Mapping Co-regulation Pathways among Ligand Binding sites in RyR1

Ryanodine receptor 1 (RyR1) is an intracellular calcium (Ca2+) release channel required for skeletal muscle contraction. RyR1 is co-regulated by multiple activators - Ca2+, ATP and caffeine (CFF), yet the mechanism of co-regulation and the action synergy of these activators is unknown. Here, we report the detailed network of allosteric connections between the three ligand sites and the pore region in (i) Ca2+ bound - closed, (ii) ATP/CFF bound - closed, (iii) Ca2+/ATP/CFF bound - closed and (iv) Ca2+/ATP/CFF bound - open RyR1 states. We find that two dominant paths mediate the communication between the Ca2+ binding site and pore region in Ca2+-only state. ATP/CFF-only occupied - closed RyR1 has two additional paths with CFF-but not ATP-occupied path using part of the closed Ca2+-only pathway. In the presence of Ca2+, ATP and CFF, major differences between the open and closed states are identified with both using part of the paths of the closed Ca2+-only and ATP/CFF-only states. We find that the three activators Ca2+, ATP, and CFF propagate their effects to the pore region through a network of partially overlapping pathways. Such coordination of allosteric pathway underlies the molecular basis of synergy of channel regulation by multiple activators.\n\nStatement of SignificanceRyRs are a group of Ca2+ channels that bind to several endogenous modulators and regulate Ca2+ release through closed-to-open gating transition. Despite the high-resolution structural data available for RyR1, the allosteric mechanism of RyR1 gating remains elusive. In this study, we employed graph-theoretical approach to demonstrate the allosteric network of synergistic interaction among various activators in RyR1. To our knowledge, for the first time we were able to identify the co-regulation among ligand sites in RyR1 to regulate the closed-to-open gating transition. The explored allosteric coupling in RyR1 may assist in designing advanced therapeutics for several debilitating diseases. Our findings in this study will assist to design new strategies for controlled allosteric regulation of RyR1 functionality in future.