Structural Modulation of RyR1 by MgATP and Free Mg2+ in Lipid Membrane using CryoEM

Abstract

Mg2+ is the most abundant divalent cation in living cells. In the skeletal muscle cytosol, out of the total Mg2+ concentration of 8 mM, around 1 mM is free Mg2+ and around 5 mM exists in the form of MgATP, the biological active form of cellular ATP. Mg2+ is constitutively bound to RyR1 in its free and complexed forms. In fact, preferential binding of Mg2+ to the acidic side chains with similar coordination geometry as Ca2+, allows it to be a potential antagonistic cation of Ca2+, which has much lower cytoplasmic concentrations (<1 μM - 100 μM). Free Mg2+ is thought to inhibit RyRs through its association with Ca2+ binding sites at cytoplasmic or luminal regions, while the effect of MgATP per se is novel in the context of excitation-contraction coupling. We studied the effect of free Mg2+ and MgATP on RyR1 bound to a POPC bilayer enclosed by membrane scaffold protein (nanodisc). CryoEM reconstructions of RyR1 nanodiscs complexed with MgAMPPCP at ∼3.8 Å resolution show that in the absence of any detergents or FKBP, the RyR1 assumes a closed conformation with a pore radius that makes impermeant to Ca2+ ions. Discernible densities for nanodisc and MgATP were observed. Binding of MgATP involves global motion of the central and cytoplasmic domains towards the T-tubule and the fourfold axis with reorganization of the P2 domain. Local motion of the TM helices, U-motif, VSC and EF hands were also observed with concomitant closure of the ATP cavity at the intersubunit interface. With the cryoEM structure of RyR1-MgATP, we seek to explain the mechanistic basis of RyR1 inhibition during the physiological resting state of skeletal muscle. Supported by grants AHA 14GRNT19660003, MDA352845, NIH R01 AR068431, and NIH U24GM116789.