Monotopic Membrane Proteins Join the Fold.

Monotopic membrane proteins, classified by topology, are proteins that embed into a single face of the membrane. These proteins are generally underrepresented in the Protein Data Bank (PDB), but the past decade of research has revealed new examples that allow the description of generalizable features. This Opinion article summarizes shared characteristics including oligomerization states, modes of membrane association, mechanisms of interaction with hydrophobic or amphiphilic substrates, and homology to soluble folds. We also discuss how associations of monotopic enzymes in pathways can be used to promote substrate specificity and product composition. These examples highlight the challenges in structure determination specific to this class of proteins, but also the promise of new understanding from future study of these proteins that reside at the interface.

Phosphoinositide-binding activity of Smad2 is essential for its function in TGF-ß signaling.

As a central player in the canonical TGF-ß signaling pathway, Smad2 transmits the activation of TGF-ß receptors at the plasma membrane (PM) to transcriptional regulation in the nucleus. Although it has been well established that binding of TGF-ß to its receptors leads to the recruitment and activation of Smad2, the spatiotemporal mechanism by which Smad2 is recruited to the activated TGF-ß receptor complex and activated is not fully understood. Here we show that Smad2 selectively and tightly binds phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the PM. The PI(4,5)P2-binding site is located in the MH2 domain that is involved in interaction with the TGF-ß receptor I that transduces TGF-ß-receptor binding to downstream signaling proteins. Quantitative optical imaging analyses show that PM recruitment of Smad2 is triggered by its interaction with PI(4,5)P2 that is locally enriched near the activated TGF-ß receptor complex, leading to its binding to the TGF-ß receptor I. The PI(4,5)P2-binding activity of Smad2 is essential for the TGF-ß-stimulated phosphorylation, nuclear transport, and transcriptional activity of Smad2. Structural comparison of all Smad MH2 domains suggests that membrane lipids may also interact with other Smad proteins and regulate their function in diverse TGF-ß-mediated biological processes.

Conformation of myelin basic protein bound to phosphatidylinositol membrane characterized by vacuum-ultraviolet circular-dichroism spectroscopy and molecular-dynamics simulations.

The 18.5-kDa isoform of myelin basic protein (MBP) interacts with the membrane surface of the myelin sheath to construct its compact multilamellar structure. This study characterized the conformation of MBP in the membrane by measuring the vacuum-ultraviolet circular-dichroism (VUVCD) spectra of MBP in the bilayer liposome comprising the following essential lipid constituents of the myelin sheath: phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2). The spectra of MBP exhibited the characteristic peaks of the helix structure in the presence of PI liposome, and the intensity increased markedly in the presence of PIP and PIP2 liposomes to show an isodichroic point. This suggests that the amount of the membrane-bound conformation of MBP enhanced due to the increased number of negative net charges on the liposome surfaces. Secondary-structure analysis revealed that MBP in the membrane comprised approximately 40% helix contents and eight helix segments. Molecular-dynamics (MD) simulations of the eight segments were conducted for 250 ns in the presence of PI membrane, which predicted two amphiphilic and three nonamphiphilic helices as the membrane-interaction sites. Further analysis of the distances of the amino-acid residues in each segment from the phosphate group suggested that the nonamphiphilic helices interact with the membrane surface electrostatically, while the amphiphilic ones invade the inside of the membrane to produce electrostatic and hydrophobic interactions. These results show that MBP can interact with the PI membrane via amphiphilic and nonamphiphilic helices under the control of a delicate balance between electrostatic and hydrophobic interactions.

A lipid-mediated conformational switch modulates the thermosensing activity of DesK.

The thermosensor DesK is a multipass transmembrane histidine-kinase that allows the bacterium Bacillus subtilis to adjust the levels of unsaturated fatty acids required to optimize membrane lipid fluidity. The cytoplasmic catalytic domain of DesK behaves like a kinase at low temperature and like a phosphatase at high temperature. Temperature sensing involves a built-in instability caused by a group of hydrophilic residues located near the N terminus of the first transmembrane (TM) segment. These residues are buried in the lipid phase at low temperature and partially "buoy" to the aqueous phase at higher temperature with the thinning of the membrane, promoting the required conformational change. Nevertheless, the core question remains poorly understood: How is the information sensed by the transmembrane region converted into a rearrangement in the cytoplasmic catalytic domain to control DesK activity? Here, we identify a "linker region" (KSRKERERLEEK) that connects the TM sensor domain with the cytoplasmic catalytic domain involved in signal transmission. The linker adopts two conformational states in response to temperature-dependent membrane thickness changes: (i) random coiled and bound to the phospholipid head groups at the water-membrane interface, promoting the phosphatase state or (ii) unbound and forming a continuous helix spanning a region from the membrane to the cytoplasm, promoting the kinase state. Our results uphold the view that the linker is endowed with a helix/random coil conformational duality that enables it to behave like a transmission switch, with helix disruption decreasing the kinase/phosphatase activity ratio, as required to modulate the DesK output response.

Guanylate Cyclase-Activating Protein-2 Undergoes Structural Changes upon Binding to Detergent Micelles and Bicelles.

GCAPs are neuronal Ca(2+)-sensors playing a central role in light adaptation. GCAPs are N-terminally myristoylated membrane-associated proteins. Although, the myristoylation of GCAPs plays an important role in light adaptation its structural and physiological roles are not yet clearly understood. The crystal-structure of GCAP-1 shows the myristoyl moiety inside the hydrophobic core of the protein, stabilizing the protein structure; but (2)H-solid-state NMR investigations on the deuterated myristoyl moiety of GCAP-2 in the presence of liposomes showed that it is inserted into the lipid bilayer. In this study, we address the question of the localization of the myristoyl group of Ca(2+)-bound GCAP-2, and the influence of CHAPS-, DPC-micelles and DMPC/DHPC-bicelles on the structure, and on the localization of the myristoyl group, of GCAP-2 by solution-state NMR. We also carried out the backbone assignment. Characteristic chemical shift differences have been observed between the myristoylated and the non-myristoylated forms of the protein. Our results support the view that in the absence of membrane forming substances the myristoyl moiety is buried inside a hydrophobic pocket of GCAP-2 similar to the crystal structure of GCAP-1. Addition of CHAPS-micelles and DMPC/DHPC-bicelles cause specific structural changes localized in and around the myristoyl binding pocket. We interpret these changes as an indication for the extrusion of the myristoyl moiety from its binding pocket and its insertion into the hydrophobic interior of the membrane mimic. On the basis of the backbone chemical shifts, we propose a structural model of myristoylated GCAP-2 in the presence of Ca(2+) and membrane mimetics.

Viral potassium channels as a robust model system for studies of membrane-protein interaction.

The viral channel KcvNTS belongs to the smallest K(+) channels known so far. A monomer of a functional homotetramer contains only 82 amino acids. As a consequence of the small size the protein is almost fully submerged into the membrane. This suggests that the channel is presumably sensitive to its lipid environment. Here we perform a comparative analysis for the function of the channel protein embedded in three different membrane environments. 1. Single-channel currents of KcvNTS were recorded with the patch clamp method on the plasma membrane of HEK293 cells. 2. They were also measured after reconstitution of recombinant channel protein into classical planar lipid bilayers and 3. into horizontal bilayers derived from giant unilamellar vesicles (GUVs). The recombinant channel protein was either expressed and purified from Pichia pastoris or from a cell-free expression system; for the latter a new approach with nanolipoprotein particles was used. The data show that single-channel activity can be recorded under all experimental conditions. The main functional features of the channel like a large single-channel conductance (80pS), high open-probability (>50%) and the approximate duration of open and closed dwell times are maintained in all experimental systems. An apparent difference between the approaches was only observed with respect to the unitary conductance, which was ca. 35% lower in HEK293 cells than in the other systems. The reason for this might be explained by the fact that the channel is tagged by GFP when expressed in HEK293 cells. Collectively the data demonstrate that the small viral channel exhibits a robust function in different experimental systems. This justifies an extrapolation of functional data from these systems to the potential performance of the channel in the virus/host interaction. This article is part of a Special Issue entitled: Viral Membrane Proteins-Channels for Cellular Networking.

Deciphering Membrane-Protein Interactions and High-Throughput Antigen Identification with Cell Doublets.

Deciphering cellular interactions is essential to both understand the mechanisms underlying a broad range of human diseases, but also to manipulate therapies targeting these diseases. Here, the formation of cell doublets resulting from specific membrane ligand-receptor interactions is discovered. Based on this phenomenon, the study developed DoubletSeeker, a novel high-throughput method for the reliable identification of ligand-receptor interactions. The study shows that DoubletSeeker can accurately identify T cell receptor (TCR)-antigen interactions with high sensitivity and specificity. Notably, DoubletSeeker effectively captured paired TCR-peptide major histocompatibility complex (pMHC) information during a highly complex library-on-library screening and successfully identified three mutant TCRs that specifically recognize the MART-1 epitope. In turn, DoubletSeeker can act as an antigen discovery platform that allows for the development of novel immunotherapy targets, making it valuable for investigating fundamental tumor immunology.

Dynamic framework for large-scale modeling of membranes and peripheral proteins.

In this chapter, we present a novel computational framework to study the dynamic behavior of extensive membrane systems, potentially in interaction with peripheral proteins, as an alternative to conventional simulation methods. The framework effectively describes the complex dynamics in protein-membrane systems in a mesoscopic particle-based setup. Furthermore, leveraging the hydrodynamic coupling between the membrane and its surrounding solvent, the coarse-grained model grounds its dynamics in macroscopic kinetic properties such as viscosity and diffusion coefficients, marrying the advantages of continuum- and particle-based approaches. We introduce the theoretical background and the parameter-space optimization method in a step-by-step fashion, present the hydrodynamic coupling method in detail, and demonstrate the application of the model at each stage through illuminating examples. We believe this modeling framework to hold great potential for simulating membrane and protein systems at biological spatiotemporal scales, and offer substantial flexibility for further development and parametrization.

A PQS-Cleaving Quorum Quenching Enzyme Targets Extracellular Membrane Vesicles of Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa uses quorum sensing to control its virulence. One of its major signal molecules, the Pseudomonas quinolone signal PQS, has high affinity to membranes and is known to be trafficked mainly via outer membrane vesicles (OMVs). We previously reported that several 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQDs) catalyze the cleavage of PQS and thus act as quorum quenching enzymes. Further analysis showed that, in contrast to other HQDs, the activity of HQD from Streptomyces bingchenggensis (HQDS.b.) was unexpectedly stabilized by culture supernatants of P. aeruginosa. Interestingly, the stabilizing effect was higher with supernatants from the strain PA14 than with supernatants from the strain PAO1. Heat treatment and lyophilization hardly affected the stabilizing effect; however, fractionation of the supernatant excluded small molecules as stabilizing agents. In a pull-down assay, HQDS.b. appeared to interact with several P. aeruginosa proteins previously found in the OMV proteome. This prompted us to probe the physical interaction of HQDS.b. with prepared extracellular membrane vesicles. Homo-FRET of fluorescently labeled HQDS.b. indeed indicated a spatial clustering of the protein on the vesicles. Binding of a PQS-cleaving enzyme to the OMVs of P. aeruginosa may enhance PQS degradation and is highly reconcilable with its function as a quorum quenching enzyme.

Interaction network of African swine fever virus structural protein p30 with host proteins.

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a lethal hemorrhagic disease that is currently threatening the global pig industry. ASFV structural protein p30 is a membrane phosphoprotein that suggests it may play a regulatory role, possibly in signal transduction. Despite its significance in internalization into host cells, the interaction between p30 and host proteins is relatively unknown. In this study, we describe the application of a DUALmembrane yeast two-hybrid assay to screen a primary porcine alveolar macrophages cDNA library and analyze the interactome of p30 protein. Our data identify seven host cellular proteins (DAB2, RPSA, OAS1, PARP9, CAPG, ARPC5, and VBP1) that putatively interact with the p30. We further verified the interaction between p30 and host proteins by laser confocal microscopy, co-immunoprecipitation, and GST-pulldown assay. To further understand the relationship between host proteins and p30, we drew the interaction network diagram and analyzed the functional enrichment of each host protein. Enrichment analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes indicated that host proteins were mainly related to endocytosis, actin cytoskeleton regulation, and innate immunity. Collectively, we identified the interaction between p30 and host cell protein using a membrane protein yeast two-hybrid system, which increases our knowledge of the interaction between ASFV and the host and informs future research on antiviral strategies.

Determining structure and action mechanism of LBF14 by molecular simulation.

The recently discovered, membrane-active peptide LBF14 contains several non-proteinogenic amino acids and is able to transform vesicles into tubule networks. The exact membrane interaction mechanism and detailed secondary structure are yet to be determined. We performed molecular dynamics simulations of LBF14 and let it fold de novo into its ensemble of native secondary structures. Histidine protonation state effects on secondary structure were investigated. An MD simulation of the peptide with a lipid bilayer was performed. Simulation results were compared to circular dichroism and electron paramagnetic resonance data of previous studies. LBF14 contains a conserved helical section in an otherwise random structure. Helical stability is influenced by histidine protonation. The peptide localized to the polar layer of the membrane, consistent with experimental results. While the overall secondary structure is unaffected by membrane interaction, Ramachandran plot analysis yielded two distinct peptide conformations during membrane interaction. This conformational change was accompanied by residue repositioning within the membrane. LBF14 only affected the local order in the membrane, and had no measurable effect on pressure. The simulation results are consistent with the previously proposed membrane interaction mechanism of LBF14 and can additionally explain the local interaction mechanism. Communicated by Ramaswamy H. Sarma.

Lipid asymmetry of a model mitochondrial outer membrane affects Bax-dependent permeabilization.

The presence of an asymmetric distribution of lipids in biological membranes was first described ca. 50 years ago. While various studies had reported the role of loss of lipid asymmetry on signaling processes, its effect on membrane physical properties and membrane-protein interactions lacks further understanding. The recent description of new technologies for the preparation of asymmetric model membranes has helped to fill part of this gap. However, the major effort so far has been on plasma membrane models. Here we describe the preparation of liposomes mimicking the mitochondria outer membrane (MOM) in regard to its lipid composition and asymmetry. By employing the methyl-ß-cyclodextrin-catalyzed lipid exchange technology and accurate quantification of lipid asymmetry with head group-specific probes we showed the successful preparation of a MOM model bearing a physiologically relevant lipid composition and asymmetry. In addition, by a direct comparison with its lipid symmetrical counterpart it is shown that asymmetric models were more resistant to tBid-promoted Bax-permeabilization, suggesting a role played by MOM lipid asymmetry on the mitochondria pathway of apoptosis. The barrier imposed by lipid asymmetry on membrane permeabilization was in part due to a decrease in the concentration of membrane-bound proteins, which was likely a consequence of the two mutually-dependent properties; i.e., the lower electrostatic surface potential and the higher molecular packing imposed by lipid asymmetry. It is proposed that MOM lipid asymmetry imparts different physical properties on the membrane and might add an additional component of regulation in intricate mitochondrial processes.

Progress of proximity labeling technology in membrane protein interaction / 中国药科大学学报

@#Abstract: Membrane proteins, which play a critical role in various life processes, particularly in regulating cell-cell contact and signal transduction, are closely linked to cell differentiation and maturation. Therefore, it is of great theoretical and practical significance to develop a variety of methods to thoroughly explore the interactions between membrane proteins. In addition to traditional techniques such as immunoprecipitation, newly developed proximity labeling (PL) techniques have gradually become important means to study membrane protein interaction. PL methods are based on engineered enzymes fused with bait protein to catalyze small molecules, label neighboring target proteins, and detect the interactions by flow cytometry, mass spectrometry, confocal microscopic imaging, etc. This paper focuses on the recent developments in PL techniques for studying membrane protein interactions, with a prospect of the potential future directions for research in this area.

Photocrosslinking Approach to Investigate Protein Interactions in the BCL-2 Family.

The Bcl-2 family of proteins regulates mitochondrial outer membrane permeability thereby making life or death decisions for cells. Most of Bcl-2 proteins contain hydrophobic regions that are embedded in intracellular membranes such as mitochondria. These membrane proteins are difficult to express and purify thereby preluding biochemical and biophysical characterizations. Here, we describe a photocrosslinking approach based on in vitro synthesis of Bcl-2 proteins with photoreactive amino acid analogs incorporated at specific locations. These photoreactive proteins are reconstituted into liposomal membranes with defined phospholipids or mitochondrial membranes isolated from animals, and their interactions with other Bcl-2 proteins are detected by photocrosslinking.

Kinetics of small molecule interactions with membrane proteins in single cells measured with mechanical amplification.

Measuring small molecule interactions with membrane proteins in single cells is critical for understanding many cellular processes and for screening drugs. However, developing such a capability has been a difficult challenge. We show that molecular interactions with membrane proteins induce a mechanical deformation in the cellular membrane, and real-time monitoring of the deformation with subnanometer resolution allows quantitative analysis of small molecule-membrane protein interaction kinetics in single cells. This new strategy provides mechanical amplification of small binding signals, making it possible to detect small molecule interactions with membrane proteins. This capability, together with spatial resolution, also allows the study of the heterogeneous nature of cells by analyzing the interaction kinetics variability between different cells and between different regions of a single cell.