Molecular Determinants of Mg-Mediated Inhibition in RyR1: Insights from Computational Approaches.

We investigated Mg-mediated inhibition of RyR1 by analyzing solvation, permeation, and binding interactions of Mg, Ca, Na, and K across three functional states: Ca-activated (opRyR1), closed (clRyR1), and Mg-inhibited (HMgRyR1). Using molecular dynamics simulations, potential of mean force (PMF) analysis, quantum mechanical calculations, and MM-GBSA binding free energy calculations, we identified the structural and energetic determinants of Mg inhibition. Our water occupancy analysis reveals that Mg binding at D4945 stabilizes the S6 helical arrangement within the cytoplasmic vestibule in the HMgRyR1 state, maintaining a narrowed pore and reducing water accessibility. PMF calculations show that Mg encounters the highest energy barriers, effectively restricting its permeation. Among the studied ions, Mg exhibits the strongest affinity at the D4945 site, particularly in the HMgRyR1 state, reinforcing its inhibitory role. Binding energy analyses reveal that Mg in HMgRyR1 has the lowest mobility and the most favorable binding free energy, indicating a highly stable ion-protein interaction and stronger retention in the closed and inhibited states. Additionally, Mg binding is primarily stabilized by electrostatic interactions, which dominate over nonpolar contributions. These findings provide a comprehensive understanding of Mg-mediated RyR1 inhibition and offer critical insights into ion-specific regulation within the channel, further supporting structural models that highlight Mg's role in stabilizing the closed conformation.