antibodies to complex multi-spanning membrane targets: Overcoming the antigen challenge and enabling discovery strategies.

Complex integral membrane proteins, which are embedded in the cell surface lipid bilayer by multiple transmembrane spanning helices, encompass families of proteins which are important target classes for drug discovery. These protein families include G protein-coupled receptors, ion channels and transporters. Although these proteins have typically been targeted by small molecule drugs and peptides, the high specificity of monoclonal antibodies offers a significant opportunity to selectively modulate these target proteins. However, it remains the case that isolation of antibodies with desired pharmacological function(s) has proven difficult due to technical challenges in preparing membrane protein antigens suitable to support antibody drug discovery. In this review recent progress in defining strategies for generation of membrane protein antigens is outlined. We also highlight antibody isolation strategies which have generated antibodies which bind the membrane protein and modulate the protein function.

Dynamics of Membrane Proteins Monitored by Single-Molecule Fluorescence Across Multiple Timescales.

Single-molecule techniques provide insights into the heterogeneity and dynamics of ensembles and enable the extraction of mechanistic information that is complementary to high-resolution structural techniques. Here, we describe the application of single-molecule Förster resonance energy transfer to study the dynamics of integral membrane protein complexes on timescales spanning sub-milliseconds to minutes (10-9-102s).

The ghrelin O-acyltransferase structure reveals a catalytic channel for transmembrane hormone acylation

Integral membrane proteins represent a large and diverse portion of the proteome and are often recalcitrant to purification, impeding studies essential for understanding protein structure and function. By combining co-evolutionary constraints and computational modeling with biochemical validation through site-directed mutagenesis and enzyme activity assays, we demonstrate here a synergistic approach to structurally model purification-resistant topologically complex integral membrane proteins. We report the first structural model of a eukaryotic membrane-bound O-acyltransferase (MBOAT), ghrelin O-acyltransferase (GOAT), which modifies the metabolism-regulating hormone ghrelin. Our structure, generated in the absence of any experimental structural data, revealed an unanticipated strategy for transmembrane protein acylation with catalysis occurring in an internal channel connecting the endoplasmic reticulum lumen and cytoplasm. This finding validated the power of our approach to generate predictive structural models for other experimentally challenging integral membrane proteins. Our results illuminate novel aspects of membrane protein function and represent key steps for advancing structure-guided inhibitor design to target therapeutically important but experimentally intractable membrane proteins.

Interfaces Between Alpha-helical Integral Membrane Proteins: Characterization, Prediction, and Docking

Protein-protein interaction (PPI) is an essential mechanism by which proteins perform their biological functions. For globular proteins, the molecular characteristics of such interactions have been well analyzed, and many computational tools are available for predicting PPI sites and constructing structural models of the complex. In contrast, little is known about the molecular features of the interaction between integral membrane proteins (IMPs) and few methods exist for constructing structural models of their complexes. Here, we analyze the interfaces from a non-redundant set of complexes of α-helical IMPs whose structures have been determined to a high resolution. We find that the interface is not significantly different from the rest of the surface in terms of average hydrophobicity. However, the interface is significantly better conserved and, on average, inter-subunit contacting residue pairs correlate more strongly than non-contacting pairs, especially in obligate complexes. We also develop a neural network-based method, with an area under the receiver operating characteristic curve of 0.75 and a Pearson correlation coefficient of 0.70, for predicting interface residues and their weighted contact numbers (WCNs). We further show that predicted interface residues and their WCNs can be used as restraints to reconstruct the structure α-helical IMP dimers through docking for fourteen out of a benchmark set of sixteen complexes. The RMSD100 values of the best-docked ligand subunit to its native structure are <2.5 Å for these fourteen cases. The structural analysis conducted in this work provides molecular details about the interface between α-helical IMPs and the WCN restraints represent an efficient means to score α-helical IMP docking candidates.

Carbohydrate Transport by Group Translocation: The Bacterial Phosphoenolpyruvate: Sugar Phosphotransferase System.

The Bacterial Phosphoenolpyruvate (PEP) : Sugar Phosphotransferase System (PTS) mediates the uptake and phosphorylation of carbohydrates, and controls the carbon- and nitrogen metabolism in response to the availability of sugars. PTS occur in eubacteria and in a few archaebacteria but not in animals and plants. All PTS comprise two cytoplasmic phosphotransferase proteins (EI and HPr) and a species-dependent, variable number of sugar-specific enzyme II complexes (IIA, IIB, IIC, IID). EI and HPr transfer phosphorylgroups from PEP to the IIA units. Cytoplasmic IIA and IIB units sequentially transfer phosphates to the sugar, which is transported by the IIC and IICIID integral membrane protein complexes. Phosphorylation by IIB and translocation by IIC(IID) are tightly coupled. The IIC(IID) sugar transporters of the PTS are in the focus of this review. There are four structurally different PTS transporter superfamilies (glucose, glucitol, ascorbate, mannose) . Crystal structures are available for transporters of two superfamilies: bcIICmal (MalT, 5IWS, 6BVG) and bcIICchb (ChbC, 3QNQ) of B. subtilis from the glucose family, and IICasc (UlaA, 4RP9, 5ZOV) of E. coli from the ascorbate superfamily . They are homodimers and each protomer has an independent transport pathway which functions by an elevator-type alternating-access mechanism. bcIICmal and bcIICchb have the same fold, IICasc has a completely different fold. Biochemical and biophysical data accumulated in the past with the transporters for mannitol (IICBAmtl) and glucose (IICBglc) are reviewed and discussed in the context of the bcIICmal crystal structures. The transporters of the mannose superfamily are dimers of protomers consisting of a IIC and a IID protein chain. The crystal structure is not known and the topology difficult to predict. Biochemical data indicate that the IICIID complex employs a different transport mechanism . Species specific IICIID serve as a gateway for the penetration of bacteriophage lambda DNA across, and insertion of class IIa bacteriocins into the inner membrane. PTS transporters are inserted into the membrane by SecYEG translocon and have specific lipid requirements. Immunoelectron- and fluorescence microscopy indicate a non-random distribution and supramolecular complexes of PTS proteins.

Lipid bilayer position and orientation of novel carprofens, modulators of γ-secretase in Alzheimer's disease

γ-Secretase is an integral membrane protein complex and is involved in the cleavage of the amyloid precursor protein APP to produce amyloid-β peptides. Amyloid-β peptides are considered causative agents for Alzheimer's disease and drugs targeted at γ-secretase are investigated as therapeutic treatments. We synthesized new carprofen derivatives, which showed γ-secretase modulating activity and determined their precise position, orientation, and dynamics in lipid membranes by combining neutron diffraction, solid-state NMR spectroscopy, and molecular dynamics simulations. Our data indicate that the carprofen derivatives are inserted into the membrane interface, where the exact position and orientation depends on the lipid phase. This knowledge will help to understand the docking of carprofen derivatives to γ-secretase and in the design of new potent drugs. The approach presented here promises to serve as a general guideline how drug/target interactions in membranes can be analyzed in a comprehensive manner.

Therapeutic Monoclonal Antibodies to Complex Membrane Protein Targets: Antigen Generation and Antibody Discovery Strategies.

Cell surface membrane proteins comprise a wide array of structurally and functionally diverse proteins involved in a variety of important physiological and homeostatic processes. Complex integral membrane proteins, which are embedded in the lipid bilayer by multiple transmembrane-spanning helices, are represented by families of proteins that are important target classes for drug discovery. Such protein families include G-protein-coupled receptors, ion channels and transporters. Although these targets have typically been the domain of small-molecule drugs, the exquisite specificity of monoclonal antibodies offers a significant opportunity to selectively modulate these target proteins. Nevertheless, the isolation of antibodies with desired pharmacological functions has proved difficult because of technical challenges in preparing membrane protein antigens for antibody drug discovery. In this review, we describe recent progress in defining strategies for the generation of membrane protein antigens. We also describe antibody-isolation strategies that identify antibodies that bind the membrane protein and modulate protein function.

Improved recombinant protein production in Arabidopsis thaliana

ABSTRACTProduction and isolation of recombinant proteins are key steps in modern Molecular Biology. Expression vectors and platforms for various hosts, including both prokaryotic and eukaryotic systems, have been used. In basic plant research, Arabidopsis thaliana is the central model for which a wealth of genetic and genomic resources is available, and enormous knowledge has been accumulated over the past years – especially since elucidation of its genome in 2000. However, until recently an Arabidopsis platform had been lacking for preparative-scale production of homologous recombinant proteins. We recently established an Arabidopsis-based super-expression system, and used it for a structural pilot study of a multi-subunit integral membrane protein complex. This review summarizes the benefits and further potential of the model plant system for protein productions.Abbreviations: Nb, Nicotiana benthamiana; OT, oligosaccharyltransferase

Primary and Higher Order Structure of the Reaction Center from the Purple Phototrophic Bacterium Blastochloris viridis: A Test for Native Mass Spectrometry.

The reaction center (RC) from the phototrophic bacterium Blastochloris viridis was the first integral membrane protein complex to have its structure determined by X-ray crystallography and has been studied extensively since then. It is composed of four protein subunits, H, M, L, and C, as well as cofactors, including bacteriopheophytin (BPh), bacteriochlorophyll (BCh), menaquinone, ubiquinone, heme, carotenoid, and Fe. In this study, we utilized mass spectrometry-based proteomics to study this protein complex via bottom-up sequencing, intact protein mass analysis, and native MS ligand-binding analysis. Its primary structure shows a series of mutations, including an unusual alteration and extension on the C-terminus of the M-subunit. In terms of quaternary structure, proteins such as this containing many cofactors serve to test the ability to introduce native-state protein assemblies into the gas phase because the cofactors will not be retained if the quaternary structure is seriously perturbed. Furthermore, this specific RC, under native MS, exhibits a strong ability not only to bind the special pair but also to preserve the two peripheral BCh's.

Sequence characterization of alpha 1 isoform (ATP1A1) of Na+/K+-ATPase gene and expression characteristics of its major isoforms across tissues of riverine buffaloes (Bubalus bubalis)

Na+/K+-ATPase is an integral plasma membrane protein complex which maintains the active transport of Na+ and K+ ions across the plasma membrane in the animal. The alpha subunit harbours the binding and catalytic site for Na+, K+ and ATP hydrolysis. The alpha (α) subunit has four known isoforms; α1, α2, α3 and α4 which are encoded by ATP1A1, ATP1A2, ATP1A3 and ATP1A4 genes respectively. The role of Na+/K+-ATPase variants has been associated with heat stress in dairy animals but the data with respect to molecular characterization and tissue expression kinetics of its alpha isoforms is lacking in riverine buffaloes (Bubalus bubalis) which is one of the major dairy species in India. Hence the present work is aimed to sequence characterize the bubaline α1 gene and also to evaluate the tissue expression pattern of all four different isoforms (α1- α4). Our ATP1A1 sequence showed a similar structural organization with other mammalian species. The predicted polypeptide has 1021 amino acids showing maximum identity with Bos taurus (99.71%) and least with Gallus gallus (92.75%). 32 variations were detected in the ATP1A1 CDS of buffalo when compared with B. taurus. qPCR expression analysis of ATP1A1, ATP1A2, ATP1A3 and ATP1A4 genes revealed tissue specific expression. ATP1A1 and ATP1A2 showed ubiquitous expression in all the tissues examined while ATP1A3 and ATP1A4 showed tissue-specific expression confined to nervous tissue and testis respectively. Hence the present study has enriched the bubaline data and also strongly points towards the critical role that these isoforms of Na+/K+-ATPase gene might be playing in maintaining the ionic concentration in the cells and tissues in which they are expressed.