Structure, mechanism, and inhibition of Hedgehog acyltransferase.

The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.

Membranes are functionalized by a proteolipid code.

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.

Plant protein-lipid interfaces studied by molecular dynamics simulations.

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.

Outer membrane vesicles as a platform for the discovery of antibodies to bacterial pathogens.

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.

Structural insights into a novel family of integral membrane siderophore reductases.

Gram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent 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 Fe-siderophore uptake.

Myelin Basic Protein Attenuates Furin-Mediated Bri2 Cleavage and Postpones Its Membrane Trafficking.

Myelin basic protein (MBP) is the second most abundant protein in the central nervous system and is responsible for structural maintenance of the myelin sheath covering axons. Previously, we showed that MBP has a more proactive role in the oligodendrocyte homeostasis, interacting with membrane-associated proteins, including integral membrane protein 2B (ITM2B or Bri2) that is associated with familial dementias. Here, we report that the molecular dynamics of the in silico-generated MBP-Bri2 complex revealed that MBP covers a significant portion of the Bri2 ectodomain, assumingly trapping the furin cleavage site, while the surface of the BRICHOS domain, which is responsible for the multimerization and activation of the Bri2 high-molecular-weight oligomer chaperone function, remains unmasked. These observations were supported by the co-expression of MBP with Bri2, its mature form, and disease-associated mutants, which showed that in mammalian cells, MBP indeed modulates the post-translational processing of Bri2 by restriction of the furin-catalyzed release of its C-terminal peptide. Moreover, we showed that the co-expression of MBP and Bri2 also leads to an altered cellular localization of Bri2, restricting its membrane trafficking independently of the MBP-mediated suppression of the Bri2 C-terminal peptide release. Further investigations should elucidate if these observations have physiological meaning in terms of Bri2 as a MBP chaperone activated by the MBP-dependent postponement of Bri2 membrane trafficking.

Amphipathic environments for determining the structure of membrane proteins by single-particle electron cryo-microscopy.

Over the past decade, the structural biology of membrane proteins (MPs) has taken a new turn thanks to epoch-making technical progress in single-particle electron cryo-microscopy (cryo-EM) as well as to improvements in sample preparation. The present analysis provides an overview of the extent and modes of usage of the various types of surfactants for cryo-EM studies. Digitonin, dodecylmaltoside, protein-based nanodiscs, lauryl maltoside-neopentyl glycol, glyco-diosgenin, and amphipols (APols) are the most popular surfactants at the vitrification step. Surfactant exchange is frequently used between MP purification and grid preparation, requiring extensive optimization each time the study of a new MP is undertaken. The variety of both the surfactants and experimental approaches used over the past few years bears witness to the need to continue developing innovative surfactants and optimizing conditions for sample preparation. The possibilities offered by novel APols for EM applications are discussed.

ATP binding and ATP hydrolysis in full-length MsbA monitored via time-resolved Fourier transform infrared spectroscopy.

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 k 1 = 0.49 ± 0.28 s-1 and k 2 = 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 (k 2 < 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.

Human myelin proteolipid protein structure and lipid bilayer stacking.

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.

A familial Danish dementia rat shows impaired presynaptic and postsynaptic glutamatergic transmission.

Familial British dementia and familial Danish dementia are neurodegenerative disorders caused by mutations in the gene integral membrane protein 2B (ITM2b) encoding BRI2, which tunes excitatory synaptic transmission at both presynaptic and postsynaptic termini. In addition, BRI2 interacts with and modulates proteolytic processing of amyloid-ß precursor protein (APP), whose mutations cause familial forms of Alzheimer's disease (AD) (familial AD). To study the pathogenic mechanisms triggered by the Danish mutation, we generated rats carrying the Danish mutation in the rat Itm2b gene (Itm2bD rats). Given the BRI2/APP interaction and the widely accepted relevance of human amyloid ß (Aß), a proteolytic product of APP, to AD, Itm2bD rats were engineered to express two humanized App alleles and produce human Aß. Here, we studied young Itm2bD rats to investigate early pathogenic changes in these diseases. We found that periadolescent Itm2bD rats not only present subtle changes in human Aß levels along with decreased spontaneous glutamate release and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated responses but also had increased short-term synaptic facilitation in the hippocampal Schaeffer-collateral pathway. These alterations in excitatory interneuronal communication can impair learning and memory processes and were akin to those observed in adult mice producing rodent Aß and carrying either the Danish or British mutations in the mouse Itm2b gene. Collectively, the data show that the pathogenic Danish mutation alters the physiological function of BRI2 at glutamatergic synapses across species and early in life. Future studies will determine whether this phenomenon represents an early pathogenic event in human dementia.

Expression, purification and preliminary characterisation of the choline transporter LicB from opportunistic bacterial pathogens.

Many opportunistic bacteria that infect the upper respiratory tract decorate their cell surface with phosphorylcholine to support colonisation and outgrowth. These surface modifications require the active import of choline from the host environment, a process thought to be mediated by a family of dedicated integral membrane proteins that act as choline permeases. Here, we present the expression and purification of the archetype of these choline transporters, LicB from Haemophilus influenzae. We show that LicB can be recombinantly produced in Escherichia coli and purified to homogeneity in a stable, folded state using the detergent n-dodecyl-ß-d-maltopyranoside. Equilibrium binding studies with the fluorescent ligand dansylcholine suggest that LicB is selective towards choline, with reduced affinity for acetylcholine and no apparent activity towards other small molecules including glycine, carnitine and betaine. We also identify a conserved sequence motif within the LicB family and show that mutations within this motif compromise protein structure and function. Our results are consistent with previous observations that LicB is a specific high-affinity choline transporter, and provide an experimental platform for further studies of this permease family.

Novel ORAI1 Mutation Disrupts Channel Trafficking Resulting in Combined Immunodeficiency.

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.

Full-length G glycoprotein directly extracted from rabies virus with detergent and then stabilized by amphipols in liquid and freeze-dried forms.

Pathogen surface antigens are at the forefront of the viral strategy when invading host organisms. These antigens, including membrane proteins (MPs), are broadly targeted by the host immune response. Obtaining these MPs in a soluble and stable form constitutes a real challenge, regardless of the application purposes (e.g. quantification/characterization assays, diagnosis, and preventive and curative strategies). A rapid process to obtain a native-like antigen by solubilization of a full-length MP directly from a pathogen is reported herein. Rabies virus (RABV) was used as a model for this demonstration and its full-length G glycoprotein (RABV-G) was stabilized with amphipathic polymers, named amphipols (APols). The stability of RABV-G trapped in APol A8-35 (RABV-G/A8-35) was evaluated under different stress conditions (temperature, agitation, and light exposure). RABV-G/A8-35 in liquid form exhibited higher unfolding temperature (+6°C) than in detergent and was demonstrated to be antigenically stable over 1 month at 5°C and 25°C. Kinetic modeling of antigenicity data predicted antigenic stability of RABV-G/A8-35 in a solution of up to 1 year at 5°C. The RABV-G/A8-35 complex formulated in an optimized buffer composition and subsequently freeze-dried displayed long-term stability for 2-years at 5, 25, and 37°C. This study reports for the first time that a natural full-length MP extracted from a virus, complexed to APols and subsequently freeze-dried, displayed long-term antigenic stability, without requiring storage under refrigerated conditions.

Characterization of the substrate binding site of an iron detoxifying membrane transporter from Plasmodium falciparum.

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.

Porphyromonas gingivalis facilitated the foam cell formation via lysosomal integral membrane protein 2 (LIMP2).

OBJECTIVE: The involvement of lysosomal integral membrane protein 2 (LIMP2) in cholesterol transport and formation of foam cells under the infection of Porphyromonas gingivalis (P. gingivalis) is yet to be elucidated. The current study verified the role and explored the mechanism of LIMP2 in promoting foam cell formation by P. gingivalis. BACKGROUND: An association between periodontitis and atherosclerosis (AS) has been established. P. gingivalis is a key pathogen of periodontitis that promotes foam cell formation by regulating activities of CD36 scavenger receptors expressed on the macrophages. LIMP2, a member of CD36 superfamily, is involved in cholesterol efflux. However, whether LIMP2 is involved in the formation of foam cells promoted by P. gingivalis remains unclear. METHODS: The formation of foam cells was examined by Oil Red O staining. The knockdown of limp2 was identified by qRT-PCR. The accumulation of cholesterol was monitored by Cholesterol Assay Kit. The location of P. gingivalis was visualized by confocal microscopy. Cathepsin L activity was monitored with Magic Red Cathepsin L Assay Kit. The key genes and pathways in P. gingivalis-infected macrophages were explored by RNA sequencing. The protein level was investigated by Western blotting. RESULTS: Porphyromonas gingivalis increases foam cells formation and upregulates the expression of LIMP2 in foam cells. The knockdown of limp2 decreases the number of foam cells and increases cholesterol export, which is related to lysosomal functions. In addition, the interaction between LIMP2 and caveolin-1(CAV1) might contribute to this process, and NF-κB and JNK activity is required for increased expression of P. gingivalis-induced LIMP2. CONCLUSIONS: This study suggested that LIMP2 is involved in the foam cells formation facilitated by P. gingivalis, which favors a close connection between periodontitis and atherosclerosis (AS).

Insights into the Role of Membrane Lipids in the Structure, Function and Regulation of Integral Membrane Proteins.

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.

Pex19 is involved in importing dually targeted tail-anchored proteins to both mitochondria and peroxisomes.

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.

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.

Mechanistic insights into transferable polymyxin resistance among gut bacteria.

Polymyxins such as colistin are antibiotics used as a final line of defense in the management of infections with multidrug-resistant Gram-negative bacteria. Although natural resistance to polymyxins is rare, the discovery of a mobilized colistin resistance gene (mcr-1) in gut bacteria has raised significant concern. As an intramembrane enzyme, MCR-1 catalyzes the transfer of phosphoethanolamine (PEA) to the 1 (or 4')-phosphate group of the lipid A moiety of lipopolysaccharide, thereby conferring colistin resistance. However, the structural and biochemical mechanisms used by this integral membrane enzyme remain poorly understood. Here, we report the modeled structure of the full-length MCR-1 membrane protein. Together with molecular docking, our structural and functional dissection of the complex of MCR-1 with its phosphatidylethanolamine (PE) substrate suggested the presence of a 12 residue-containing cavity for substrate entry, which is critical for both enzymatic activity and its resultant phenotypic resistance to colistin. More importantly, two periplasm-facing helices (PH2 and PH2') of the trans-membrane domain were essential for MCR-1 activity. MALDI-TOF MS and thin-layer chromatography assays provide both in vivo and in vitro evidence that MCR-1 catalyzes the transfer of PEA from the PE donor substrate to its recipient substrate lipid A. Also, the chemical modification of lipid A species was detected in clinical species of bacteria carrying mcr-1 Our results provide mechanistic insights into transferable MCR-1 polymyxin resistance, raising the prospect of rational design of small molecules that reverse bacterial polymyxin resistance, as a last-resort clinical option to combat pathogens with carbapenem resistance.

The glycan-dependent ERAD machinery degrades topologically diverse misfolded proteins.

Many soluble and integral membrane proteins fold in the endoplasmic reticulum (ER) with the help of chaperones and folding factors. Despite these efforts, protein folding is intrinsically error prone and amino acid changes, alterations in post-translational modifications or cellular stress can cause protein misfolding. Folding-defective non-native proteins are cleared from the ER and typically undergo ER-associated degradation (ERAD). Here, we investigated whether different misfolded glycoproteins require the same set of ERAD factors and are directed to HRD1 complex-mediated degradation in plants. We generated a series of glycoprotein ERAD substrates harboring a misfolded domain from Arabidopsis STRUBBELIG or the BRASSINOSTEROID INSENSITVE 1 receptor fused to different membrane anchoring regions. We show that single pass and multispanning ERAD substrates are subjected to glycan-dependent degradation by the HRD1 complex. However, the presence of a powerful ER exit signal in the multispanning ERAD substrates causes competition with ER quality control and targeting of misfolded glycoproteins to the vacuole. Our results demonstrate that the same machinery is used for degradation of topologically different misfolded glycoproteins in the ER of plants.