Voltage-gated sodium (Nav) channels are essential for the rapid depolarization of nerve and muscle1, and are important drug targets2. A family of bacterial Nav channels, exemplified by NaChBac (Na+-selective Channel of Bacteria)3, provides a good model system for structure-function analysis. Here we report the crystal structure of NavAP, a NaChBac orthologue from marine bacteria alpha proteobacterium HIMB114, at 3.05 Å resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr178 and Leu179 constitute an inner site within the selectivity filter (178TLSSWE183) where a Ca2+ can bind and resides in the crystal structure. The outer mouth of the Na+ selectivity filter, defined by Ser181 and Glu183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation with all the gating charges exposing to the extracellular side. We hypothesize that NavAP is captured in an inactivated conformation. Comparison of NavAP with NavAb4 reveals significant conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.

The major facilitator superfamily glucose transporters, exemplified by human GLUT1-4, have been central to the study of solute transport. Using lipidic cubic phase crystallization and microfocus X-ray diffraction, we determined the structure of human GLUT3 in complex with D-glucose at 1.5 Å resolution in an outward-occluded conformation. The high-resolution structure allows discrimination of both α- and β-anomers of D-glucose. Two additional structures of GLUT3 bound to the exofacial inhibitor maltose were obtained at 2.6 Å in the outward-open and 2.4 Å in the outward-occluded states. In all three structures, the ligands are predominantly coordinated by polar residues from the carboxy terminal domain. Conformational transition from outward-open to outward-occluded entails a prominent local rearrangement of the extracellular part of transmembrane segment TM7. Comparison of the outward-facing GLUT3 structures with the inward-open GLUT1 provides insights into the alternating access cycle for GLUTs, whereby the C-terminal domain provides the primary substrate-binding site and the amino-terminal domain undergoes rigid-body rotation with respect to the C-terminal domain. Our studies provide an important framework for the mechanistic and kinetic understanding of GLUTs and shed light on structure-guided ligand design.

The pandemic of COVID-19, caused by SARS-CoV-2, is a major global health threat. Epidemiological studies suggest that bats (Rhinolophus affinis) are the natural zoonotic reservoir for SARS-CoV-2. However, the host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. The interaction of coronavirus with its host receptor is a key genetic determinant of host range and cross-species transmission. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the receptor to enter host cells in a species-dependent manner. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. By analyzing the conservation of five residues in two virus-binding hotspots of ACE2 (hotspot 31Lys and hotspot 353Lys), we predicted 80 ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 ACE2 orthologs among them for functional analysis, and showed that 44 of these orthologs—including domestic animals, pets, livestock, and animals commonly found in zoos and aquaria—could bind the SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind the SARS-CoV-2 spike protein and support viral entry. We further identified the genetic determinant of New World monkey ACE2 that restricts viral entry using genetic and functional analyses. These findings highlight a potentially broad host tropism of SARS-CoV-2 and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, underscoring the necessity to monitor susceptible hosts to prevent future outbreaks.

SARS-CoV-2 variants continue to emerge during the global pandemic and may facilitate escape from current antibody therapies and vaccine protection. Here, we showed that the South African variant B.1.351 was the most resistant to current monoclonal antibodies and convalescent plasma from COVID-19-infected individuals, followed by the Brazilian variant P.1 and the UK variant B.1.1.7. This resistance hierarchy corresponded with Y144del and 242-244del mutations in the N-terminal domain and K417N/T, E484K and N501Y mutations in the receptor binding domain (RBD) of SARS-CoV-2. Crystal structural analysis of B.1.351 triple mutant (417N-484K-501Y) RBD complexed with monoclonal antibody P2C-1F11 revealed the molecular basis for antibody neutralization and escape. B.1.351 and P.1 also acquired the ability to use mouse and mink ACE2 receptor for entry. Our results demonstrate major antigenic shifts and potential broadening of the host range for B.1.351 and P.1 variants, which pose serious challenges to our current antibody therapies and vaccine protection.