Jian Li

The SARS-CoV-2 main protease (M ^pro ) has a pivotal role in mediating viral genome replication and transcription of the coronavirus, making it a prime target for antiviral drug development. This study presents four high-resolution crystal structures of SARS-CoV-2 M ^pro in the apo state. Among these structures, we identify a previously unobserved, stable inactive conformation, characterized by a major displacement of the oxyanion loop (especially Phe140, Leu141 and Asn142), the rotation of some important residues (including His163 and His172) and the loss of many key interactions around the active site, which provides new insights into the conformational dynamics of the enzyme. We also report structures of M ^pro in complex with the covalent inhibitor pomotrelvir (PBI-0451) for the wild-type enzyme and three clinical variant-associated mutants (G15S, K90R and P132H). PBI-0451 adopts a virtually identical mode when bound to wild-type M ^pro and the K90R and G15S M ^pro mutants. However, the crystal structure of PBI-0451 bound to SARS-CoV-2 M ^pro P132H reveals a discernible shift of the Gln189-bearing loop and incomplete electron density for the Gln189 side chain, which may be responsible for the subtle modest reduction in PBI-0451 susceptibility. These findings expand the conformational landscape of M ^pro and offer structural guidance for the design of M ^pro -targeted antiviral drugs.

The nucleocapsid (N) protein of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) functions in viral RNA binding, genome packaging and ribonucleoprotein (RNP) assembly. Crystal structures were determined of the N-terminal domain (NTD) from crystals grown at pH 8.0 to a resolution of 1.94 Å and of the C-terminal domain (CTD) from crystals grown at pH 5.0 and 8.5 to resolutions of 1.59 and 1.30 Å, respectively. The NTD adopts the canonical coronavirus NTD fold and contains a positively charged cleft corresponding to a previously proposed RNA-binding region. Comparison of the apo NTD structure determined in this study with a previously reported RNA-bound NTD structure revealed local differences in the side-chain conformations of RNA-contacting residues, including Thr49, Arg88, Arg92, Arg107, Tyr109 and Tyr111, whereas the overall domain fold remained essentially unchanged. These observations suggest that RNA binding may be accommodated by local side-chain rearrangements within a pre-existing positively charged cleft rather than by a large-scale conformational change. The CTD forms a domain-swapped dimer mediated by β-hairpins, with a positively charged groove extending across the dimer interface. Superposition of the two CTD structures yielded a root-mean-square deviation (r.m.s.d.) of 0.129 Å, and 11 intersubunit hydrogen bonds were observed at the dimer interface in each structure, indicating that the dimer architecture is conserved under the crystallization conditions examined. Together, these structures provide a framework for further investigation of NTD–RNA recognition, the possible role of the CTD positively charged groove in nucleic acid interactions and N-protein assembly.

Human trichromatic color vision relies on three cone opsins [long-, middle-, and short-wavelength-sensitive opsins (LWS-, MWS-, and SWS-opsins, respectively)], whereas scotopic rod vision is mediated by rhodopsin. Although the structure of rhodopsin was solved more than 20 years ago, cone opsin structures have been lacking. Here, we present cryo–electron microscopy structures of the three human cone opsins, each bound to a G protein and all- trans retinal in the presumed active state. All three cone opsins differ markedly from rhodopsin. Within the retinal binding pocket, we identified a distinct counterion site (LWS- and MWS-opsins) and a ring of serines around the retinal (SWS-opsin). The active cone opsin structures explain how amino acid substitutions fine-tune spectral sensitivity and help clarify the molecular basis of color vision deficiencies and key differences in rod versus cone activation.

The TRPC1/5 heteromer exhibits electrophysiological and ligand-binding properties distinct from TRPC5 homomers, enabling tissue-specific cellular regulation. Here we present the cryo-EM structure of the TRPC1/5 heterotetramer at 2.8 Å resolution, revealing an asymmetric assembly of three TRPC5 subunits around one TRPC1 subunit. TRPC1 contributes a unique pore-loop configuration and specialized inter-subunit interfaces that sculpt an asymmetrical ion conduction pathway, altering gating and ion selectivity. The heteromer harbors a ligand-binding pocket at the TRPC1-TRPC5 interface absent in homomeric channels. Using this insight, we design JD03-02, a high-affinity antagonist preferentially targeting this pocket with >10,000-fold selectivity for TRPC1/5 over TRPC5 homomers. In mouse models, JD03-02 produces potent anxiolytic and antidepressant effects with reduced off-target activity. These findings elucidate the structural basis of TRPC1/5 function and can guide precision drug design targeting heteromeric ion channels in neuropsychiatric disorders.