Class B G protein–coupled receptors, an important class of therapeutic targets, signal mainly through the Gs class of heterotrimeric G proteins, although they do display some promiscuity in G protein binding. Using cryo–electron microscopy, we determined the structures of the human glucagon receptor (GCGR) bound to glucagon and distinct classes of heterotrimeric G proteins, Gs or Gi1. These two structures adopt a similar open binding cavity to accommodate Gs and Gi1. The Gs binding selectivity of GCGR is explained by a larger interaction interface, but there are specific interactions that affect Gi more than Gs binding. Conformational differences in the receptor intracellular loops were found to be key selectivity determinants. These distinctions in transducer engagement were supported by mutagenesis and functional studies.

The metabotropic glutamate receptors (mGlus) are involved in modulation of synaptic transmission and neuronal excitability in the central nervous system 1 . These receptors likely exist as both homo-and heterodimers with unique pharmacological and functional properties 2-4 . Here we report four cryo-electron microscopy structures of the human mGlus, including inactive mGlu2 and mGlu7 homodimers, agonist/PAM-bound mGlu2 homodimer, and inactive mGlu2-7 heterodimer. A subtype-dependent dimerization mode of mGlus was observed, as a unique dimer interface mediated by helix IV important to limit receptor activity exists in the inactive mGlu2 structure only. The structures provide molecular details of inter-and intrasubunit conformational changes that are required for receptor activation, which distinguish class C G-proteincoupled receptors (GPCRs) from classes A and B receptors. Furthermore, the mGlu2-7 heterodimer structure and functional studies suggest that the mGlu7 subunit plays a dominant role in controlling dimeric association and G protein activation in the heterodimer. These insights into mGlu homo-and heterodimers highlight the complex landscape of mGlu dimerization and activation.The mGlus contain a large extracellular domain (ECD) composed of the Venus flytrap (VFT) domain that binds agonist and a cysteine-rich domain (CRD) connected to a seven-helical transmembrane domain (TMD) responsible for G protein coupling 4 . It has been acknowledged that homodimerization is mandatory for the function of the mGlus, making them complex allosteric proteins with two subunits influencing each other 3 . In addition, there is increasing evidence suggesting that different mGlu subunits can associate to form multiple types of heterodimers, adding complexity of function modulation in this receptor family 2,4 . However, conformational difference between the mGlu homo-and heterodimerization and how these dimers control mGlu function remain unclear. Thus, we performed single-particle

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important target for vaccine and drug development. However, the rapid emergence of variant strains with mutated S proteins has rendered many treatments ineffective. Cleavage of the S protein by host proteases is essential for viral infection. Here, we discovered that the S protein contains two previously unidentified Cathepsin L (CTSL) cleavage sites (CS-1 and CS-2). Both sites are highly conserved among all known SARS-CoV-2 variants. Our structural studies revealed that CTSL cleavage promoted S to adopt receptor-binding domain (RBD) “up” activated conformations, facilitating receptor-binding and membrane fusion. We confirmed that CTSL cleavage is essential during infection of all emerged SARS-CoV-2 variants (including the recently emerged Omicron variant) by pseudovirus (PsV) infection experiment. Furthermore, we found CTSL-specific inhibitors not only blocked infection of PsV/live virus in cells but also reduced live virus infection of ex vivo lung tissues of both human donors and human ACE2-transgenic mice. Finally, we showed that two CTSL-specific inhibitors exhibited excellent In vivo effects to prevent live virus infection in human ACE2-transgenic mice. Our work demonstrated that inhibition of CTSL cleavage of SARS-CoV-2 S protein is a promising approach for the development of future mutation-resistant therapy.