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Although prions are known as protein-only infectious particles, they exhibit lipid specificities, cofactor dependencies and membrane-dependent activities. Such membrane interactions play key roles in how prions are processed, presented and regulated, and hence have significant functional consequences. The expansive literature related to prion protein interactions with lipids and native nanodiscs is discussed, and provides a unique opportunity to re-evaluate the molecular composition and mechanisms of its infectious and cellular states. A family of crystal and solution structures of prions are analyzed here for the first time using the membrane optimal docking area (MODA) program, revealling the presence of structured binding elements that could mediate specific lipid recognition. A set of motifs centerred around W99, L125, Y169 and Y226 are consistently predicted as being membrane interactive and form an exposed surface which includes α helical, β strand and loop elements involving the prion protein (PrP) structural domain, while the scrapie form is radically different and doubles the size of the membrane interactive site into an extensible surface. These motifs are highly conserved throughout mammalian evolution, suggesting that prions have long been intrinsically attached to membranes at central and N- and C-terminal points, providing several opportunities for stable and specific bilayer interactions as well as multiple complexed orientations. Resistance or susceptibility to prion disease correlates with increased or decreased membrane binding propensity by mutant forms, respectively, indicating a protective role by lipids. The various prion states found in vivo are increasingly resolvable using native nanodiscs formed by styrene maleic acid (SMA) and stilbene maleic acid (STMA) copolymers rather than classical detergents, allowing the endogenous states to be tackled. These copolymers spontaneously fragment intact membranes into water-soluble discs holding a section of native bilayer, and can accommodate prion multimers and mini-fibrils. Such nanodiscs have also proven useful for understanding how β amyloid and α synuclein proteins contribute to Alzheimer's and Parkinson's diseases, providing further biomedical applications. Structural and functional insights of such proteins in styrene maleic acid lipid particles (SMALPs) can be resolved at high resolution by methods including cryo-electron microscopy (cEM), motivating continued progress in polymer design to resolve biological and pathological mechanisms., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Rho proteins are signalling molecules that control cellular dynamics, movement and morphological changes. They are activated by Rho guanine-nucleotide exchange factors (Rho GEFs) that transduce upstream signals into Rho-mediated activation of downstream processes. Fgd5 is a Rho GEF involved in angiogenesis and its target Rho protein for this process has been linked to Cdc42 activation. Here, we examined the function of purified Fgd5, specifically, which Rho proteins it activates and pinpoint the structural domains required for enzymatic activity. Using a GEF enzyme assay, we found that purified Fgd5 showed preferential activation of Rac1 and direct binding of Rac1 in pull-down and co-immunoprecipitation assays. Structural comparisons showed that the Fgd5 DH domain is highly similar to the Rac1 GEF, TrioN, supporting a role for Fgd5 as a Rac1 GEF. Compounds that bind to purified Fgd5 DH-PH protein were identified by screening a small molecule library via surface plasmon resonance. The effects of eleven ligands were further examined for their ability to inhibit the Fgd5 GEF enzymatic activity and Rac1 interaction. From these studies, we found that the compound aurintricarboxylic acid, and to a lesser extent mitoxantrone dihydrochloride, inhibited both Fgd5 GEF activation of Rac1 and their interaction. Aurintricarboxylic acid had no effect on the activity or binding of the Rac1 GEF, TrioN, thus demonstrating the feasibility of selectively disrupting Rho GEF activators. Abbreviations : a.a.: amino acid; ATA: aurintricarboxylic acid; DH: Dbl homology; DOCK: dictator of cytokinesis; Fgd: faciogenital dysplasia; GEF: guanine-nucleotide exchange factor; GST: glutathione S -transferase; LOPAC: library of pharmacologically active compounds; PH: pleckstrin homology; PDB: protein data bank; s.e.m.: standard error of the mean; SPR: surface plasmon resonance.

The Gram-negative outer-membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents, and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here, we reveal DolP is a lipoprotein functionally conserved amongst Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for Escherichia coli DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds anionic phospholipids through an extensive membrane:protein interface. This interaction is essential for DolP function and is required for sub-cellular localisation of the protein to the cell division site, providing evidence of subcellular localisation of these phospholipids within the outer membrane. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope., Competing Interests: JB, FM, TK, RM, EH, GB, DA, AC, PW, KS, MJ, DB, YS, TW, AR, VB, PS, DW, ZC, EG, CI, AT, SC, DR, TL, AC, MB, MO, IH No competing interests declared, (© 2020, Bryant et al.)

The plakin family of cytolinkers interacts with intermediate filaments (IFs) through plakin repeat domain (PRD) and linker modules. Recent structure/function studies have established the molecular basis of envoplakin-PRD and periplakin-linker interactions with vimentin. Both plakin modules share a broad basic groove which recognizes acidic rod elements on IFs, a mechanism that is applicable to other plakin family members. This review postulates a universal IF engagement mechanism that illuminates the specific effects of pathogenic mutations associated with diseases including arrhythmogenic right ventricular cardiomyopathy, and reveals how diverse plakin proteins offer tailored IF tethering to ensure stable, dynamic and regulated cellular structures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The CaMK subfamily of Ser/Thr kinases are regulated by calmodulin interactions with their C-terminal regions. They are exemplified by Ca 2+ /calmodulin dependent protein kinase 1δ which is known as CaMK1D, CaMKIδ or CKLiK. CaMK1D mediates intracellular signalling downstream of Ca 2+ influx and thereby exhibits amplifications of Ca 2+ signals and polymorphisms that have been implicated in breast cancer and diabetes. Here we report the backbone 1 H, 13 C, 15 N assignments of the 38 kDa human CaMK1D protein in its free state, including both the canonical bi-lobed kinase fold as well as the autoinhibitory and calmodulin binding domains.

The development of amphipathic polymers, including various formulations of styrene-maleic acid (SMA) copolymers, has allowed the purification of increasing sizes and complexities of biological membrane protein assemblies in native nanodiscs. However, the factors determining the sizes and shapes of the resulting bio-nano particles remain unclear. Here, we show how grafting on short alkyl amine sidechains onto the polar residues leads to a broad set of nanoparticle sizes with improved solution behavior. The solubilization of lipid vesicles occurs over a wide range of pH levels and calcium concentrations, providing utility across the physiologically relevant range of solution conditions. Furthermore, the active SMA derivatives can contain strictly alternating monomers, which have inherently lower sequence polydispersity. Pronounced differences in the shapes of native nanoparticles were formed from Escherichia coli bacterial outer membrane containing PagP protein using methyl, ethyl and propylamine derivatives of styrene-maleic anhydride. In particular, the methylamine-substituted polymer forms smaller, monodispersed nanodiscs, while the longer alkyl derivatives form worm-like nanostructures. Thus the introduction of hydrophobicity onto the polar sidechains of amphipathic polymers has profound effects on morphology of native nanodisc, with shorter methyl moieties offering more uniformity and utility for structural biology studies., Competing Interests: Declaration of competing interest The authors M.E. and M.O. have a competing financial interest. They have filed a patent on the polymers presented in this manuscript., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Methylstilbene-alt-maleic acid copolymers spontaneously convert biological membranes into bilayer discs with ∼20 nm diameters. This readily functionalizable class of copolymers has the compositional homogeneity, hydrophobicity, dynamics, and charge that may help to achieve optimal structural resolution, membrane dissolution, stability, and broad utility.

Polymorphisms in the region of the calmodulin-dependent kinase isoform D (CaMK1D) gene are associated with increased incidence of diabetes, with the most common polymorphism resulting in increased recognition by transcription factors and increased protein expression. While reducing CaMK1D expression has a potentially beneficial effect on glucose processing in human hepatocytes, there are no known selective inhibitors of CaMK1 kinases that can be used to validate or translate these findings. Here we describe the development of a series of potent, selective, and drug-like CaMK1 inhibitors that are able to provide significant free target cover in mouse models and are therefore useful as in vivo tool compounds. Our results show that a lead compound from this series improves insulin sensitivity and glucose control in the diet-induced obesity mouse model after both acute and chronic administration, providing the first in vivo validation of CaMK1D as a target for diabetes therapeutics.

Chaperones in the endoplasmic reticulum (ER) control the flux of Ca 2+ ions into mitochondria, thereby increasing or decreasing the energetic output of the oxidative phosphorylation pathway. An example is the abundant ER lectin calnexin, which interacts with sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA). We found that calnexin stimulated the ATPase activity of SERCA by maintaining its redox state. This function enabled calnexin to control how much ER Ca 2+ was available for mitochondria, a key determinant for mitochondrial bioenergetics. Calnexin-deficient cells compensated for the loss of this function by partially shifting energy generation to the glycolytic pathway. These cells also showed closer apposition between the ER and mitochondria. Calnexin therefore controls the cellular energy balance between oxidative phosphorylation and glycolysis., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Prions are lipidated proteins that interact with endogenous lipids and metal ions. They also assemble into multimers and propagate into the infectious scrapie form known as PrP Sc The high-resolution structure of the infectious PrP Sc state remains unknown, and its analysis largely relies on detergent-based preparations devoid of endogenous ligands. Here we designed polymers that allow isolation of endogenous membrane:protein assemblies in native nanodiscs without exposure to conventional detergents that destabilize protein structures and induce fibrillization. A set of styrene-maleic acid (SMA) polymers including a methylamine derivative facilitated gentle release of the infectious complexes for resolution of multimers, and a thiol-containing version promoted crystallization. Polymer extraction from brain homogenates from Syrian hamsters infected with Hyper prions and WT mice infected with Rocky Mountain Laboratories prions yielded infectious prion nanoparticles including oligomers and microfilaments bound to lipid vesicles. Lipid analysis revealed the brain phospholipids that associate with prion protofilaments, as well as those that are specifically enriched in prion assemblies captured by the methylamine-modified copolymer. A comparison of the infectivity of PrP Sc attached to SMA lipid particles in mice and hamsters indicated that these amphipathic polymers offer a valuable tool for high-yield production of intact, detergent-free prions that retain in vivo activity. This native prion isolation method provides an avenue for producing relevant prion:lipid targets and potentially other proteins that form multimeric assemblies and fibrils on membranes., Competing Interests: Conflict of interest—M. O. and M. E. have filed a patent on related SMA copolymers and methods., (© 2020 Esmaili et al.)

Daptomycin is a lipopeptide antibiotic that is important in the treatment of infections with Gram-positive bacteria. In the presence of calcium, daptomycin binds to phosphatidylglycerol in the bacterial cytoplasmic membrane and then forms oligomers that mediate its bactericidal effect. The structure of these bactericidal oligomers has not been elucidated. We here explore the feasibility of structural studies on the oligomer by solution-state NMR. To this end, we use nanodiscs that contain DMPC and DMPG, stabilized with a styrene-maleic acid copolymer that has been modified to minimize calcium chelation. We show that these nanodiscs bind daptomycin and induce the formation of stable oligomers under physiologically relevant conditions. The findings suggest that this membrane model is suitable for structural and functional characterization of oligomeric daptomycin, and possibly of other calcium-dependent lipopeptide antibiotics. We show that these nanodiscs bind daptomycin and induce the formation of stable oligomers, under conditions that are suitable for biomolecular NMR. The findings suggest that this membrane model is suitable for structural elucidation of oligomeric daptomycin, and possibly of other calcium-dependent lipopeptide antibiotics., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The plant plasma membrane Na + /H + antiporter SOS1 (Salt Overlay Sensitive 1) of Arabidopsis thaliana is the major transporter extruding Na + out of cells in exchange for an intracellular H + . The sodium extrusion process maintains a low intracellular Na + concentration and thereby facilitates salt tolerance. A. thaliana SOS1 consists of 1146 amino acids, with the first 450 in a N-terminal membrane transport domain and the balance forming a cytosolic regulatory domain. For studies on characterization of the protein, two different constructs of SOS1 comprising of the residues 28 to 460 and 28 to 990 were cloned and overexpressed in methylotropic yeast strain of Pichia pastoris with a C-terminal histidine tag using the expression vector pPICZA. Styrene malic acid copolymers (SMA) were used as a cost-effective alternative to detergent for solubilization and isolation of this membrane protein. Immobilized Ni 2+ -ion affinity chromatography was used to purify the expressed protein resulting in a yield of ~0.6-2 mg of SOS1 per liter of Pichia pastoris culture. The SMA purified protein containing amino acids 28 to 990 was directly reconstituted into liposomes for determination of Na + transport activity and was functionally active. However, similar reconstitution with amino acids 28-460 did not yield a functional protein. Other results have shown that the truncated SOS1 protein at amino acid 481 is active, which infers the presence of an element between residues 461-481 which is necessary for SOS1 activity. This region contains several conserved segments that may be important in SOS1 structure and function., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Plakin proteins form connections that link the cell membrane to the intermediate filament cytoskeleton. Their interactions are mediated by a highly conserved linker domain through an unresolved mechanism. Here analysis of the human periplakin linker domain structure reveals a bi-lobed module transected by an electropositive groove. Key basic residues within the periplakin groove are vital for co-localization with vimentin in human cells and compromise direct binding which also requires acidic residues D176 and E187 in vimentin. We propose a model whereby basic periplakin linker domain residues recognize acidic vimentin side chains and form a complementary binding groove. The model is shared amongst diverse linker domains and can be used to investigate the effects of pathogenic mutations in the desmoplakin linker associated with arrhythmogenic right ventricular cardiomyopathy. Linker modules either act solely or collaborate with adjacent plakin repeat domains to create strong and adaptable tethering within epithelia and cardiac muscle.

Transmembrane proteins function within a continuous layer of biologically relevant lipid molecules that stabilizes their structures and modulates their activities. Structures and interactions of biological membrane-protein complexes or "memteins" can now be elucidated using native nanodiscs made by poly(styrene co-maleic anhydride) derivatives. These linear polymers contain a series of hydrophobic and polar subunits that gently fragment membranes into water-soluble discs with diameters of 5-50 nm known as styrene maleic acid lipid particles (SMALPs). High-resolution structures of memteins that include endogenous lipid ligands and posttranslational modifications can be resolved without resorting to synthetic detergents or artificial lipids. The resulting ex situ structures better recapitulate the in vivo situation and can be visualized by methods including cryo-electron microscopy (cryoEM), electron paramagnetic resonance (EPR), mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, small angle x-ray scattering (SAXS), and x-ray diffraction (XRD). Recent progress including 3D structures of biological bilayers illustrates how polymers and native nanodiscs expose previously inaccessible membrane assemblies at atomic resolution and suggest ways in which the SMALP system could be exploited for drug discovery.

FYVE domains are highly conserved protein modules that typically bind phosphatidylinositol 3-phosphate (PI3P) on the surface of early endosomes. Along with pleckstrin homology (PH) and phox homology (PX) domains, FYVE domains are the principal readers of the phosphoinositide (PI) code that mediate specific recognition of eukaryotic organelles. Of all the human FYVE domain containing proteins, those within the faciogenital dysplasia (Fgd) subfamily are particularly divergent and couple with GTPases to exert unique cellular functions. The subcellular distributions and functions of these evolutionarily conserved signal transducers, which also include Dbl homology (DH) and two PH domains, are discussed here to better understand the biological range of processes that such multidomain proteins engage in. Determinants of their various functions include specific multidomain architectures, posttranslational modifications including PIP stops that have been discovered in sorting nexins, PI recognition motifs, and phospholipid-binding surfaces as defined by the Membrane Optimal Docking Area (MODA) program. How these orchestrate Fgd function remains unclear but has implications for developmental diseases including Aarskog-Scott syndrome, which is also known as faciogenital dysplasia, and forms of cancer that are associated with mutations and amplifications of Fgd genes.

The concept of a memtein as the minimal unit of membrane function is proposed here, and refers to the complex of a membrane protein together with a continuous layer of biological lipid molecules. The elucidation of the atomic resolution structures and specific interactions within memteins remains technically challenging. Nonetheless, we argue that these entities are critical endpoints for the postgenomic era, being essential units of cellular function that mediate signal transduction and trafficking. Their biological mechanisms and molecular compositions can be resolved using native nanodiscs formed by poly(styrene-co-maleic acid) (SMA) copolymers. These amphipathic polymers rapidly and spontaneously fragment membranes into water-soluble discs holding a section of bilayer. This allows structures of complexes found in vivo to be prepared without resorting to synthetic detergents or artificial lipids. The ex situ structures of memteins can be resolved by methods including cryo-electron microscopy (cEM), X-ray crystallography (XRC), NMR spectroscopy and mass spectrometry (MS). Progress in the field demonstrates that memteins are better representations of how biology actually works in membranes than naked proteins devoid of lipid, spurring on further advances in polymer chemistry to resolve their details., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin γ-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15 N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Mutations in σ E -regulated lipoproteins have previously been shown to impact bacterial viability under conditions of stress and during in vivo infection. YraP is conserved across a number of Gram-negative pathogens, including Neisseria meningitidis , where the homolog is a component of the Bexsero meningococcal group B vaccine. Investigations using laboratory-adapted Escherichia coli K-12 have shown that yraP mutants have elevated sensitivity to a range of compounds, including detergents and normally ineffective antibiotics. In this study, we investigate the role of the outer membrane lipoprotein YraP in the pathogenesis of Salmonella enterica serovar Typhimurium. We show that mutations in S Typhimurium yraP result in a defective outer membrane barrier with elevated sensitivity to a range of compounds. This defect is associated with attenuated virulence in an oral infection model and during the early stages of systemic infection. We show that this attenuation is not a result of defects in lipopolysaccharide and O-antigen synthesis, changes in outer membrane protein levels, or the ability to adhere to and invade eukaryotic cell lines in vitro ., (Copyright © 2018 American Society for Microbiology.)

The immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B has emerged as a key regulator of platelet homeostasis. However, it remains unclear how it mediates its effects. Tyrosine phosphorylation of ITIM and immunoreceptor tyrosine-based switch motif (ITSM) within the cytoplasmic tail of G6b-B provides a docking site for Src homology 2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2, which are also critical regulators of platelet production and function. In this study, we investigate the physiological consequences of uncoupling G6b-B from Shp1 and Shp2. To address this, we generated a transgenic mouse model expressing a mutant form of G6b-B in which tyrosine residues 212 and 238 within ITIM and ITSM were mutated to phenylalanine. Mice homozygous for the mutation ( G6b-B diY/F ) were macrothrombocytopenic, as a result of the reduction in platelet production, and had large clusters of megakaryocytes and myelofibrosis at sites of hematopoiesis, similar to those observed in G6b -deficient mice and patients. Platelets from G6b-B diY/F mice were hyporesponsive to collagen, as a result of the significant reduction in the expression of the immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor complex GPVI-FcR γ-chain, as well as thrombin, which could be partially rescued by costimulating the platelets with adenosine diphosphate. In contrast, platelets from G6b-B diY/F , G6b KO , and megakaryocyte-specific Shp2 KO mice were hyperresponsive to antibody-mediated cross-linking of the hemi-ITAM-containing podoplanin receptor CLEC-2, suggesting that G6b-B inhibits CLEC-2-mediated platelet activation through Shp2. Findings from this study demonstrate that G6b-B must engage with Shp1 and Shp2 to mediate its regulatory effects on platelet homeostasis., (© 2018 by The American Society of Hematology.)

Sorting nexins anchor trafficking machines to membranes by binding phospholipids. The paradigm of the superfamily is sorting nexin 3 (SNX3), which localizes to early endosomes by recognizing phosphatidylinositol 3-phosphate (PI3P) to initiate retromer-mediated segregation of cargoes to the trans-Golgi network (TGN). Here we report the solution structure of full length human SNX3, and show that PI3P recognition is accompanied by bilayer insertion of a proximal loop in its extended Phox homology (PX) domain. Phosphoinositide (PIP) binding is completely blocked by cancer-linked phosphorylation of a conserved serine beside the stereospecific PI3P pocket. This "PIP-stop" releases endosomal SNX3 to the cytosol, and reveals how protein kinases control membrane assemblies. It constitutes a widespread regulatory element found across the PX superfamily and throughout evolution including of fungi and plants. This illuminates the mechanism of a biological switch whereby structured PIP sites are phosphorylated to liberate protein machines from organelle surfaces.

Discovering how membrane proteins recognize signals and passage molecules remains challenging. Life depends on compartmentalizing these processes into dynamic lipid bilayers that are technically difficult to work with. Several polymers have proven adept at separating the responsible machines intact for detailed analysis of their structures and interactions. Styrene maleic acid (SMA) co-polymers efficiently solubilize membranes into native nanodiscs and, unlike amphipols and membrane scaffold proteins, require no potentially destabilizing detergents. Here we review progress with the SMA lipid particle (SMALP) system and its impacts including three dimensional structures and biochemical functions of peripheral and transmembrane proteins. Polymers systems are emerging to tackle the remaining challenges for wider use and future applications including in membrane proteomics, structural biology of transient or unstable states, and discovery of ligand and drug-like molecules specific for native lipid-bound states., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The mechanisms behind Aβ-peptide accumulation in non-familial Alzheimer's disease (AD) remain elusive. Proteins of the tetraspanin family modulate Aβ production by interacting to γ-secretase., Methods: We searched for tetraspanins with altered expression in AD brains. The function of the selected tetraspanin was studied in vitro and the physiological relevance of our findings was confirmed in vivo., Results: Tetraspanin-6 (TSPAN6) is increased in AD brains and overexpression in cells exerts paradoxical effects on Amyloid Precursor Protein (APP) metabolism, increasing APP-C-terminal fragments (APP-CTF) and Aβ levels at the same time. TSPAN6 affects autophagosome-lysosomal fusion slowing down the degradation of APP-CTF. TSPAN6 recruits also the cytosolic, exosome-forming adaptor syntenin which increases secretion of exosomes that contain APP-CTF., Conclusions: TSPAN6 is a key player in the bifurcation between lysosomal-dependent degradation and exosome mediated secretion of APP-CTF. This corroborates the central role of the autophagosomal/lysosomal pathway in APP metabolism and shows that TSPAN6 is a crucial player in APP-CTF turnover.

Uncontrolled activation of Rho signaling by RhoGEFs, in particular AKAP13 (Lbc) and its close homologs, is implicated in a number of human tumors with poor prognosis and resistance to therapy. Structure predictions and alanine scanning mutagenesis of Lbc identified a circumscribed hot region for RhoA recognition and activation. Virtual screening targeting that region led to the discovery of an inhibitor of Lbc-RhoA interaction inside cells. By interacting with the DH domain, the compound inhibits the catalytic activity of Lbc, halts cellular responses to activation of oncogenic Lbc pathways, and reverses a number of prostate cancer cell phenotypes such as proliferation, migration, and invasiveness. This study provides insights into the structural determinants of Lbc-RhoA recognition. This is a successful example of structure-based discovery of a small protein-protein interaction inhibitor able to halt oncogenic Rho signaling in cancer cells with therapeutic implications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Human papillomaviruses enter host cells via a clathrin-independent endocytic pathway involving tetraspanin proteins. However, post-endocytic trafficking required for virus capsid disassembly remains unclear. Here we demonstrate that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63, syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive endosomes recruiting syntenin-1, a CD63-interacting adaptor protein. Electron microscopy and immunofluorescence experiments indicate that the CD63-syntenin-1 complex controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types 16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly. Thus, our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking.

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.

Rho GTPases regulate cellular morphology and dynamics, and some are key drivers of cancer progression. This superfamily offers attractive potential targets for therapeutic intervention, with RhoA, Rac1 and Cdc42 being prime examples. The challenges in developing agents that act on these signaling enzymes include the lack of obvious druggable pockets and their membrane-bound activities. However, progress in targeting the similar Ras protein is illuminating new strategies for specifically inhibiting oncogenic GTPases. The structures of multiple signaling and regulatory states of Rho proteins have been determined, and the post-translational modifications including acylation and phosphorylation points have been mapped and their functional effects examined. The development of inhibitors to probe the significance of overexpression and mutational hyperactivation of these GTPases underscores their importance in cancer progression. The ability to integrate in silico, in vitro, and in vivo investigations of drug-like molecules indicates the growing tractability of GTPase systems for lead optimization. Although no Rho-targeted drug molecules have yet been clinically approved, this family is clearly showing increasing promise for the development of precision medicine and combination cancer therapies.

Dendritic epidermal T cells (DETC) form a skin-resident γδ T cell population that makes key contributions to cutaneous immune stress surveillance, including non-redundant contributions to protection from cutaneous carcinogens. How DETC become uniquely associated with the epidermis was in large part solved by the identification of Skint-1, the prototypic member of a novel B7-related multigene family. Expressed only by thymic epithelial cells and epidermal keratinocytes, Skint-1 drives specifically the development of DETC progenitors, making it the first clear candidate for a selecting ligand for non-MHC/CD1-restricted T cells. However, the molecular mechanisms underpinning Skint-1 activity are unresolved. Here, we provide evidence that DETC selection requires Skint-1 expression on the surface of thymic epithelial cells, and depends upon specific residues on the CDR3-like loop within the membrane-distal variable domain of Skint-1 (Skint-1 DV). Nuclear magnetic resonance of Skint-1 DV revealed a core tertiary structure conserved across the Skint family, but a highly distinct surface charge distribution, possibly explaining its unique function. Crucially, the CDR3-like loop formed an electrostatically distinct surface, featuring key charged and hydrophobic solvent-exposed residues, at the membrane-distal tip of DV. These results provide the first structural insights into the Skint family, identifying a putative receptor binding surface that directly implicates Skint-1 in receptor-ligand interactions crucial for DETC selection., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The plakin repeat domain is a distinctive hallmark of the plakin superfamily of proteins, which are found within all epithelial tissues. Plakin repeat domains mediate the interactions of these proteins with the cell cytoskeleton and are critical for the maintenance of tissue integrity. Despite their biological importance, no solution state resonance assignments are available for any homologue. Here we report the essentially complete (1)H, (13)C and (15)N backbone chemical shift assignments of the singular 22 kDa plakin repeat domain of human envoplakin, providing the means to investigate its interactions with ligands including intermediate filaments.

Plakin proteins form critical connections between cell junctions and the cytoskeleton; their disruption within epithelial and cardiac muscle cells cause skin-blistering diseases and cardiomyopathies. Envoplakin has a single plakin repeat domain (PRD) which recognizes intermediate filaments through an unresolved mechanism. Herein we report the crystal structure of envoplakin's complete PRD fold, revealing binding determinants within its electropositive binding groove. Four of its five internal repeats recognize negatively charged patches within vimentin via five basic determinants that are identified by nuclear magnetic resonance spectroscopy. Mutations of the Lys1901 or Arg1914 binding determinants delocalize heterodimeric envoplakin from intracellular vimentin and keratin filaments in cultured cells. Recognition of vimentin is abolished when its residues Asp112 or Asp119 are mutated. The latter slot intermediate filament rods into basic PRD domain grooves through electrosteric complementarity in a widely applicable mechanism. Together this reveals how plakin family members form dynamic linkages with cytoskeletal frameworks.

Membrane proteins are essential for the flow of signals, nutrients and energy between cells and between compartments of the cell. Their mechanisms can only be fully understood once the precise structures, dynamics and interactions involved are defined at atomic resolution. Through advances in solution and solid state NMR spectroscopy, this information is now available, as demonstrated by recent studies of stable peripheral and transmembrane proteins. Here we highlight recent cases of G-protein coupled receptors, outer membrane proteins, such as VDAC, phosphoinositide sensors, such as the FAPP-1 pleckstrin homology domain, and enzymes including the metalloproteinase MMP-12. The studies highlighted have resulted in the determination of the 3D structures, dynamical properties and interaction surfaces for membrane-associated proteins using advanced isotope labelling strategies, solubilisation systems and NMR experiments designed for very high field magnets. Solid state NMR offers further insights into the structure and multimeric assembly of membrane proteins in lipid bilayers, as well as into interactions with ligands and targets. Remaining challenges for wider application of NMR to membrane structural biology include the need for overexpression and purification systems for the production of isotope-labelled proteins with fragile folds, and the availability of only a few expensive perdeuterated detergents.Step changes that may transform the field include polymers, such as styrene maleic acid, which obviate the need for detergent altogether, and allow direct high yield purification from cells or membranes. Broader demand for NMR may be facilitated by MODA software, which instantly predicts membrane interactive residues that can subsequently be validated by NMR. In addition, recent developments in dynamic nuclear polarization NMR instrumentation offer a remarkable sensitivity enhancement from low molarity samples and cell surfaces. These advances illustrate the current capabilities and future potential of NMR for membrane protein structural biology and ligand discovery.

The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

Sorting nexin 3 (SNX3) belongs to a sub-family of sorting nexins that primarily contain a single Phox homology domain capable of binding phosphoinositides and membranes. We report the complete (1)H, (13)C and (15)N resonance assignments of the full-length human SNX3 protein and identification of its secondary structure elements, revealing a canonical fold and unstructured termini.

BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA are found in all Gram-negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the outer membrane. BamA binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains at its N-terminus. The C-terminus of BamA folds into a β-barrel domain, which tethers BamA to the outer membrane and is involved in OMP insertion. BamA orthologues are found in all Gram-negative bacteria and appear to function in a species-specific manner. Here we investigate the nature of this species-specificity by examining whether chimeric Escherichia coli BamA fusion proteins, carrying either the β-barrel or POTRA domains from various BamA orthologues, can functionally replace E. coli BamA. We demonstrate that the β-barrel domains of many BamA orthologues are functionally interchangeable. We show that defects in the orthologous POTRA domains can be rescued by compensatory mutations within the β-barrel. These data reveal that the POTRA and barrel domains must be precisely aligned to ensure efficient OMP insertion., (© 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (~5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([(3)H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.

In the past few years there has been a growth in the use of nano-particles for stabilizing lipid membranes with embedded proteins. These bionanoparticles provide a solution to the challenging problem of membrane protein isolation by maintaining a lipid bilayer essential to protein integrity and activity. We have described the use of an amphipathic polymer (Poly(styrene-co-maleic acid); SMA) to produce discoidal nanoparticles that contain a lipid bilayer with embedded protein. However the structure of the nanoparticle itself has not yet been determined. This leaves a major gap in understanding how the SMA stabilizes the encapsulated bilayer and how the bilayer relates physically and structurally to an unecapsulated lipid bilayer. In this paper we address this issue by describing the structure of the SMA Lipid Particle (SMALP) using data from small angle neutron scattering (SANS), electron microscopy (EM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and nuclear magnetic resonance spectroscopy (NMR). We show that the particle is disc shaped containing a polymer "bracelet" encircling the lipid bilayer. The structure and orientation of the individual components within the bilayer and polymer are determined showing that styrene moieties within SMA intercalate between the lipid acyl chains. The dimensions of the encapsulated bilayer are also determined and match those measured for a natural membrane. Taken together, the description of structure of the SMALP forms the foundation of future development and applications of SMALPs in membrane protein production and analysis.

Glycosphingolipid metabolism relies on selective recruitment of the pleckstrin homology (PH) domains of FAPP proteins to the trans-Golgi network. The mechanism involved is unclear but requires recognition of phosphatidylinositol-4-phosphate (PI4P) within the Golgi membrane. We investigated the molecular basis of FAPP1-PH domain interactions with PI4P bilayers in liposome sedimentation and membrane partitioning assays. Our data reveals a mechanism in which FAPP-PH proteins preferentially target PI4P-containing liquid disordered membranes, while liquid ordered membranes were disfavored. Additionally, NMR spectroscopy was used to identify the binding determinants responsible for recognizing trans-Golgi network-like bicelles including phosphoinositide and neighboring lipid molecules. Membrane penetration by the FAPP1-PH domain was mediated by an exposed, conserved hydrophobic wedge next to the PI4P recognition site and ringed by a network of complementary polar residues and basic charges. Our data illuminates how insertion of a structured loop provides selectivity for sensing membrane fluidity and targeting to defined membrane zones and organelles. The determinants of this membrane sensing process are conserved across the CERT, OSBP and FAPP family. Hence, lipid gradients not only result in differential membrane ordering along the secretory pathway but also specifically localize diverse proteins through recognition of ensembles of lipid ligands in dynamic and deformable bilayers in order to promote anterograde trafficking., (Copyright © 2015. Published by Elsevier Ltd.)

The function of a protein is determined by its intrinsic activity in the context of its subcellular distribution. Membranes localize proteins within cellular compartments and govern their specific activities. Discovering such membrane-protein interactions is important for understanding biological mechanisms and could uncover novel sites for therapeutic intervention. We present a method for detecting membrane interactive proteins and their exposed residues that insert into lipid bilayers. Although the development process involved analysis of how C1b, C2, ENTH, FYVE, Gla, pleckstrin homology (PH), and PX domains bind membranes, the resulting membrane optimal docking area (MODA) method yields predictions for a given protein of known three-dimensional structures without referring to canonical membrane-targeting modules. This approach was tested on the Arf1 GTPase, ATF2 acetyltransferase, von Willebrand factor A3 domain, and Neisseria gonorrhoeae MsrB protein and further refined with membrane interactive and non-interactive FAPP1 and PKD1 pleckstrin homology domains, respectively. Furthermore we demonstrate how this tool can be used to discover unprecedented membrane binding functions as illustrated by the Bro1 domain of Alix, which was revealed to recognize lysobisphosphatidic acid (LBPA). Validation of novel membrane-protein interactions relies on other techniques such as nuclear magnetic resonance spectroscopy (NMR), which was used here to map the sites of micelle interaction. Together this indicates that genome-wide identification of known and novel membrane interactive proteins and sites is now feasible and provides a new tool for functional annotation of the proteome.

Matrix metalloproteinases (MMPs) regulate tissue remodelling, inflammation and disease progression. Some soluble MMPs are inexplicably active near cell surfaces. Here we demonstrate the binding of MMP-12 directly to bilayers and cellular membranes using paramagnetic NMR and fluorescence. Opposing sides of the catalytic domain engage spin-labelled membrane mimics. Loops project from the β-sheet interface to contact the phospholipid bilayer with basic and hydrophobic residues. The distal membrane interface comprises loops on the other side of the catalytic cleft. Both interfaces mediate MMP-12 association with vesicles and cell membranes. MMP-12 binds plasma membranes and is internalized to hydrophobic perinuclear features, the nuclear membrane and inside the nucleus within minutes. While binding of TIMP-2 to MMP-12 hinders membrane interactions beside the active site, TIMP-2-inhibited MMP-12 binds vesicles and cells, suggesting compensatory rotation of its membrane approaches. MMP-12 association with diverse cell membranes may target its activities to modulate innate immune responses and inflammation.

The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). The onco-Lbc protein is a hyperactive Rho-specific guanine nucleotide exchange factor (GEF), but its structural mechanism has not been reported despite its involvement in cardiac hypertrophy and cancer causation. The pleckstrin homology (PH) domain of Lbc is located at the C-terminal end of the protein and is shown here to specifically recognize activated RhoA rather than lipids. The isolated dbl homology (DH) domain can function as an independent activator with an enhanced activity. However, the DH domain normally does not act as a solitary Lbc interface with RhoA-GDP. Instead it is negatively controlled by the PH domain. In particular, the DH helical bundle is coupled to the structurally dependent PH domain through a helical linker, which reduces its activity. Together the two domains form a rigid scaffold in solution as evidenced by small angle x-ray scattering and (1)H,(13)C,(15)N-based NMR spectroscopy. The two domains assume a "chair" shape with its back possessing independent GEF activity and the PH domain providing a broad seat for RhoA-GTP docking rather than membrane recognition. This provides structural and dynamical insights into how DH and PH domains work together in solution to support regulated RhoA activity. Mutational analysis supports the bifunctional PH domain mediation of DH-RhoA interactions and explains why the tandem domain is required for controlled GEF signaling., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up a wide range of possibilities for the future study of their structure and function.

Protein tyrosine phosphatases have been the focus of considerable research efforts aimed at developing novel therapeutics; however, these targets are often characterized as being 'undruggable' due to the challenge of achieving selectivity, potency and cell permeability. More recently, there has been renewed interest in developing inhibitors of the tyrosine phosphatase SHP2 (PTPN11) in the light of its broad role in cancer, specifically juvenile myelomonocytic leukemia, and recent studies that implicate SHP2 as a key factor in breast cancer progression. Recent significant advances in the field of SHP2 inhibitor development raise the question: are we on the verge of a new era of protein tyrosine phosphatase-directed therapeutics? This article critically appraises recent developments, assesses ongoing challenges and presents a perspective on possible future directions.

The multi-protein β-barrel assembly machine (BAM) of Escherichia coli is responsible for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the bacterial outer membrane. An essential component of this complex is the BamA protein, which binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains in its N-terminus. The C-terminus of BamA contains a β-barrel domain, which tethers BamA to the outer membrane and is also thought to be involved in OMP insertion. Here we mutagenize BamA using linker scanning mutagenesis and demonstrate that all five POTRA domains are essential for BamA protein function in our experimental system. Furthermore, we generate a homology based model of the BamA β-barrel and test our model using insertion mutagenesis, deletion analysis and immunofluorescence to identify β-strands, periplasmic turns and extracellular loops. We show that the surface-exposed loops of the BamA β-barrel are essential.

Desmosomes are dynamic junctions between cells that maintain the structural integrity of skin and heart tissues by withstanding shear forces. Mutations in component genes cause life-threatening conditions including arrhythmogenic right ventricular cardiomyopathy, and desmosomal proteins are targeted by pathogenic autoantibodies in skin blistering diseases such as pemphigus. Here, we review a set of newly discovered pathogenic alterations and discuss the structural repercussions of debilitating mutations on desmosomal proteins. The architectures of native desmosomal assemblies have been visualized by cryo-electron microscopy and cryo-electron tomography, and the network of protein domain interactions is becoming apparent. Plakophilin and desmoplakin mutations have been discovered to alter binding interfaces, structures, and stabilities of folded domains that have been resolved by X-ray crystallography and NMR spectroscopy. The flexibility within desmoplakin has been revealed by small-angle X-ray scattering and fluorescence assays, explaining how mechanical stresses are accommodated. These studies have shown that the structural and functional consequences of desmosomal mutations can now begin to be understood at multiple levels of spatial and temporal resolution. This review discusses the recent structural insights and raises the possibility of using modeling for mechanism-based diagnosis of how deleterious mutations alter the integrity of solid tissues., (© 2013. Published by Elsevier Ltd. All rights reserved.)

Envoplakin, periplakin and desmoplakin are cytoskeletal proteins that provide structural integrity within the skin and heart by resisting shear forces. Here we reveal the nature of unique hinges within their plakin domains that provides divergent degrees of flexibility between rigid long and short arms composed of spectrin repeats. The range of mobility of the two arms about the hinge is revealed by applying the ensemble optimization method to small-angle X-ray scattering data. Envoplakin and periplakin adopt 'L' shaped conformations exhibiting a 'helicopter propeller'-like mobility about the hinge. By contrast desmoplakin exhibits essentially unrestricted mobility by 'jack-knifing' about the hinge. Thus the diversity of molecular jointing that can occur about plakin hinges includes 'L' shaped bends, 'U' turns and fully extended 'I' orientations between rigid blocks of spectrin repeats. This establishes specialised hinges in plakin domains as a key source of flexibility that may allow sweeping of cellular spaces during assembly of cellular structures and could impart adaptability, so preventing irreversible damage to desmosomes and the cell cytoskeleton upon exposure to mechanical stress.

ALIX plays a role in nucleocapsid release during viral infection, as does lysobisphosphatidic acid (LBPA). However, the mechanism remains unclear. Here we report that LBPA is recognized within an exposed site in ALIX Bro1 domain predicted by MODA, an algorithm for discovering membrane-docking areas in proteins. LBPA interactions revealed a strict requirement for a structural calcium tightly bound near the lipid interaction site. Unlike other calcium- and phospholipid-binding proteins, the all-helical triangle-shaped fold of the Bro1 domain confers selectivity for LBPA via a pair of hydrophobic residues in a flexible loop, which undergoes a conformational change upon membrane association. Both LBPA and calcium binding are necessary for endosome association and virus infection, as are ALIX ESCRT binding and dimerization capacity. We conclude that LBPA recruits ALIX onto late endosomes via the calcium-bound Bro1 domain, triggering a conformational change in ALIX to mediate the delivery of viral nucleocapsids to the cytosol during infection., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The Golgi apparatus is a sorting platform that exchanges extensively with the endoplasmic reticulum (ER), endosomes (Es) and plasma membrane (PM) compartments. The last compartment of the Golgi, the trans-Golgi Network (TGN) is a large complex of highly deformed membranes from which vesicles depart to their targeted organelles but also are harbored from retrograde pathways. The phosphoinositide (PI) composition of the TGN is marked by an important contingent of phosphatidylinositol-4-phosphate (PtdIns(4)P). Although this PI is present throughout the Golgi, its proportion grows along the successive cisternae and peaks at the TGN. The levels of this phospholipid are controlled by a set of kinases and phosphatases that regulate its concentrations in the Golgi and maintain a dynamic gradient that determines the cellular localization of several interacting proteins. Though not exclusive to the Golgi, the synthesis of PtdIns(4)P in other membranes is relatively marginal and has unclear consequences. The significance of PtdIns(4)P within the TGN has been demonstrated for numerous cellular events such as vesicle formation, lipid metabolism, and membrane trafficking.