Identification of a Calsequestrin-1 Mutation in a Human Vacuolar Myopathy

Abstract

b-SBD-a-lid interfaces. We used pulsed dipolar ESR and ion mobility mass spectroscopy technologies to characterize the conformational ensembles of DnaK in its allosteric states to better understand how the nucleotide and substrates modulate the allosteric landscape. We found that DnaK has a narrow distance distribution in ATP-bound state, but broader distance distributions in all other states exhibit multiple coexisting components. The ATPþsubstrate ensemble reflects the tug-of-war between the forces of NBD-SBD interaction driven by the binding of nucleotides, and the force of b-SBD-a-lid interaction driven by the binding of substrates. The ATPþsubstrate state contains 24% of docked and 76% of undocked conformers. The ADPþsubstrate state has a smaller fraction of docked conformers and an additional species, which we may represent as a ''domain rotamer'' around the unbound linker. Rotation of the NBD and SBD around the interdomain linker may play an important role in the allosteric mechanism. The ATPþsubstrate state releases the SBD's helical lid from the NBD bound in the ATP-bound state to an SBD bound position and a ''free'' position; and the ADPþsubstrate state pushes the equilibrium from the free position to the SBD bound position. The allosteric states can be modulated by mutations to dissect the energetic contributions.