Role of membrane environment and membrane-spanning protein regions in assembly and function of the Class II Major Histocompatibility complex

Class II Major Histocompatibility complex (MHC-II) is a polymorphic heterodimer that binds antigen-derived peptides and presents them on the surface of antigen presenting cells. This mechanism of antigen presentation leads to recognition by CD4 T-cells and T-cell activation, making it a critical element of adaptive immune response. For this reason, the structural determinants of MHC-II function have been of great interest for the past 30 years, resulting in a robust structural understanding of the extracellular regions of the complex. However, the membrane-localized regions have also been strongly implicated in protein-protein and protein-lipid interactions that facilitate Class II assembly, transport and function, and it is these regions that are the focus of this review. Here we describe studies that reveal the strong and selective interactions between the transmembrane domains of the MHC α, and invariant chains which, when altered, have broad reaching impacts on antigen presentation and Class II function. We also summarize work that clearly demonstrates the link between membrane lipid composition (particularly the presence of cholesterol) and MHC-II conformation, subsequent peptide binding, and downstream T-cell activation. We have integrated these studies into a comprehensive view of Class II transmembrane domain biology. [Abstract copyright: Copyright © 2018. Published by Elsevier Inc.

Thermodynamic competition between membrane protein oligomeric states

Self-assembly of protein monomers into distinct membrane protein oligomers provides a general mechanism for diversity in the molecular architectures, and resulting biological functions, of membrane proteins. We develop a general physical framework describing the thermodynamic competition between different oligomeric states of membrane proteins. Using the mechanosensitive channel of large conductance as a model system, we show how the dominant oligomeric states of membrane proteins emerge from the interplay of protein concentration in the cell membrane, protein-induced lipid bilayer deformations, and direct monomer-monomer interactions. Our results suggest general physical mechanisms and principles underlying regulation of protein function via control of membrane protein oligomeric state.Comment: 7 pages, 5 figure

The MemProtMD database : a resource for membrane-embedded protein structures and their lipid interactions

Integral membrane proteins fulfil important roles in many crucial biological processes, including cell signalling, molecular transport and bioenergetic processes. Advancements in experimental techniques are revealing high resolution structures for an increasing number of membrane proteins. Yet, these structures are rarely resolved in complex with membrane lipids. In 2015, the MemProtMD pipeline was developed to allow the automated lipid bilayer assembly around new membrane protein structures, released from the Protein Data Bank (PDB). To make these data available to the scientific community, a web database (http://memprotmd.bioch.ox.ac.uk) has been developed. Simulations and the results of subsequent analysis can be viewed using a web browser, including interactive 3D visualizations of the assembled bilayer and 2D visualizations of lipid contact data and membrane protein topology. In addition, ensemble analyses are performed to detail conserved lipid interaction information across proteins, families and for the entire database of 3506 PDB entries. Proteins may be searched using keywords, PDB or Uniprot identifier, or browsed using classification systems, such as Pfam, Gene Ontology annotation, mpstruc or the Transporter Classification Database. All files required to run further molecular simulations of proteins in the database are provided

Membrane-protein interactions in mechanosensitive channels

In this paper, we examine the mechanical role of the lipid bilayer in ion channel conformation and function with specific reference to the case of the mechanosensitive channel of large conductance (MscL). In a recent paper (Wiggins and Phillips, 2004), we argued that mechanotransduction very naturally arises from lipid-protein interactions by invoking a simple analytic model of the MscL channel and the surrounding lipid bilayer. In this paper, we focus on improving and expanding this analytic framework for studying lipid-protein interactions with special attention to MscL. Our goal is to generate simple scaling relations which can be used to provide qualitative understanding of the role of membrane mechanics in protein function and to quantitatively interpret experimental results. For the MscL channel, we find that the free energies induced by lipid-protein interaction are of the same order as the free energy differences between conductance states measured by Sukharev et al. (1999). We therefore conclude that the mechanics of the bilayer plays an essential role in determining the conformation and function of the channel. Finally, we compare the predictions of our model to experimental results from the recent investigations of the MscL channel by Perozo et al. (2002), Powl et al. (2003), Yoshimura et al. (2004), and others and suggest a suite of new experiments

Insights into membrane protein–lipid interactions from free energy calculations

Integral membrane proteins are regulated by specific interactions with lipids from the surrounding bilayer. The structures of protein–lipid complexes can be determined through a combination of experimental and computational approaches, but the energetic basis of these interactions is difficult to resolve. Molecular dynamics simulations provide the primary computational technique to estimate the free energies of these interactions. We demonstrate that the energetics of protein–lipid interactions may be reliably and reproducibly calculated using three simulation-based approaches: potential of mean force calculations, alchemical free energy perturbation, and well-tempered metadynamics. We employ these techniques within the framework of a coarse-grained force field and apply them to both bacterial and mammalian membrane protein–lipid systems. We demonstrate good agreement between the different techniques, providing a robust framework for their automated implementation within a pipeline for annotation of newly determined membrane protein structures

A saposin-lipoprotein nanoparticle system for membrane proteins.

A limiting factor in membrane protein research is the ability to solubilize and stabilize such proteins. Detergents are used most often for solubilizing membrane proteins, but they are associated with protein instability and poor compatibility with structural and biophysical studies. Here we present a saposin-lipoprotein nanoparticle system, Salipro, which allows for the reconstitution of membrane proteins in a lipid environment that is stabilized by a scaffold of saposin proteins. We demonstrate the applicability of the method on two purified membrane protein complexes as well as by the direct solubilization and nanoparticle incorporation of a viral membrane protein complex from the virus membrane. Our approach facilitated high-resolution structural studies of the bacterial peptide transporter PeptTSo2 by single-particle cryo-electron microscopy (cryo-EM) and allowed us to stabilize the HIV envelope glycoprotein in a functional state

The role of interfacial lipids in stabilizing membrane protein oligomers

Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways1 but is often difficult to define2 or predict3. Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT4, one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET5, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na+/H+ antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors

Membrane protein structure determination and characterisation by solution and solid-state nmr

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Biological membranes define the interface of life and its basic unit, the cell. Membrane proteins play key roles in membrane functions, yet their structure and mechanisms remain poorly understood. Breakthroughs in crystallography and electron microscopy have invigorated structural analysis while failing to characterise key functional interactions with lipids, small molecules and membrane modulators, as well as their conformational polymorphism and dynamics. NMR is uniquely suited to resolving atomic environments within complex molecular assemblies and reporting on membrane organisation, protein structure, lipid and polysaccharide composition, conformational variations and molecular interactions. The main challenge in membrane protein studies at the atomic level remains the need for a membrane environment to support their fold. NMR studies in membrane mimetics and membranes of increasing complexity offer close to native environments for structural and molecular studies of membrane proteins. Solution NMR inherits high resolution from small molecule analysis, providing insights from detergent solubilised proteins and small molecular assemblies. Solid-state NMR achieves high resolution in membrane samples through fast sample spinning or sample alignment. Recent developments in dynamic nuclear polarisation NMR allow signal enhancement by orders of magnitude opening new opportunities for expanding the applications of NMR to studies of native membranes and whole cells

ABCC6 is a basolateral plasma membrane protein

RATIONALE:: ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseudoxanthoma elasticum and general arterial calcification of infancy. To elucidate the role of ABCC6 in cellular physiology and disease, it is crucial to establish the exact subcellular localization of the native ABCC6 protein. OBJECTIVE:: In a recent article in Circulation Research, ABCC6 was reported to localize to the mitochondria-associated membrane and not the plasma membrane. As the suggested mitochondrial localization is inconsistent with published data and the presumed role of ABCC6, we performed experiments to determine the cellular localization of ABCC6 in its physiological environment. METHODS AND RESULTS:: We performed immunofluorescent labeling of frozen mouse and human liver sections, as well as primary hepatocytes. We used several different antibodies recognizing human and mouse ABCC6. Our results unequivocally show that ABCC6 is in the basolateral membrane of hepatocytes and is not associated with the mitochondria, mitochondria-associated membrane, or the endoplasmic reticulum. CONCLUSIONS:: Our findings support the model that ABCC6 is in the basolateral membrane, mediating the sinusoidal efflux of a metabolite from the hepatocytes to systemic circulation. © 2013 American Heart Association, Inc