Mapping co-regulatory interactions among ligand-binding sites in ryanodine receptor 1

Ryanodine receptor 1 (RyR1) is an intracellular calcium ion (Ca²⁺) release channel required for skeletal muscle contraction. Although cryo-electron microscopy identified binding sites of three coactivators Ca²⁺, ATP, and caffeine (CFF), the mechanism of co-regulation and synergy of these activators is unknown. Here, we report allosteric connections among the three ligand-binding sites and 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 identified two dominant networks of interactions that mediate communication between the Ca²⁺-binding site and pore region in Ca²⁺ bound-closed state, which partially overlapped with the pore communications in ATP/CFF bound-closed RyR1 state. In Ca²⁺/ATP/CFF bound-closed and -open RyR1 states, co-regulatory interactions were analogous to communications in the Ca²⁺ bound-closed and ATP/CFF bound-closed states. Both ATP- and CFF-binding sites mediate communication between the Ca²⁺-binding site and the pore region in Ca²⁺/ATP/CFF bound—open RyR1 structure. We conclude that Ca²⁺, ATP, and CFF propagate their effects to the pore region through a network of overlapping interactions that mediate allosteric control and molecular synergy in channel regulation.