Structures of human SGLT in the occluded state reveal conformational changes during sugar transport.

Sodium-Glucose Cotransporters (SGLT) mediate the uphill uptake of extracellular sugars and play fundamental roles in sugar metabolism. Although their structures in inward-open and outward-open conformations are emerging from structural studies, the trajectory of how SGLTs transit from the outward-facing to the inward-facing conformation remains unknown. Here, we present the cryo-EM structures of human SGLT1 and SGLT2 in the substrate-bound state. Both structures show an occluded conformation, with not only the extracellular gate but also the intracellular gate tightly sealed. The sugar substrate are caged inside a cavity surrounded by TM1, TM2, TM3, TM6, TM7, and TM10. Further structural analysis reveals the conformational changes associated with the binding and release of substrates. These structures fill a gap in our understanding of the structural mechanisms of SGLT transporters.

The Protein Fusion Strategy Facilitates the Structure Determination of Small Membrane Proteins by Cryo-EM.

Despite the resolution revolution of cryo-EM, structures of small membrane proteins (<80 kDa) are still understudied. These proteins are notoriously reluctant to structure determination by single-particle cryo-EM. Protein fusion might represent a plausible strategy to overcome such difficulties. This Perspective enumerates recent exemplary progress and discusses the future potential of the protein fusion strategy.

Structural mechanism of SGLT1 inhibitors.

Sodium glucose co-transporters (SGLT) harness the electrochemical gradient of sodium to drive the uphill transport of glucose across the plasma membrane. Human SGLT1 (hSGLT1) plays a key role in sugar uptake from food and its inhibitors show promise in the treatment of several diseases. However, the inhibition mechanism for hSGLT1 remains elusive. Here, we present the cryo-EM structure of the hSGLT1-MAP17 hetero-dimeric complex in the presence of the high-affinity inhibitor LX2761. LX2761 locks the transporter in an outward-open conformation by wedging inside the substrate-binding site and the extracellular vestibule of hSGLT1. LX2761 blocks the putative water permeation pathway of hSGLT1. The structure also uncovers the conformational changes of hSGLT1 during transitions from outward-open to inward-open states.

Cryo-EM structure of human MG53 homodimer.

MG53 is a tripartite motif (TRIM) family E3 ligase and plays important biological functions. Here we present the cryo-EM structure of human MG53, showing that MG53 is a homodimer consisting of a 'body' and two 'wings'. Intermolecular interactions are mainly distributed in the 'body' which is relatively stable, while two 'wings' are more dynamic. The overall architecture of MG53 is distinct from those of TRIM20 and TRIM25, illustrating the broad structural diversity of this protein family.

Structural basis of inhibition of the human SGLT2-MAP17 glucose transporter.

Human sodium-glucose cotransporter 2 (hSGLT2) mediates the reabsorption of the majority of filtrated glucose in the kidney1. Pharmacological inhibition of hSGLT2 by oral small-molecule inhibitors, such as empagliflozin, leads to enhanced excretion of glucose and is widely used in the clinic to manage blood glucose levels for the treatment of type 2 diabetes1. Here we determined the cryogenic electron microscopy structure of the hSGLT2-MAP17 complex in the empagliflozin-bound state to an overall resolution of 2.95 Å. Our structure shows eukaryotic SGLT-specific structural features. MAP17 interacts with transmembrane helix 13 of hSGLT2. Empagliflozin occupies both the sugar-substrate-binding site and the external vestibule to lock hSGLT2 in an outward-open conformation, thus inhibiting the transport cycle. Our work provides a framework for understanding the mechanism of SLC5A family glucose transporters and also develops a foundation for the future rational design and optimization of new inhibitors targeting these transporters.

Structural insights into the inhibition mechanism of human sterol O-acyltransferase 1 by a competitive inhibitor.

Sterol O-acyltransferase 1 (SOAT1) is an endoplasmic reticulum (ER) resident, multi-transmembrane enzyme that belongs to the membrane-bound O-acyltransferase (MBOAT) family. It catalyzes the esterification of cholesterol to generate cholesteryl esters for cholesterol storage. SOAT1 is a target to treat several human diseases. However, its structure and mechanism remain elusive since its discovery. Here, we report the structure of human SOAT1 (hSOAT1) determined by cryo-EM. hSOAT1 is a tetramer consisted of a dimer of dimer. The structure of hSOAT1 dimer at 3.5 Å resolution reveals that a small molecule inhibitor CI-976 binds inside the catalytic chamber and blocks the accessibility of the active site residues H460, N421 and W420. Our results pave the way for future mechanistic study and rational drug design targeting hSOAT1 and other mammalian MBOAT family members.

Threonine 80 phosphorylation of non-structural protein 1 regulates the replication of influenza A virus by reducing the binding affinity with RIG-I.

Influenza A virus evades host antiviral defense through hijacking innate immunity by its non-structural protein 1 (NS1). By using mass spectrometry, threonine 80 (T80) was identified as a novel phosphorylated residue in the NS1 of the influenza virus A/WSN/1933(H1N1). By generating recombinant influenza viruses encoding NS1 T80 mutants, the roles of this phosphorylation site were characterized during viral replication. The T80E (phosphomimetic) mutant attenuated virus replication, whereas the T80A (non-phosphorylatable) mutant did not. Similar phenotypes were observed for these mutants in a mouse model experiment. In further study, the T80E mutant decreased the binding capacity between NS1 and viral nucleoprotein (NP), leading to impaired viral ribonucleoprotein (vRNP)-mediated viral transcription. The T80E mutant was also unable to inhibit interferon (IFN) production by reducing the binding affinity between NS1 and retinoic acid-induced gene 1 protein (RIG-I), causing attenuation of virus replication. Taken together, the present study reveals that T80 phosphorylation of NS1 reduced influenza virus replication through controlling RIG-I-mediated IFN production and vRNP activity.

Immunogenicity of multi-epitope-based vaccine candidates administered with the adjuvant Gp96 against rabies.

Rabies, a zoonotic disease, causes > 55,000 human deaths globally and results in at least 500 million dollars in losses every year. The currently available rabies vaccines are mainly inactivated and attenuated vaccines, which have been linked with clinical diseases in animals. Thus, a rabies vaccine with high safety and efficacy is urgently needed. Peptide vaccines are known for their low cost, simple production procedures and high safety. Therefore, in this study, we examined the efficacy of multi-epitope-based vaccine candidates against rabies virus. The ability of various peptides to induce epitope-specific responses was examined, and the two peptides that possessed the highest antigenicity and conservation, i.e., AR16 and hPAB, were coated with adjuvant canine-Gp96 and used to prepare vaccines. The peptides were prepared as an emulsion of oil in water (O/W) to create three batches of bivalent vaccine products. The vaccine candidates possessed high safety. Virus neutralizing antibodies were detected on the day 14 after the first immunization in mice and beagles, reaching 5-6 IU/mL in mice and 7-9 IU/mL in beagles by day 28. The protective efficacy of the vaccine candidates was about 70%-80% in mice challenged by a virulent strain of rabies virus. Thus, a novel multi-epitope-based rabies vaccine with Gp96 as an adjuvant was developed and validated in mice and dogs. Our results suggest that synthetic peptides hold promise for the development of novel vaccines against rabies.