Molecular Determinants of Mg²⁺-Mediated Inhibition in RyR1: Insights from Computational Approaches

We investigated Mg²⁺-mediated inhibition of RyR1 by analyzing solvation, permeation, and binding interactions of Mg²⁺, Ca²⁺, Na⁺, and K⁺ across three functional states: Ca²⁺-activated (opRyR1), closed (clRyR1), and Mg²⁺-inhibited (HMg²⁺RyR1). 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 Mg²⁺ inhibition. Our water occupancy analysis reveals that Mg²⁺ binding at D4945 stabilizes the S6 helical arrangement within the cytoplasmic vestibule in the HMg²⁺RyR1 state, maintaining a narrowed pore and reducing water accessibility. PMF calculations show that Mg²⁺ encounters the highest energy barriers, effectively restricting its permeation. Among the studied ions, Mg²⁺ exhibits the strongest affinity at the D4945 site, particularly in the HMg²⁺RyR1 state, reinforcing its inhibitory role. Binding energy analyses reveal that Mg²⁺ in HMg²⁺RyR1 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, Mg²⁺ binding is primarily stabilized by electrostatic interactions, which dominate over nonpolar contributions. These findings provide a comprehensive understanding of Mg²⁺-mediated RyR1 inhibition and offer critical insights into ion-specific regulation within the channel, further supporting structural models that highlight Mg²⁺'s role in stabilizing the closed conformation.