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Abstract Membranes are protein and lipid structures that surround cells and other biological compartments. We present a conceptual model wherein all membranes are organized into structural and functional zones. The assembly of zones such as receptor clusters, protein-coated pits, lamellipodia, cell junctions, and membrane fusion sites is explained to occur through a protein-lipid code. This challenges the theory that lipids sort proteins after forming stable membrane subregions independently of proteins.

Membranes are protein and lipid structures that surround cells and other biological compartments. We present a conceptual model wherein all membranes are organized into structural and functional zones. The assembly of zones such as receptor clusters, protein-coated pits, lamellipodia, cell junctions, and membrane fusion sites is explained to occur through a protein-lipid code. This challenges the theory that lipids sort proteins after forming stable membrane subregions independently of proteins. [ABSTRACT FROM AUTHOR]

Bacterial outer membrane vesicles (OMVs) are nanosized spheroidal particles shed by gram-negative bacteria that contain biomolecules derived from the periplasmic space, the bacterial outer membrane, and possibly other compartments. OMVs can be purified from bacterial culture supernatants, and by genetically manipulating the bacterial cells that produce them, they can be engineered to harbor cargoes and/or display molecules of interest on their surfaces including antigens that are immunogenic in mammals. Since OMV bilayer-embedded components presumably maintain their native structures, OMVs may represent highly useful tools for generating antibodies to bacterial outer membrane targets. OMVs have historically been utilized as vaccines or vaccine constituents. Antibodies that target bacterial surfaces are increasingly being explored as antimicrobial agents either in unmodified form or as targeting moieties for bactericidal compounds. Here, we review the properties of OMVs, their use as immunogens, and their ability to elicit antibody responses against bacterial antigens. We highlight antigens from bacterial pathogens that have been successfully targeted using antibodies derived from OMV-based immunization and describe opportunities and limitations for OMVs as a platform for antimicrobial antibody development. Key points: • Outer membrane vesicles (OMVs) of gram-negative bacteria bear cell-surface molecules • OMV immunization allows rapid antibody (Ab) isolation to bacterial membrane targets • Review and analysis of OMV-based immunogens for antimicrobial Ab development [ABSTRACT FROM AUTHOR]

NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.

The essential Escherichia coli ATPase MsbA is a lipid flippase that serves as a prototype for multi drug resistant ABC transporters. Its physiological function is the transport of lipopolisaccharides to build up the outer membranes of Gram-negative bacteria. Although several structural and biochemical studies of MsbA have been conducted previously, a detailed picture of the dynamic processes that link ATP hydrolysis to allocrit transport remains elusive. We report here for the first time time-resolved Fourier transform infrared (FTIR) spectroscopic measurements of the ATP binding and ATP hydrolysis reaction of full-length MsbA and determined reaction rates at 288 K of k1 = 0.49 ± 0.28 s−1 and k2 = 0.014 ± 0.003 s−1, respectively. We further verified these rates with photocaged NPEcgAppNHp where only nucleotide binding was observable and the negative mutant MsbA-H537A that showed slow hydrolysis (k2 < 2 × 10−4 s−1). Besides single turnover kinetics, FTIR measurements also deliver IR signatures of all educts, products and the protein. ADP remains protein-bound after ATP hydrolysis. In addition, the spectral changes observed for the two variants MsbA-S378A and MsbA-S482A correlated with the loss of hydrogen bonding to the γ-phosphate of ATP. This study paves the way for FTIR-spectroscopic investigations of allocrite transport in full-length MsbA. [ABSTRACT FROM AUTHOR]

The myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, α-helical conformation for both PLP and DM20 in detergent complexes, and pinpoint structural variations between the isoforms and their influence on protein function. In phosphatidylcholine membranes, reconstituted PLP and DM20 spontaneously induced formation of multilamellar myelin-like membrane assemblies. Cholesterol and sphingomyelin enhanced the membrane organization but were not crucial for membrane stacking. Electron cryomicroscopy, atomic force microscopy, and X-ray diffraction experiments for membrane-embedded PLP/DM20 illustrated effective membrane stacking and ordered organization of membrane assemblies with a repeat distance in line with CNS myelin. Our results shed light on the 3D structure of myelin PLP and DM20, their structure–function differences, as well as fundamental protein–lipid interplay in CNS compact myelin. [ABSTRACT FROM AUTHOR]

Integral membrane transporters play essential roles in the movement of substrates across biological membranes. One approach to produce transporters suitable for structural studies is to introduce mutations that reduce conformational flexibility and increase stability. However, it can be difficult to predict which mutations will result in a more stable protein. Previously, we stabilised the uric acid‐xanthine transporter, UapA, a member of the SLC23 family, through introduction of a single‐point mutation, G411V, trapping the protein in the inward‐facing conformation. Here, we attempted to stabilise the structurally related BOR1 transporter from Arabidopsis thaliana, a member of the SLC4 family, by introducing the equivalent substitution. We identified possible residues, P362 and M363, in AtBOR1, likely to be equivalent to the G411 of UapA, and generated four mutants, P362V or L and M363F or Y. Stability analysis using heated Fluorescent Size Exclusion Chromatography indicated that the M363F/Y mutants were more stable than the WT AtBOR1 and P362V/L mutants. Furthermore, functional complementation analysis revealed that the M363F/Y mutants exhibited reduced transport activity compared to the P362V/L and WT proteins. Purification and crystallisation of the M363F/Y proteins yielded crystals that diffracted better than WT (5.5 vs 7 Å). We hypothesise that the increased bulk of the F and Y substitutions limits the ability of the protein to undergo the conformational rearrangements associated with transport. These proteins represent a basis for future studies on AtBOR1.

Abstract Background Plasmodium species are entirely dependent upon their host as a source of essential iron. Although it is an indispensable micronutrient, oxidation of excess ferrous iron to the ferric state in the cell cytoplasm can produce reactive oxygen species that are cytotoxic. The malaria parasite must therefore carefully regulate the processes involved in iron acquisition and storage. A 273 amino acid membrane transporter that is a member of the vacuolar iron transporter (VIT) family and an orthologue of the yeast Ca2+-sensitive cross complementer (CCC1) protein plays a major role in cytosolic iron detoxification of Plasmodium species and functions in transport of ferrous iron ions into the endoplasmic reticulum for storage. While this transporter, termed PfVIT, is not critical for viability of the parasite evidence from studies of mice infected with VIT-deficient Plasmodium suggests it could still provide an efficient target for chemoprophylactic treatment of malaria. Individual amino acid residues that constitute the Fe2+ binding site of the protein were identified to better understand the structural basis of substrate recognition and binding by PfVIT. Methods Using the crystal structure of a recently published plant VIT as a template, a high-quality homology model of PfVIT was constructed to identify the amino acid composition of the transporter’s substrate binding site and to act as a guide for subsequent mutagenesis studies. To test the effect of mutation of the substrate binding-site residues on PfVIT function a yeast complementation assay assessed the ability of overexpressed, recombinant wild type and mutant PfVIT to rescue an iron-sensitive deletion strain (ccc1∆) of Saccharomyces cerevisiae yeast from the toxic effects of a high concentration of extracellular iron. Results The combined in silico and mutagenesis approach identified a methionine residue located within the cytoplasmic metal binding domain of the transporter as essential for PfVIT function and provided insight into the structural basis for the Fe2+-selectivity of the protein. Conclusion The structural model of the metal binding site of PfVIT opens the door for rational design of therapeutics to interfere with iron homeostasis within the malaria parasite.

The photosynthetic reaction center of the purple nonsulfur bacterium Cereibacter sphaeroides is a useful model for the study of mechanisms of photoinduced electron transfer and a promising component for photo-bio-electrocatalytic systems. The basic research and technological applications of this membrane pigment-protein complex require effective approaches to increase its structural stability. In this work, a rational design approach to genetically modify the reaction centers by introducing disulfide bonds is used. This resulted in significantly increasing the thermal stability of some of the mutant pigment-protein complexes. The formation of the S-S bonds was confirmed by X-ray crystallography as well as SDS-PAGE, and the optical properties of the reaction centers were studied. The genetically modified reaction centers presented here preserved their ability for photochemical charge separation and could be of interest for basic science and biotechnology.

Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

The delineation of protein-lipid interfaces is essential for understanding the mechanisms of various membrane-associated processes crucial to plant development and growth, including signalling, trafficking, and membrane transport. Due to their highly dynamic nature, the precise characterization of lipid-protein interactions is challenging by experimental techniques. Molecular dynamics (MD) simulations provide a powerful computational alternative with a spatial-temporal resolution allowing the atomistic-level description. In this review, we aim to introduce plant scientists to the MD simulations. We describe different steps of performing the MD simulations and provide a broad survey of the MD studies investigating plant protein-lipid interfaces. Our aim is also to illustrate that combining the MD simulations with artificial intelligence-based protein structure determination opens unprecedented possibilities for future investigations of dynamic plant protein-lipid interfaces., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae , can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2's requirement for activation., Competing Interests: IP, AV, MT, AH, FD, JD, VC, JM, SS, FF No competing interests declared, (© 2024, Petit-Hartlein et al.)

Gram-negative bacteria take up the essential ion Fe3+ as ferricsiderophore complexes through their outer membrane using TonBdependent transporters. However, the subsequent route through the inner membrane differs across many bacterial species and siderophore chemistries and is not understood in detail. Here, we report the crystal structure of the inner membrane protein FoxB (from Pseudomonas aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a fold with two tightly bound heme molecules. In combination with in vitro reduction assays and in vivo iron uptake studies, these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fesiderophore uptake. [ABSTRACT FROM AUTHOR]

Store-operated Ca2+ entry (SOCE) represents a predominant Ca2+ influx pathway in non-excitable cells. SOCE is required for immune cell activation and is mediated by the plasma membrane (PM) channel ORAI1 and the endoplasmic reticulum (ER) Ca2+ sensor STIM1. Mutations in the Orai1 or STIM1 genes abolish SOCE leading to combined immunodeficiency (CID), muscular hypotonia, and anhidrotic ectodermal dysplasia. Here, we identify a novel autosomal recessive mutation in ORAI1 in a child with CID. The patient is homozygous for p.C126R mutation in the second transmembrane domain (TM2) of ORAI1, a region with no previous loss-of-function mutations. SOCE is suppressed in the patient's lymphocytes, which is associated with impaired T cell proliferation and cytokine production. Functional analyses demonstrate that the p.C126R mutation does not alter protein expression but disrupts ORAI1 trafficking. Orai1-C126R does not insert properly into the bilayer resulting in ER retention. Insertion of an Arg on the opposite face of TM2 (L135R) also results in defective folding and trafficking. We conclude that positive side chains within ORAI1 TM2 are not tolerated and result in misfolding, defective bilayer insertion, and channel trafficking thus abolishing SOCE and resulting in CID. [ABSTRACT FROM AUTHOR]

Background: Plasmodium species are entirely dependent upon their host as a source of essential iron. Although it is an indispensable micronutrient, oxidation of excess ferrous iron to the ferric state in the cell cytoplasm can produce reactive oxygen species that are cytotoxic. The malaria parasite must therefore carefully regulate the processes involved in iron acquisition and storage. A 273 amino acid membrane transporter that is a member of the vacuolar iron transporter (VIT) family and an orthologue of the yeast Ca2+-sensitive cross complementer (CCC1) protein plays a major role in cytosolic iron detoxification of Plasmodium species and functions in transport of ferrous iron ions into the endoplasmic reticulum for storage. While this transporter, termed PfVIT, is not critical for viability of the parasite evidence from studies of mice infected with VIT-deficient Plasmodium suggests it could still provide an efficient target for chemoprophylactic treatment of malaria. Individual amino acid residues that constitute the Fe2+ binding site of the protein were identified to better understand the structural basis of substrate recognition and binding by PfVIT. Methods: Using the crystal structure of a recently published plant VIT as a template, a high-quality homology model of PfVIT was constructed to identify the amino acid composition of the transporter's substrate binding site and to act as a guide for subsequent mutagenesis studies. To test the effect of mutation of the substrate binding-site residues on PfVIT function a yeast complementation assay assessed the ability of overexpressed, recombinant wild type and mutant PfVIT to rescue an iron-sensitive deletion strain (ccc1∆) of Saccharomyces cerevisiae yeast from the toxic effects of a high concentration of extracellular iron. Results: The combined in silico and mutagenesis approach identified a methionine residue located within the cytoplasmic metal binding domain of the transporter as essential for PfVIT function and provided insight into the structural basis for the Fe2+-selectivity of the protein. Conclusion: The structural model of the metal binding site of PfVIT opens the door for rational design of therapeutics to interfere with iron homeostasis within the malaria parasite. [ABSTRACT FROM AUTHOR]

Integral membrane transporters play essential roles in the movement of substrates across biological membranes. One approach to produce transporters suitable for structural studies is to introduce mutations that reduce conformational flexibility and increase stability. However, it can be difficult to predict which mutations will result in a more stable protein. Previously, we stabilised the uric acid‐xanthine transporter, UapA, a member of the SLC23 family, through introduction of a single‐point mutation, G411V, trapping the protein in the inward‐facing conformation. Here, we attempted to stabilise the structurally related BOR1 transporter from Arabidopsis thaliana, a member of the SLC4 family, by introducing the equivalent substitution. We identified possible residues, P362 and M363, in AtBOR1, likely to be equivalent to the G411 of UapA, and generated four mutants, P362V or L and M363F or Y. Stability analysis using heated Fluorescent Size Exclusion Chromatography indicated that the M363F/Y mutants were more stable than the WT AtBOR1 and P362V/L mutants. Furthermore, functional complementation analysis revealed that the M363F/Y mutants exhibited reduced transport activity compared to the P362V/L and WT proteins. Purification and crystallisation of the M363F/Y proteins yielded crystals that diffracted better than WT (5.5 vs 7 Å). We hypothesise that the increased bulk of the F and Y substitutions limits the ability of the protein to undergo the conformational rearrangements associated with transport. These proteins represent a basis for future studies on AtBOR1. [ABSTRACT FROM AUTHOR]

Solution NMR spectroscopy of large protein systems is hampered by rapid signal decay, so most multidimensional studies focus on long-lived 1H-13C magnetization in methyl groups and/or backbone amide 1H-15N magnetization in an otherwise perdeuterated environment. Herein we demonstrate that it is possible to biosynthetically incorporate additional 1H-12C groups that possess long-lived magnetization using cost-effective partially deuterated or unlabeled amino acid precursors added to Escherichia coli growth media. This approach is applied to the outer membrane enzyme PagP in membrane-mimetic dodecylphosphocholine micelles. We were able to obtain chemical shift assignments for a majority of side chain 1H positions in PagP using nuclear Overhauser enhancements (NOEs) to connect them to previously assigned backbone 1H-15N groups and newly assigned 1H-13C methyl groups. Side chain methyl-to-aromatic NOEs were particularly important for confirming that the amphipathic α-helix of PagP packs against its eight-stranded β-barrel, as indicated by previous X-ray crystal structures. Interestingly, aromatic NOEs suggest that some aromatic residues in PagP that are buried in the membrane bilayer are highly mobile in the micellar environment, like Phe138 and Phe159. In contrast, Tyr87 in the middle of the bilayer is quite rigid, held in place by a hydrogen bonded network extending to the surface that resembles a classic catalytic triad: Tyr87-His67-Asp61. This hydrogen bonded arrangement of residues is not known to have any catalytic activity, but we postulate that its role is to immobilize Tyr87 to facilitate packing of the amphipathic α-helix against the β-barrel. The solution NMR structure of bacterial outer membrane protein PagP (red) agrees with the X-ray crystal structure (blue) in the packing of the amphipathic helix against the β-barrel. An important structural feature that enables this packing is the immobilization of barrel residues via a network of hydrogen bonds comprised of D61, H67, and Y87 that superficially resembles a classic catalytic triad. [Display omitted] • Selective deuteration of aromatic residues for solution 1H NMR of big proteins • Biosynthetic incorporation of deuterated aromatic precursors in E. coli • NOEs between methyl 1H-13C, backbone amide 1H-15N, and side chain 1H-12C • In the membrane, side chain immobilization facilitates protein packing. [ABSTRACT FROM AUTHOR]

Transmembrane (TM) proteins interact closely with the surrounding membrane lipids. Lipids in the vicinity of TM proteins were reported to have hindered mobility, which has been associated with lipids being caught up in the rough surface of the TM domains. These reports, however, neglect one important factor that largely influences the membrane behavior — electrostatics of the TM peptides that are usually positively charged at their cytosolic end. Here, we study on the example of a neutral and a positively charged WALP peptide, how the charge of a TM peptide influences the membrane. We investigate both its dynamics and mechanics by: (i) time dependent fluorescent shift in combination with classical and FRET generalized polarization to evaluate the mobility of lipids at short and long-range distance from the peptide, (ii) atomic force microscopy to observe the mechanical stability of the peptide-containing membranes, and (iii) molecular dynamics simulations to analyze the peptide-lipid interactions. We show that both TM peptides lower lipid mobility in their closest surroundings. The peptides cause lateral heterogeneity in lipid mobility, which in turn prevents free lipid rearrangement and lowers the membrane ability to seal ruptures after mechanical indentations. Introduction of a positive charge to the peptide largely enhances these effects, affecting the whole membrane. We thus highlight that unspecific peptide-lipid interactions, especially the electrostatics, should not be overlooked as they have a great impact on the mechanics and dynamics of the whole membrane. [Display omitted] • TM proteins are known to hinder mobility of lipids in their closest surrounding. • Positive charge of the TM peptides hinders the lipids mobility on a long range. • Lateral heterogeneity in mobility of lipids can hamper their free rearrangement. • Hindered rearrangement of lipids lowers the membrane ability to seal ruptures. • Electrostatics enhances the effects of TM peptides on the whole membrane. [ABSTRACT FROM AUTHOR]

Electrogenic bacteria deliver excess respiratory electrons to externally located metal oxide particles and electrodes. The biochemical basis for this process is arguably best understood for species of Shewanella where the integral membrane complex termed MtrCAB is key to electron transfer across the bacterial outer membranes. A crystal structure was recently resolved for MtrCAB from S. baltica OS185. However, X-ray diffraction did not resolve the N-terminal residues so that the lipidation status of proteins in the mature complex was poorly described. Here we report liquid chromatography mass spectrometry revealing the intact mass values for all three proteins in the MtrCAB complexes purified from Shewanella oneidensis MR-1 and S. baltica OS185. The masses of MtrA and MtrB are consistent with both proteins being processed by Signal Peptidase I and covalent attachment of ten c -type hemes to MtrA. The mass of MtrC is most reasonably interpreted as arising from protein processed by Signal Peptidase II to produce a diacylated lipoprotein containing ten c -type hemes. Our two-step protocol for liquid-chromatography mass spectrometry used a reverse phase column to achieve on-column detergent removal prior to gradient protein resolution and elution. We envisage the method will be capable of simultaneously resolving the intact mass values for multiple proteins in other membrane protein complexes. [Display omitted] • An LC-MS method defining the intact mass of integral membrane proteins is presented. • On-line detergent removal occurs prior to membrane protein resolution and ionization. • Proteins of outer membrane MtrCAB complexes from Shewanella species are resolved. • The intact mass of MtrC proteins reveals a diacylated decaheme c -type cytochrome. • The method is readily optimized for application to other membrane proteins. [ABSTRACT FROM AUTHOR]

Chemical cross-linking would conceivably cause structural disruption of a protein, but few cross-linkers have been fully evaluated in this aspect. Furthermore, integral membrane proteins may differ from soluble proteins in the selection of suitable cross-linkers, which has never been investigated. In this study, we systematically evaluated the impact of five conventional cross-linkers targeting Lys, Asp and Glu, and two Arg-reactive cross-linkers on the structural and functional integrity of two G protein-coupled receptors (GPCRs). Perturbation of the receptor structure and ligand-binding activity was observed, depending on the receptor and cross-linking conditions. In particular, our study demonstrated that the concentrations of PDH and KArGO need to be fine-tuned in order to minimize the structural and functional disturbance of specific GPCRs. A set of amenable cross-linkers was selected to acquire the most comprehensive cross-link maps for two GPCRs. Our in-depth cross-linking mass spectrometry (CXMS) analysis has revealed dynamic features of structural regions in GPCRs that are not observable in the crystal structures. Thus, CXMS analysis of GPCRs using the expanded toolkit would facilitate structural modeling of uncharacterized receptors and gain new insights into receptor-ligand interactions. Image 1 • Assess the impact of cross-linkers on GPCR structural integrity with three orthogonal assays. • Evaluate and optimize seven cross-linkers for CXMS analysis of GPCRs. • Arg-reactive cross-linkers are particularly useful for increasing GPCR cross-linking coverage. • Acquire the most comprehensive cross-link maps for two GPCRs to reveal structural dynamics. [ABSTRACT FROM AUTHOR]

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

α-crystallin is a major protein found in the mammalian eye lens that works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress in the eye lens. These functions of α-crystallin are significant for maintaining lens transparency. However, with age and cataract formation, the concentration of α-crystallin in the eye lens cytoplasm decreases with a corresponding increase in the membrane-bound α-crystallin, accompanied by increased light scattering. The purpose of this review is to summarize previous and recent findings of the role of the: (1) lens membrane components, i.e., the major phospholipids (PLs) and sphingolipids, cholesterol (Chol), cholesterol bilayer domains (CBDs), and the integral membrane proteins aquaporin-0 (AQP0; formally MIP26) and connexins, and (2) α-crystallin mutations and post-translational modifications (PTMs) in the association of α-crystallin to the eye lens’s fiber cell plasma membrane, providing thorough insights into a molecular basis of such an association. Furthermore, this review highlights the current knowledge and need for further studies to understand the fundamental molecular processes involved in the association of α-crystallin to the lens membrane, potentially leading to new avenues for preventing cataract formation and progression.

Many microbes and fungi acquire the essential ion Fe3+ through the synthesis and secretion of high-affinity chelators termed siderophores. In Gram-negative bacteria, these ferric-siderophore complexes are actively taken up using highly specific TonB-dependent transporters (TBDTs) located in the outer bacterial membrane (OM). However, the detailed mechanism of how the inner-membrane protein TonB connects to the transporters in the OM as well as the interplay between siderophore- and TonB-binding to the transporter is still poorly understood. Here, we present three crystal structures of the TBDT FoxA from Pseudomonas aeruginosa (containing a signalling domain) in complex with the siderophore ferrioxamine B and TonB and combine them with a detailed analysis of binding constants. The structures show that both siderophore and TonB-binding is required to form a translocation-competent state of the FoxA transporter in a two-step TonB-binding mechanism. The complex structure also indicates how TonB-binding influences the orientation of the signalling domain.

The bimolecular fluorescence complementation (BiFC) assay has been widely used to examine interactions between integral and peripheral proteins within putative plasma membrane (PM) microdomains. In the course of using BiFC assays to examine the co-localization of plasma membrane (PM) targeted receptor-like kinases (RLKs), such as FLS2, with PM micro-domain proteins such as remorins, we unexpectedly observed heterogeneous distribution patterns of fluorescence on the PM of Nicotiana benthamiana leaf cortical cells. These patterns appeared to co-localize with the endoplasmic reticulum (ER) and with ER-PM contact sites, and closely resembled patterns caused by over-expression of the ER-PM tether protein Synaptotagmin1 (SYT1). Using domain swap experiments with SYT1, we inferred that non-specific dimerization between FLS2-VenusN and VenusC-StRem1.3 could create artificial ER-PM tether proteins analogous to SYT1. The same patterns of ER-PM tethering were produced when a representative set of integral membrane proteins were partnered in BiFC complexes with PM-targeted peripheral membrane proteins, including PtdIns(4)P-binding proteins. We inferred that spontaneous formation of mature fluorescent proteins caused the BiFC complexes to trap the integral membrane proteins in the ER during delivery to the PM, producing a PM-ER tether. This phenomenon could be a useful tool to deliberately manipulate ER-PM tethering or to test protein membrane localization. However, this study also highlights the risk of using the BiFC assay to study membrane protein interactions in plants, due to the possibility of alterations in cellular structures and membrane organization, or misinterpretation of protein-protein interactions. A number of published studies using this approach may therefore need to be revisited.

Tail‐anchored (TA) proteins are embedded into their corresponding membrane via a single transmembrane segment at their C‐terminus whereas the majority of the protein is facing the cytosol. So far, cellular factors that mediate the integration of such proteins into the mitochondrial outer membrane were not found. Using budding yeast as a model system, we identified the cytosolic Hsp70 chaperone Ssa1 and the peroxisome import factor Pex19 as import mediators for a subset of mitochondrial TA proteins. Accordingly, deletion of PEX19 results in: (1) growth defect under respiration conditions, (2) alteration in mitochondrial morphology, (3) reduced steady‐state levels of the mitochondrial TA proteins Fis1 and Gem1, and (4) hampered in organello import of the TA proteins Fis1 and Gem1. Furthermore, recombinant Pex19 can bind directly the TA proteins Fis1 and Gem1. Collectively, this work identified the first factors that are involved in the biogenesis of mitochondrial TA proteins and uncovered an unexpected function of Pex19. [ABSTRACT FROM AUTHOR]

Pannexin 1 (Panx1) is a ubiquitously expressed hexameric integral membrane protein known to function as an adenosine triphosphate (ATP) release channel. Panx1 proteins exist in unglycosylated core form (Gly0). They undergo critical post-translational modifications forming the high mannose glycosylation state (Gly1) in the endoplasmic reticulum (ER) and the complex glycosylation state (Gly2) in the Golgi apparatus. The regulation of transition from the ER to the cell membrane is not fully understood. Using site-specific mutagenesis, dye uptake assays, and interaction testing, we identified two conserved aromatic residues, Trp123 and Tyr205, in the transmembrane domains 2 and 3 of the zebrafish panx1a protein. Results suggest that both residues primarily govern the assembly of panx1a subunits into channels, with mutant proteins failing to interact. The results provide insight into a mechanism enabling regulation of Panx1 oligomerization, glycosylation, and trafficking.