C-Glycosyltransferases (CGTs) catalyze the formation of C-glycosidic bonds for the biosynthesis of C-glycosides, but the underlying mechanism is unclear. This process improves the solubility and bioavailability of specialized metabolites, which play important roles in plant growth and development and represent rich resources for drug discovery. Here, we performed functional and structural studies of the C-glycosyltransferase UGT708C1 from buckwheat (Fagopyrum esculentum). Enzymatic analysis showed that UGT708C1 is capable of utilizing both uridine‐59 diphosphate galactose (UDP-galactose) and uridine‐59‐diphosphate glucose (UDP-glucose) as sugar donors. Our structural studies of UGT708C1 complexed with UDP-glucose and uridine‐59‐diphosphate (UDP) identified the key roles of Asp382, Gln383, Thr151, and Thr150 in recognizing the sugar moiety of the donor substrate and Phe130, Tyr102, and Phe198 in binding and stabilizing the acceptor. A systematic site-directed mutagenesis study confirmed the important roles of these residues. Further structural analysis combined with molecular dynamics simulations revealed that phloretin binds to the acceptor-binding pocket in a bent state with a precise spatial disposition and complementarity. These findings provide insights into a catalytic mechanism for C-glycosyltransferases.

The full activation process of G protein–coupled receptor (GPCR) plays an important role in cellular signal transduction. However, it remains challenging to simulate the whole process in which the GPCR is recognized and activated by a ligand and then couples to the G protein on a reasonable simulation timescale. Here, we developed a molecular dynamics (MD) approach named supervised (Su) Gaussian accelerated MD (GaMD) by incorporating a tabu-like supervision algorithm into a standard GaMD simulation. By using this Su-GaMD method, from the active and inactive structure of adenosine A ₁ receptor (A ₁ R), we successfully revealed the full activation mechanism of A ₁ R, including adenosine (Ado)–A ₁ R recognition, preactivation of A ₁ R, and A ₁ R–G protein recognition, in hundreds of nanoseconds of simulations. The binding of Ado to the extracellular side of A ₁ R initiates conformational changes and the preactivation of A ₁ R. In turn, the binding of G _i2 to the intracellular side of A ₁ R causes a decrease in the volume of the extracellular orthosteric site and stabilizes the binding of Ado to A ₁ R. Su-GaMD could be a useful tool to reconstruct or even predict ligand–protein and protein–protein recognition pathways on a short timescale. The intermediate states revealed in this study could provide more detailed complementary structural characterizations to facilitate the drug design of A ₁ R in the future.

Mitochondrial RNA splicing 2 (Mrs2), a eukaryotic CorA ortholog, enables Mg²⁺ to permeate the inner mitochondrial membrane and plays an important role in mitochondrial metabolic function. However, the mechanism by which Mrs2 permeates Mg²⁺ remains unclear. Here, we report four cryo-electron microscopy (cryo-EM) reconstructions of Homo sapiens Mrs2 (hMrs2) under various conditions. All of these hMrs2 structures form symmetrical pentamers with very similar pentamer and protomer conformations. A special structural feature of Cl⁻-bound R-ring, which consists of five Arg332 residues, was found in the hMrs2 structure. Molecular dynamics simulations and mitochondrial Mg²⁺ uptake assays show that the R-ring may function as a charge repulsion barrier, and Cl⁻ may function as a ferry to jointly gate Mg²⁺ permeation in hMrs2. In addition, the membrane potential is likely to be the driving force for Mg²⁺ permeation. Our results provide insights into the channel assembly and Mg²⁺ permeation of hMrs2.

The human glucagon receptor (GCGR) is a class B G-protein-coupled receptor (GPCR). The GCGR can be activated by glucagon and regulates the release of glucose. The GCGR has been proposed to be an important drug target for type 2 diabetes. Based on the structural model of a full-length glucagon-bound GCGR (glu-GCGR), we performed accelerated molecular dynamics (aMD) simulations, potential of mean force (PMF) calculations, cross-correlation analysis and community network analysis to study the activation mechanism and the conformational dynamics during the activation process. The PMF map depicts three different conformational states of the GCGR: the inactive, intermediate and active states. The activation of the GCGR is characterized by the outward movement of the intracellular side of helix VI. In the active state of the GCGR, the Arg173^2.46–Ser350^6.41 and Glu245^3.50–Thr351^6.42 hydrogen bonds break, and the χ₁ rotamer of Phe322^5.54 changes from perpendicular to parallel to helix VI. The binding of the agonist glucagon decreases the correlated motions of the extracellular loops (ELCs) and the helices around the glucagon-binding site. During the activation of the GCGR, the connections between the intracellular sides of helices become weaker, and the connections between glucagon and ECLs and the extracellular sides of helices become stronger. These facilitate G-protein coupling on the intracellular side and glucagon binding on the extracellular side, and stabilize the GCGR in the active state. We expect that this study can provide useful information on the activation mechanism of the GCGR and facilitate the future design of GCGR inhibitors.

The human P2Y₁ receptor (P2Y₁R) is a purinergic G-protein-coupled receptor (GPCR) that functions as a receptor for adenosine 5′-diphosphate (ADP). An antagonist of P2Y₁R might potentially have antithrombotic effects, whereas agonists might serve as antidiabetic agents. On the basis of the antagonist-bound MRS2500-P2Y₁R crystal structure, we constructed computational models of apo-P2Y₁R and the agonist-receptor complex 2MeSADP-P2Y₁R. We then performed conventional molecular dynamics (cMD) and accelerated molecular dynamics (aMD) simulations to study the conformational dynamics after binding with agonist/antagonist as well as the P2Y₁R activation mechanism. We identified a new agonist-binding site of P2Y₁R that is consistent with previous mutagenesis data. This new site is deeper than those of the agonist ADP in the recently simulated ADP-P2Y₁R structure and the antagonist MRS2500 in the MRS2500-P2Y₁R crystal structure. During P2Y₁R activation, the cytoplasmic end of helix VI shifts outward 9.1 Å, the Ser146^3.47-Tyr237^5.58 hydrogen bond breaks, a Tyr237^5.58-Val262^6.37 hydrogen bond forms, and the conformation of the χ1 rotamer of Phe269^6.44 changes from parallel to perpendicular to helix VI. The apo-P2Y₁R system and the MRS2500-P2Y₁R system remain inactive. The newly identified agonist binding site and activation mechanism revealed in this study may aid in the design of P2Y₁R antagonists/agonists as antithrombotic/antidiabetic agents, respectively.