Melatonin receptors (MT₁and MT₂in humans) are family A G protein–coupled receptors that respond to the neurohormone melatonin to regulate circadian rhythm and sleep. Numerous efforts have been made to develop drugs targeting melatonin receptors for the treatment of insomnia, circadian rhythm disorder, and cancer. However, designing subtype-selective melatonergic drugs remains challenging. Here, we report the cryo-EM structures of the MT₁–G_isignaling complex with 2-iodomelatonin and ramelteon and the MT₂–G_isignaling complex with ramelteon. These structures, together with the reported functional data, reveal that although MT₁and MT₂possess highly similar orthosteric ligand-binding pockets, they also display distinctive features that could be targeted to design subtype-selective drugs. The unique structural motifs in MT₁and MT₂mediate structural rearrangements with a particularly wide opening on the cytoplasmic side. G_iis engaged in the receptor core shared by MT₁and MT₂and presents a conformation deviating from those in other G_icomplexes. Together, our results provide new clues for designing melatonergic drugs and further insights into understanding the G protein coupling mechanism.

Histamine receptors are a group of G protein-coupled receptors (GPCRs) that play important roles in various physiological and pathophysiological conditions. Antihistamines that target the histamine H₁ receptor (H₁R) have been widely used to relieve the symptoms of allergy and inflammation. Here, to uncover the details of the regulation of H₁R by the known second-generation antihistamines, thereby providing clues for the rational design of newer antihistamines, we determine the cryo-EM structure of H₁R in the apo form and bound to different antihistamines. In addition to the deep hydrophobic cavity, we identify a secondary ligand-binding site in H₁R, which potentially may support the introduction of new derivative groups to generate newer antihistamines. Furthermore, these structures show that antihistamines exert inverse regulation by utilizing a shared phenyl group that inserts into the deep cavity and block the movement of the toggle switch residue W428^6.48. Together, these results enrich our understanding of GPCR modulation and facilitate the structure-based design of novel antihistamines.

The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and β-arrestin pathways. Structural and spectroscopic analysis suggest that LY211620 stabilizes distinct intracellular conformational ensembles from agonist-bound M2R, which may enhance β-arrestin recruitment while impairing G-protein activation. These results highlight the role of conformational dynamics in the complex signaling behavior of GPCRs, and could facilitate design of better drugs.

The major challenge to controlling the COVID pandemic is the rapid mutation rate of the SARS-CoV-2 virus, leading to the escape of the protection of vaccines and most of the neutralizing antibodies to date. Thus, it is essential to develop neutralizing antibodies with broad-spectrum activity targeting multiple SARS-CoV-2 variants. Here, we report a synthetic nanobody (named C5G2) obtained by phage display and subsequent antibody engineering. C5G2 has a single-digit nanomolar binding affinity to the RBD domain and inhibits its binding to ACE2 with an IC₅₀ of 3.7 nM. Pseudovirus assays indicated that monovalent C5G2 could protect the cells from infection with SARS-CoV-2 wild-type virus and most of the viruses of concern, i.e., Alpha, Beta, Gamma and Omicron variants. Strikingly, C5G2 has the highest potency against Omicron BA.1 among all the variants, with an IC₅₀ of 4.9 ng/mL. The cryo-EM structure of C5G2 in complex with the spike trimer showed that C5G2 binds to RBD mainly through its CDR3 at a conserved region that does not overlap with the ACE2 binding surface. Additionally, C5G2 binds simultaneously to the neighboring NTD domain of the spike trimer through the same CDR3 loop, which may further increase its potency against viral infection. Third, the steric hindrance caused by FR2 of C5G2 could inhibit the binding of ACE2 to RBD as well. Thus, this triple-function nanobody may serve as an effective drug for prophylaxis and therapy against Omicron as well as future variants.

Significance: To adapt to the changing environment, bacteria must quickly transduce extracellular information into appropriate cellular response pathways. Two-component systems consisting of a membrane-embedded histidine kinase (HK) and a cytoplasmic response regulator often perform this function. In this work, we present structural and functional data showing that HptRSA, a G6P sensor from Staphylococcus aureus , undergoes conformational changes after G6P binding with HptA that causes it to switch its interface with the periplasmic domain of HptS. As a result, a rotation and closure occur in the periplasmic side of the HK. This previously unreported mode of activation not only expands our understanding of HK signal perception in kinase activation but also provides a framework for designing new antimicrobial drugs.