Investigating dual Ca ²⁺ modulation of the ryanodine receptor 1 by molecular dynamics simulation

The ryanodine receptors (RyR) are essential to calcium signaling in striated muscles. A deep understanding of the complex Ca 2+ -activation/inhibition mechanism of RyRs requires detailed structural and dynamic information for RyRs in different functional states (e.g., with Ca 2+ bound to activating or inhibitory sites). Recently, high-resolution structures of the RyR isoform 1 (RyR1) were solved by cryo-electron microscopy, revealing the location of a Ca 2+ binding site for activation. Toward elucidating the Ca 2+ -modulation mechanism of RyR1, we performed extensive molecular dynamics simulation of the core RyR1 structure in the presence and absence of bound and solvent Ca 2+ (total simulation time is > 5 microseconds). In the presence of solvent Ca 2+ , Ca 2+ binding to the activating site enhanced dynamics of RyR1 with higher inter-subunit flexibility, asymmetric inter-subunit motions, outward domain motions and partial pore dilation, which may prime RyR1 for subsequent channel opening. In contrast, the solvent Ca 2+ alone reduced dynamics of RyR1 and led to inward domain motions and pore contraction, which may cause inhibition. Combining our simulation with the map of disease mutation sites in RyR1, we constructed a wiring diagram of key domains coupled via specific hydrogen bonds involving the mutation sites, some of which were modulated by Ca 2+ binding. The rich structural and dynamic information gained from this study will guide future mutational and functional studies of RyR1 activation and inhibition by Ca 2+ . Statement of Significance The ryanodine receptors (RyR) are key players in calcium signaling, and make prominent targets for drug design owning to their association with many diseases of cardiac and skeletal muscles. However, the molecular mechanism of their activation and inhibition by Ca 2+ remains elusive for the lack of high-resolution structural and dynamic information. Recent solutions of RyR1 structures by cryo-EM have paved the way for structure-based investigation of this important receptor by atomistic molecular simulation. This study presented, to our knowledge, the most extensive MD simulation of RyR1 core structure. Our simulation has offered new insights to the dual modulation mechanism of Ca 2+ , in which Ca 2+ binding to the activating site primes RyR1 activation by elevating its dynamics while solvent Ca 2+ inhibits RyR1 by reducing its dynamics. Additionally, our simulation has yielded a new wiring diagram of the allosterically coupled RyR1 domains informed by disease mutations.