Activation and Purification of ß-Glucocerebrosidase by Exploiting its Transporter LIMP-2 - Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease.

Genetic variants of GBA1 can cause the lysosomal storage disorder Gaucher disease and are among the highest genetic risk factors for Parkinson's disease (PD). GBA1 encodes the lysosomal enzyme beta-glucocerebrosidase (GCase), which orchestrates the degradation of glucosylceramide (GluCer) in the lysosome. Recent studies have shown that GluCer accelerates α-synuclein aggregation, exposing GCase deficiency as a major risk factor in PD pathology and as a promising target for treatment. This study investigates the interaction of GCase and three disease-associated variants (p.E326K, p.N370S, p.L444P) with their transporter, the lysosomal integral membrane protein 2 (LIMP-2). Overexpression of LIMP-2 in HEK 293T cells boosts lysosomal abundance of wt, E326K, and N370S GCase and increases/rescues enzymatic activity of the wt and E326K variant. Using a novel purification approach, co-purification of untagged wt, E326K, and N370S GCase in complex with His-tagged LIMP-2 from cell supernatant of HEK 293F cells is achieved, confirming functional binding and trafficking for these variants. Furthermore, a single helix in the LIMP-2 ectodomain is exploited to design a lysosome-targeted peptide that enhances lysosomal GCase activity in PD patient-derived and control fibroblasts. These findings reveal LIMP-2 as an allosteric activator of GCase, suggesting a possible therapeutic potential of targeting this interaction.

Cryo-EM structure of cell-free synthesized human histamine 2 receptor/Gs complex in nanodisc environment.

Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (H2R) in an active conformation with bound histamine and in complex with Gs heterotrimeric protein at an overall resolution of 3.4 Å. The complex was generated by cotranslational insertion of the receptor into preformed nanodisc membranes using cell-free synthesis in E. coli lysates. Structural comparison with the inactive conformation of H2R and the inactive and Gq-coupled active state of H1R together with structure-guided functional experiments reveal molecular insights into the specificity of ligand binding and G protein coupling for this receptor family. We demonstrate lipid-modulated folding of cell-free synthesized H2R, its agonist-dependent internalization and its interaction with endogenously synthesized H1R and H2R in HEK293 cells by applying a recently developed nanotransfer technique.

Tracing the substrate translocation mechanism in P-glycoprotein.

P-glycoprotein (Pgp) is a prototypical ATP-binding cassette (ABC) transporter of great biological and clinical significance.Pgp confers cancer multidrug resistance and mediates the bioavailability and pharmacokinetics of many drugs (Juliano and Ling, 1976; Ueda et al., 1986; Sharom, 2011). Decades of structural and biochemical studies have provided insights into how Pgp binds diverse compounds (Loo and Clarke, 2000; Loo et al., 2009; Aller et al., 2009; Alam et al., 2019; Nosol et al., 2020; Chufan et al., 2015), but how they are translocated through the membrane has remained elusive. Here, we covalently attached a cyclic substrate to discrete sites of Pgp and determined multiple complex structures in inward- and outward-facing states by cryoEM. In conjunction with molecular dynamics simulations, our structures trace the substrate passage across the membrane and identify conformational changes in transmembrane helix 1 (TM1) as regulators of substrate transport. In mid-transport conformations, TM1 breaks at glycine 72. Mutation of this residue significantly impairs drug transport of Pgp in vivo, corroborating the importance of its regulatory role. Importantly, our data suggest that the cyclic substrate can exit Pgp without the requirement of a wide-open outward-facing conformation, diverting from the common efflux model for Pgp and other ABC exporters. The substrate transport mechanism of Pgp revealed here pinpoints critical targets for future drug discovery studies of this medically relevant system.

Structure of the ceramide-bound SPOTS complex.

Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in sphingolipid biogenesis. Its activity is tightly regulated through multiple binding partners, including Tsc3, Orm proteins, ceramides, and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1. The structural organization and regulatory mechanisms of this complex are not yet understood. Here, we report the high-resolution cryo-EM structures of the yeast SPT in complex with Tsc3 and Orm1 (SPOT) as dimers and monomers and a monomeric complex further carrying Sac1 (SPOTS). In all complexes, the tight interaction of the downstream metabolite ceramide and Orm1 reveals the ceramide-dependent inhibition. Additionally, observation of ceramide and ergosterol binding suggests a co-regulation of sphingolipid biogenesis and sterol metabolism within the SPOTS complex.

The ABC transporter MsbA adopts the wide inward-open conformation in E. coli cells.

Membrane proteins are currently investigated after detergent extraction from native cellular membranes and reconstitution into artificial liposomes or nanodiscs, thereby removing them from their physiological environment. However, to truly understand the biophysical properties of membrane proteins in a physiological environment, they must be investigated within living cells. Here, we used a spin-labeled nanobody to interrogate the conformational cycle of the ABC transporter MsbA by double electron-electron resonance. Unexpectedly, the wide inward-open conformation of MsbA, commonly considered a nonphysiological state, was found to be prominently populated in Escherichia coli cells. Molecular dynamics simulations revealed that extensive lateral portal opening is essential to provide access of its large natural substrate core lipid A to the binding cavity. Our work paves the way to investigate the conformational landscape of membrane proteins in cells.

Structure of the HOPS tethering complex, a lysosomal membrane fusion machinery.

Lysosomes are essential for cellular recycling, nutrient signaling, autophagy, and pathogenic bacteria and viruses invasion. Lysosomal fusion is fundamental to cell survival and requires HOPS, a conserved heterohexameric tethering complex. On the membranes to be fused, HOPS binds small membrane-associated GTPases and assembles SNAREs for fusion, but how the complex fulfills its function remained speculative. Here, we used cryo-electron microscopy to reveal the structure of HOPS. Unlike previously reported, significant flexibility of HOPS is confined to its extremities, where GTPase binding occurs. The SNARE-binding module is firmly attached to the core, therefore, ideally positioned between the membranes to catalyze fusion. Our data suggest a model for how HOPS fulfills its dual functionality of tethering and fusion and indicate why it is an essential part of the membrane fusion machinery.

Our cells break down the nutrients that they receive from the body to create the building blocks needed to keep us alive. This is done by compartments called lysosomes that are filled with a cocktail of proteins called enzymes, which speed up the breakdown process. Lysosomes are surrounded by a membrane, a barrier of fatty molecules that protects the rest of the cell from being digested. When new nutrients reach the cell, they travel to the lysosome packaged in vesicles, which have their own fatty membrane. To allow the nutrients to enter the lysosome without creating a leak, the membranes of the vesicles and the lysosome must fuse. The mechanism through which these membranes fuse is not fully clear. It is known that both fusing membranes must contain proteins called SNAREs, which wind around each other when they interact. However, this alone is not enough. Other proteins are also required to tether the membranes together before they fuse. To understand how these tethers play a role, Shvarev, Schoppe, König et al. studied the structure of the HOPS complex from yeast. This assembly of six proteins is vital for lysosomal fusion and, has a composition similar to the equivalent complex in humans. Using cryo-electron microscopy, a technique that relies on freezing purified proteins to image them with an electron microscope and reveal their structure, allowed Shvarev, Schoppe, König et al. to provide a model for how HOPS interacts with SNAREs and membranes. In addition to HOPS acting as a tether to bring the membranes together, it can also bind directly to SNAREs. This creates a bridge that allows the proteins to wrap around each other, driving the membranes to fuse. HOPS is a crucial component in the cellular machinery, and mutations in the complex can cause devastating neurological defects. The complex is also targeted by viruses ­ such as SARS-CoV-2 ­ that manipulate HOPS to reduce its activity. Shvarev, Schoppe, König et al.'s findings could help researchers to develop drugs to maintain or recover the activity of HOPS. However, this will require additional information about its structure and how the complex acts in the biological environment of the cell.

Frozen motion: how cryo-EM changes the way we look at ABC transporters.

ATP-binding cassette (ABC) transporters are widely present molecular machines that transfer substrates across the cell membrane. ABC transporters are involved in numerous physiological processes and are often clinical targets. Structural biology is fundamental to obtain the molecular details underlying ABC transporter function and suggest approaches to modulate it. Until recently, X-ray crystallography has been the only method capable of providing high-resolution structures of ABC transporters. However, modern cryo-electron microscopy (cryo-EM) opens entirely new ways of studying these dynamic membrane proteins. Cryo-EM enables analyses of targets that resist X-ray crystallography, challenging multicomponent complexes, and the exploration of conformational dynamics. These unique capacities have turned cryo-EM into the dominant technique for structural studies of membrane proteins, including ABC transporters.

Single-Particle Cryo-EM of Membrane Proteins.

In the recent years, the protein databank has been fueled by the exponential growth of high-resolution electron cryo-microscopy (cryo-EM) structures. This trend will be further accelerated through the continuous software and method developments and the increasing availability of imaging centers, which will open cryo-EM to a wide array of researchers with their diverse scientific goals and questions. Especially for structural biology of membrane proteins, cryo-EM offers significant advantages as it can overcome multiple limitations of classical methods. Most importantly, in cryo-EM, the sample is prepared as a vitrified suspension, which abolishes the need for crystallization, reduces the required sample amount and allows usage of a wide arsenal of hydrophobic environments. Despite recent improvements, high-resolution cryo-EM still poses some significant challenges, and standardized procedures, especially for the characterization of membrane proteins, are missing. While there can be no ultimate recipe toward a high-resolution cryo-EM structure for every membrane protein, certain factors seem to be universally relevant. Here, we share the protocols that have been successfully used in our laboratory. We hope that this may be a useful resource to other researchers in the field and may increase their chances of success.

Cryo-EM of ABC transporters: an ice-cold solution to everything?

High-resolution cryo-EM has revolutionized how we look at ABC transporters and membrane proteins in general. An ever-increasing number of software tools and faster processing now allow dissecting the molecular details of nanomachines at atomic precision. Considering the further benefits of significantly reduced sample demands and increased speed, cryo-EM will dominate the structure determination of membrane proteins in the near future without compromising on data quality or detail. Moreover, improved and new algorithms make it now possible to resolve the conformational spectrum of macromolecular machines under turnover conditions and to analyze heterogeneous samples at high resolution. The future of cryo-EM is, therefore, bright, and the growing number of imaging facilities and groups active in this field will amplify this trend even further. Nevertheless, expectations have to be managed, as cryo-EM alone cannot provide an ultimate answer to all scientific questions. In this review, we discuss the capabilities and limitations of cryo-EM together with possible solutions for studies of ABC transporters.

Structure of Merkel Cell Polyomavirus Capsid and Interaction with Its Glycosaminoglycan Attachment Receptor.

Merkel cell polyomavirus (MCPyV) is a human double-stranded DNA tumor virus. MCPyV cell entry is unique among members of the polyomavirus family as it requires the engagement of two types of glycans, sialylated oligosaccharides and sulfated glycosaminoglycans (GAGs). Here, we present crystallographic and cryo-electron microscopic structures of the icosahedral MCPyV capsid and analysis of its glycan interactions via nuclear magnetic resonance (NMR) spectroscopy. While sialic acid binding is specific for α2-3-linked sialic acid and mediated by the exposed apical loops of the major capsid protein VP1, a broad range of GAG oligosaccharides bind to recessed regions between VP1 capsomers. Individual VP1 capsomers are tethered to one another by an extensive disulfide network that differs in architecture from previously described interactions for other PyVs. An unusual C-terminal extension in MCPyV VP1 projects from the recessed capsid regions. Mutagenesis experiments show that this extension is dispensable for receptor interactions.IMPORTANCE The MCPyV genome was found to be clonally integrated in 80% of cases of Merkel cell carcinoma (MCC), a rare but aggressive form of human skin cancer, strongly suggesting that this virus is tumorigenic. In the metastasizing state, the course of the disease is often fatal, especially in immunocompromised individuals, as reflected by the high mortality rate of 33 to 46% and the low 5-year survival rate (<45%). The high seroprevalence of about 60% makes MCPyV a serious health care burden and illustrates the need for targeted treatments. In this study, we present the first high-resolution structural data for this human tumor virus and demonstrate that the full capsid is required for the essential interaction with its GAG receptor(s). Together, these data can be used as a basis for future strategies in drug development.

Conformation space of a heterodimeric ABC exporter under turnover conditions.

Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action1-3. ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol4-6 and thereby contribute to essential cellular processes, adaptive immunity and multidrug resistance7,8. Despite their importance, the coupling of nucleotide binding, hydrolysis and release to the conformational dynamics of these proteins remains poorly resolved, especially for heterodimeric and/or asymmetric ABC exporters that are abundant in humans. Here we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in a lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations-one of them with a bound peptide substrate-and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains and a closed extracellular gate. By decreasing the rate of ATP hydrolysis, we could capture an outward-facing (OF) open conformation-an otherwise transient state vulnerable to substrate re-entry. The ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent; both comprise co-existing OF conformations with open and closed extracellular gates. By contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ATP and ADP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental steps of the substrate translocation cycle of asymmetric ABC transporters.

Structure and autoregulation of a P4-ATPase lipid flippase.

Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active transport of phospholipids from exoplasmic or luminal leaflets to cytosolic leaflets of eukaryotic membranes. The molecular architecture of P4-ATPases and the mechanism through which they recognize and transport lipids have remained unknown. Here we describe the cryo-electron microscopy structure of the P4-ATPase Drs2p-Cdc50p, a Saccharomyces cerevisiae lipid flippase that is specific to phosphatidylserine and phosphatidylethanolamine. Drs2p-Cdc50p is autoinhibited by the C-terminal tail of Drs2p, and activated by the lipid phosphatidylinositol-4-phosphate (PtdIns4P or PI4P). We present three structures that represent the complex in an autoinhibited, an intermediate and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of autoinhibition and PI4P-dependent activation. We also observe a putative lipid translocation pathway in this flippase that involves a conserved PISL motif in transmembrane segment 4 and polar residues of transmembrane segments 2 and 5, in particular Lys1018, in the centre of the lipid bilayer.

Know your detergents: A case study on detergent background in negative stain electron microscopy.

Electron cryo-microscopy (cryo-EM) of purified macromolecular complexes is now providing 3D-structures at near-atomic resolution (Kühlbrandt, 2014). Cryo-EM can tolerate heterogeneous specimens, however, high-resolution efforts demand highly optimized samples. Therefore, significant pre-screening and evaluation is essential before a final dataset can be obtained. While cryo-EM is comparably slow and requires access to expensive high-end electron microscopes, room temperature negative stain EM is fast, inexpensive and provides immediate feedback. This has made it a popular approach for sample quality control in the early phases of a project. Optimization in negative stain can be critical not only for cryo-EM, but also for X-ray crystallography, as highlighted for example by studies on GPCR complexes (Kang et al., 2015; Rasmussen et al., 2012). However, when not done carefully and interpreted correctly, negative stain can be prone to artifacts. A typical problem, which is often overlooked in the interpretation of EM data of small membrane proteins, is the background, caused by empty detergent micelles, as it can be easily confused with detergent embedded protein samples. To counteract this ubiquitous problem, we present a case study on commonly used detergents.We show that most detergents produce significant background in negative stain EM, even below nominal critical micelle concentration (CMC). Unawareness of such artefacts can lead to misinterpretation of sample quality and homogeneity. We hope that this study can serve as a template to evaluate images in the early phases of a project.

Acidic Environment Induces Dimerization and Ligand Binding Site Collapse in the Vps10p Domain of Sortilin.

Sortilin is a neuronal receptor involved in transmembrane signaling, endocytosis, and intracellular sorting of proteins. It cycles through a number of cellular compartments where it encounters various acidic conditions. The crystal structure of the sortilin ectodomain has previously been determined at neutral pH. Here, we present the 3.5-Å resolution crystal structure of sortilin at pH 5.5, which represents an environment similar to that of late endosomes, where ligands are released. The structure reveals an overall distortion of the 10-bladed ß-propeller domain. This distortion and specific conformational changes, caused by protonation of a number of histidine residues, render the currently known binding sites unavailable for ligand binding. Access to the binding sites is furthermore blocked by a reversible and pH-dependent formation of tight sortilin dimers, also confirmed by electron microscopy, size-exclusion chromatography, and mutational studies. This study reveals how sortilin binding sites are disrupted and explains pH-dependent ligand affinity.

Structure of the human MHC-I peptide-loading complex.

The peptide-loading complex (PLC) is a transient, multisubunit membrane complex in the endoplasmic reticulum that is essential for establishing a hierarchical immune response. The PLC coordinates peptide translocation into the endoplasmic reticulum with loading and editing of major histocompatibility complex class I (MHC-I) molecules. After final proofreading in the PLC, stable peptide-MHC-I complexes are released to the cell surface to evoke a T-cell response against infected or malignant cells. Sampling of different MHC-I allomorphs requires the precise coordination of seven different subunits in a single macromolecular assembly, including the transporter associated with antigen processing (TAP1 and TAP2, jointly referred to as TAP), the oxidoreductase ERp57, the MHC-I heterodimer, and the chaperones tapasin and calreticulin. The molecular organization of and mechanistic events that take place in the PLC are unknown owing to the heterogeneous composition and intrinsically dynamic nature of the complex. Here, we isolate human PLC from Burkitt's lymphoma cells using an engineered viral inhibitor as bait and determine the structure of native PLC by electron cryo-microscopy. Two endoplasmic reticulum-resident editing modules composed of tapasin, calreticulin, ERp57, and MHC-I are centred around TAP in a pseudo-symmetric orientation. A multivalent chaperone network within and across the editing modules establishes the proofreading function at two lateral binding platforms for MHC-I molecules. The lectin-like domain of calreticulin senses the MHC-I glycan, whereas the P domain reaches over the MHC-I peptide-binding pocket towards ERp57. This arrangement allows tapasin to facilitate peptide editing by clamping MHC-I. The translocation pathway of TAP opens out into a large endoplasmic reticulum lumenal cavity, confined by the membrane entry points of tapasin and MHC-I. Two lateral windows channel the antigenic peptides to MHC-I. Structures of PLC captured at distinct assembly states provide mechanistic insight into the recruitment and release of MHC-I. Our work defines the molecular symbiosis of an ABC transporter and an endoplasmic reticulum chaperone network in MHC-I assembly and provides insight into the onset of the adaptive immune response.

Hidden Twins: SorCS Neuroreceptors Form Stable Dimers.

SorCS1, SorCS2 and SorCS3 belong to the Vps10p-domain family of multiligand receptors. Genetic and functional studies have linked SorCS receptors to psychiatric disorders, Alzheimer's disease and type 2 diabetes, demonstrating critical roles in neuronal functionality and metabolic control. Surprisingly, their structural composition has so far not been studied. Here we have characterized SorCS1, SorCS2 and SorCS3 using biochemical methods and electron microscopy. We found that their purified extracellular domains co-exist in stable dimeric and monomeric populations. This was supported by co-immunoprecipitation experiments, where membrane-bound dimers were successfully pulled down from cell lysate. While dimers were virtually unbreakable, dimerization of the monomeric population was promoted through enzymatic deglycosylation. We conclude that post-translational modifications, specifically the degree and pattern of glycosylation, regulate the oligomeric state of the protein. Hence, cells may dictate ligand specificity by controlling the ratio between monomers and dimers and, therefore, regulate the multiple functions of SorCS receptors.