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Tetraspanins play critical roles in various physiological processes, ranging from cell adhesion to virus infection. The members of the tetraspanin family have four membrane-spanning domains and short and large extracellular loops, and associate with a broad range of other functional proteins to exert cellular functions. Here we report the crystal structure of CD9 and the cryo-electron microscopic structure of CD9 in complex with its single membrane-spanning partner protein, EWI-2. The reversed cone-like molecular shape of CD9 generates membrane curvature in the crystalline lipid layers, which explains the CD9 localization in regions with high membrane curvature and its implications in membrane remodeling. The molecular interaction between CD9 and EWI-2 is mainly mediated through the small residues in the transmembrane region and protein/lipid interactions, whereas the fertilization assay revealed the critical involvement of the LEL region in the sperm-egg fusion, indicating the different dependency of each binding domain for other partner proteins. Tetraspanins play critical roles in various physiological processes, ranging from cell adhesion to virus infection. Here authors report the crystal structure of CD9 and the cryo-electron microscopic structure of CD9 in complex with its single membrane-spanning partner protein, EWI-2.

The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9’s ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.Cryo-EM and liposome assays reveal that Atg9 functions as a lipid scramblase, transporting phospholipids between inner and outer liposome leaflets. Analyses of mutants in yeast support a role for this activity in autophagy.

Many drugs target the serotonin 2A receptor (5-HT2AR), including second-generation antipsychotics that also target the dopamine D2 receptor (D2R). These drugs often produce severe side effects due to non-selective binding to other aminergic receptors. Here, we report the structures of human 5-HT2AR in complex with the second-generation antipsychotics risperidone and zotepine. These antipsychotics effectively stabilize the inactive conformation by forming direct contacts with the residues at the bottom of the ligand-binding pocket, the movements of which are important for receptor activation. 5-HT2AR is structurally similar to 5-HT2CR but possesses a unique side-extended cavity near the orthosteric binding site. A docking study and mutagenic studies suggest that a highly 5-HT2AR-selective antagonist binds the side-extended cavity. The conformation of the ligand-binding pocket in 5-HT2AR significantly differs around extracellular loops 1 and 2 from that in D2R. These findings are beneficial for the rational design of safer antipsychotics and 5-HT2AR-selective drugs.Structures of human 5-HT2AR in complex with several drugs reveal a side-extended cavity that is unique for this receptor, while molecular docking suggests that a highly 5-HT2AR-selective antagonist binds residues within this cavity.

Chronic infection with hepatitis B virus (HBV) affects more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma, and results in an estimated 820,000 deaths annually 1 , 2 . For HBV infection to be established, a molecular interaction is required between the large glycoproteins of the virus envelope (known as LHBs) and the host entry receptor sodium taurocholate co-transporting polypeptide (NTCP), a sodium-dependent bile acid transporter from the blood to hepatocytes 3 . However, the molecular basis for the virus–transporter interaction is poorly understood. Here we report the cryo-electron microscopy structures of human, bovine and rat NTCPs in the apo state, which reveal the presence of a tunnel across the membrane and a possible transport route for the substrate. Moreover, the cryo-electron microscopy structure of human NTCP in the presence of the myristoylated preS1 domain of LHBs, together with mutation and transport assays, suggest a binding mode in which preS1 and the substrate compete for the extracellular opening of the tunnel in NTCP. Our preS1 domain interaction analysis enables a mechanistic interpretation of naturally occurring HBV-insusceptible mutations in human NTCP. Together, our findings provide a structural framework for HBV recognition and a mechanistic understanding of sodium-dependent bile acid translocation by mammalian NTCPs. Cryo-electron microscopy structures of the bile acid transporter NTCP in the apo state and in complex with the preS1 domain of hepatitis B virus (HBV) provide insight into NTCP substrate transport and HBV recognition mechanisms.

The coronavirus membrane protein (M) is the most abundant viral structural protein and plays a central role in virus assembly and morphogenesis. However, the process of M protein-driven virus assembly are largely unknown. Here, we report the cryo-electron microscopy structure of the SARS-CoV-2 M protein in two different conformations. M protein forms a mushroom-shaped dimer, composed of two transmembrane domain-swapped three-helix bundles and two intravirion domains. M protein further assembles into higher-order oligomers. A highly conserved hinge region is key for conformational changes. The M protein dimer is unexpectedly similar to SARS-CoV-2 ORF3a, a viral ion channel. Moreover, the interaction analyses of M protein with nucleocapsid protein (N) and RNA suggest that the M protein mediates the concerted recruitment of these components through the positively charged intravirion domain. Our data shed light on the M protein-driven virus assembly mechanism and provide a structural basis for therapeutic intervention targeting M protein. M protein plays essential roles in virus assembly and morphogenesis. Here, authors reveal two cryo-EM structures of M protein from SARS-CoV-2 that suggest conformational dynamics of M protein and its role in virus assembly.

Melatonin receptors (MT 1 and MT 2 ) transduce inhibitory signaling by melatonin ( N -acetyl-5-methoxytryptamine), which is associated with sleep induction and circadian rhythm modulation. Although recently reported crystal structures of ligand-bound MT 1 and MT 2 elucidated the basis of ligand entry and recognition, the ligand-induced MT 1 rearrangement leading to G i -coupling remains unclear. Here we report a cryo-EM structure of the human MT 1 –G i signaling complex at 3.3 Å resolution, revealing melatonin-induced conformational changes propagated to the G-protein-coupling interface during activation. In contrast to other G i -coupled receptors, MT 1 exhibits a large outward movement of TM6, which is considered a specific feature of G s -coupled receptors. Structural comparison of G i and G s complexes demonstrated conformational diversity of the C-terminal entry of the G i protein, suggesting loose and variable interactions at the end of the α5 helix of G i protein. These notions, together with our biochemical and computational analyses, highlight variable binding modes of Gα i and provide the basis for the selectivity of G-protein signaling. A cryo-EM structure of the active human melatonin receptor in complex with G i reveals conformational changes upon activation and the molecular basis for G-protein selectivity.

In addition to the serotonin 5-HT 2A receptor (5-HT 2A R), the dopamine D 2 receptor (D 2 R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D 2 R have been described in complex with the inverse agonists risperidone (D 2 R ris ) and haloperidol (D 2 R hal ). Here we describe the structure of human D 2 R in complex with spiperone (D 2 R spi ). In D 2 R spi , the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D 2 R ris and D 2 R hal , demonstrating that ECL2 in D 2 R is highly dynamic. Moreover, D 2 R spi exhibited an extended binding pocket to accommodate spiperone’s phenyl ring, which probably contributes to the selectivity of spiperone to D 2 R and 5-HT 2A R. Together with D 2 R ris and D 2 R hal , the structural information of D 2 R spi should be of value for designing novel antipsychotics with improved safety and efficacy. The dopamine D 2 receptor (D 2 R) is a GPCR and an important drug target for schizophrenia treatment. Here, the authors present the crystal structure of human D 2 R in complex with the antipsychotic drug spiperone, which is of interest for designing antipsychotics with improved receptor selectivity.

The sodium/proton exchanger NHA2, also known as SLC9B2, is important for insulin secretion, renal blood pressure regulation, and electrolyte retention. Recent structures of bison NHA2 has revealed its unique 14-transmembrane helix architecture, which is different from SLC9A/NHE members made up from 13-TM helices. Sodium/proton exchangers are functional homodimers, and the additional N-terminal helix in NHA2 was found to alter homodimer assembly. Here, we present the cryo-electron microscopy structures of apo human NHA2 in complex with a Fab fragment and also with the inhibitor phloretin bound at 2.8 and 2.9 Å resolution, respectively. We show how phosphatidic acid (PA) lipids bind to the homodimer interface of NHA2 on the extracellular side, which we propose has a regulatory role linked to cell volume regulation. The ion binding site of human NHA2 has a salt bridge interaction between the ion binding aspartate D278 and R432, an interaction previously broken in the bison NHA2 structure, and these differences suggest a possible ion coupling mechanism. Lastly, the human NHA2 structure in complex with phloretin offers a template for structure-guided drug design, potentially leading to the development of more selective and potent NHA2 inhibitors.

The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward- and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a ‘gated-pore’ transport mechanism in such monosaccharide transporters. This study has determined the X-ray crystal structures of GLUT5 from Rattus norvegicus in an open, outward-facing conformation and GLUT5 from Bos taurus in an open, inward-facing conformation; comparison of these structures with previously published structures of the related Escherichia coli d -xylose:H + symporter XylE suggests that transport in GLUT5 is controlled by both a global ‘rocker-switch’-type motion and a local ‘gated-pore’-type transport mechanism. Structure of fructose transporter GLUT5 SLC2 family glucose transporters (GLUTs) facilitate the transport of glucose and other monosaccharides across biological membranes. GLUT5, which is fructose-specific, has been linked to disorders such as type 2 diabetes and obesity and is overexpressed in certain tumour cells. The authors solve X-ray crystal structures of GLUT5 from the brown rat in an open, outward-facing conformation and GLUT5 from cattle in an open, inward-facing conformation. Comparison of these structures with previously published structures of the related XylE, a proton-coupled sugar transporter from Escherichia coli , suggest that transport in GLUT5 is controlled by both 'rocker-switch' and 'gated-pore' type transport mechanisms. Also in this issue of Nature , Dong Deng et al . solve the X-ray crystal structures of human GLUT3 in outward-open and outward-occluded conformations.

The Na + /H + exchanger SLC9B2, also known as NHA2, correlates with the long-sought-after Na + /Li + exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite the functional importance of NHA2, structural information and the molecular basis for its ion-exchange mechanism have been lacking. Here we report the cryo-EM structures of bison NHA2 in detergent and in nanodiscs, at 3.0 and 3.5 Å resolution, respectively. The bison NHA2 structure, together with solid-state membrane-based electrophysiology, establishes the molecular basis for electroneutral ion exchange. NHA2 consists of 14 transmembrane (TM) segments, rather than the 13 TMs previously observed in mammalian Na + /H + exchangers (NHEs) and related bacterial antiporters. The additional N-terminal helix in NHA2 forms a unique homodimer interface with a large intracellular gap between the protomers, which closes in the presence of phosphoinositol lipids. We propose that the additional N-terminal helix has evolved as a lipid-mediated remodeling switch for the regulation of NHA2 activity. NHA2 exchanges sodium ions for protons across cell membranes, and its activity is linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Drew et al. report the cryo-EM structure of NHA2 in detergent and nanodiscs.