Revealing the conformational dynamics of UDP-GlcNAc recognition by O-GlcNAc transferase via Markov state model.

The O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is a critical post-translational modification and closely linked to various physiological and pathological conditions. The O-GlcNAc transferase (OGT) functions as the only glycosyltransferase of O-GlcNAc glycosylation by transferring GlcNAc from UDP-GlcNAc to serine or threonine residues on protein substrates. The interaction mode of UDP-GlcNAc against OGT has been preliminarily revealed by the crystal structures, yet an atomic-level comprehension for the conformational dynamics of the recognition process remains elusive. Here, we construct the Markov state model based on extensive all-atom molecular dynamics (MD) simulations with an aggregated simulation time of ∼9 µs, and reveal that the UDP-GlcNAc recognition process by OGT encompasses four key metastable states, occurring within an estimated timescale of ∼10 µs. During UDP-GlcNAc recognition process, we find the pyrophosphate moiety (P2O52-) initially anchors to the active pocket via salt bridge and hydrogen bonds, facilitating subsequent binding of the uridine and GlcNAc moieties. Furthermore, the functional roles of K842 involved in the salt bridge with P2O52- were evaluated through extra mutant MD simulations. Overall, our study provides valuable insights into the UDP-GlcNAc recognition mechanism by OGT, which could further aid in mechanistic studies of O-GlcNAc glycosylation and drug development targeting on OGT.

Investigation of ECD conformational transition mechanism of GLP-1R by molecular dynamics simulations and Markov state model.

As a member of the class B G protein-coupled receptors (GPCRs), the glucagon-like peptide-1 (GLP-1) can regulate the blood glucose level by binding to the glucagon-like peptide-1 receptor (GLP-1R). Since the extracellular domain (ECD) of GLP-1R is considered as one of the binding sites of GLP-1, the open and closed states of ECD play an important role in the binding process of GLP-1. To investigate the transition path of GLP-1R ECD, the crystal structures of GLP-1R in its bound and unbound states (apo-state) are chosen to perform a total of 1.6 µs of molecular dynamics simulations. The simulated results show that the ECD of GLP-1R closes in the GLP-1 bound state and opens in the GLP-1 unbound state. To determine the critical role that GLP-1 played in regulating the open and closed states of the ECD, we applied the independent gradient model (IGM) to the simulation trajectories. We found that the "hand-like" N-terminal of the GLP-1R ECD plays an important role in the GLP-1 binding. In contrast, the apo-state GLP-1R ECD opens and exposes the two ligand binding domains of GLP-1 after 200 ns of simulations. To elucidate the open and closed mechanisms of GLP-1R ECD in the apo-state and GLP-1 bound state, the Markov state model (MSM) is performed on the MD simulation trajectories. Our results provide possible transition pathways from the closed state to open state of the apo-state GLP-1R ECD. Each pathway contains several intermediate states that correspond to different local minima in deep wells. The dynamical relationships and the most possible conversion pathway between two states are detailed through the MSM analysis. Our results profile the conformation transition mechanism of the GLP-1R ECD and will help in hypoglycemic peptide design of GLP-1R.