Here, we report the identification of three novel missense mutations in the calsequestrin-1 (CASQ1) gene in four patients with tubular aggregate myopathy. These CASQ1 mutations affect conserved amino acids in position 44 (p.(Asp44Asn)), 103 (p.(Gly103Asp)), and 385 (p.(Ile385Thr)). Functional studies, based on turbidity and dynamic light scattering measurements at increasing Ca concentrations, showed a reduced Ca -dependent aggregation for the CASQ1 protein containing p.Asp44Asn and p.Gly103Asp mutations and a slight increase in Ca -dependent aggregation for the p.Ile385Thr. Accordingly, limited trypsin proteolysis assay showed that p.Asp44Asn and p.Gly103Asp were more susceptible to trypsin cleavage in the presence of Ca in comparison with WT and p.Ile385Thr. Analysis of single muscle fibers of a patient carrying the p.Gly103Asp mutation showed a significant reduction in response to caffeine stimulation, compared with normal control fibers. Expression of CASQ1 mutations in eukaryotic cells revealed a reduced ability of all these CASQ1 mutants to store Ca and a reduced inhibitory effect of p.Ile385Thr and p.Asp44Asn on store operated Ca entry. These results widen the spectrum of skeletal muscle diseases associated with CASQ1 and indicate that these mutations affect properties critical for correct Ca handling in skeletal muscle fibers.

The sarcoplasmic reticulum (SR) of striated muscles is specialized for releasing Ca(2+) following sarcolemma depolarization in order to activate muscle contraction. To this end, the SR forms a network of longitudinal tubules and cisternae that surrounds the myofibrils and, at the same time, participates to the assembly of the triadic junctional membrane complexes formed by the close apposition of one t-tubule, originated from the sarcolemma, and two SR terminal cisternae. Advancements in understanding the molecular basis of the SR structural organization have identified an interaction between sAnk1, a transmembrane protein located on the longitudinal SR (l-SR) tubules, and obscurin, a myofibrillar protein. The direct interaction between these two proteins results in molecular contacts that have the overall effect to stabilize the l-SR tubules along myofibrils in skeletal muscle fibers. Less known is the structural organization of the sites in the SR that are specialized for Ca(2+) release and are positioned at the junctional SR (j-SR), i.e. the region of the terminal cisternae that faces the t-tubule at triads. At the j-SR, several trans-membrane proteins like triadin, junctin, or intra-luminal SR proteins like calsequestrin, are assembled together with the ryanodine receptor, the SR Ca(2+) release channel, into a macromolecular complex specialized in releasing Ca(2+). At triads, the 12 nm-wide gap between the t-tubule and the j-SR allows the ryanodine receptor on the j-SR to be functionally coupled with the voltage-gated L-type calcium channel on the t-tubule in order to allow the transduction of the voltage-induced signal into Ca(2+) release through the ryanodine receptor channels. The muscle-specific junctophilin isoforms (JPH1 and JPH2) are anchored to the j-SR with a trans-membrane segment present at the C-terminus and are capable to bind the sarcolemma with a series of phospholipid-binding motifs localized at the N-terminus. Accordingly, through this dual interaction, JPH1 and JPH2 are responsible for the assembly of the triadic junctional membrane complexes. Recent data indicate that junctophilins seem also to interact with other proteins of the excitation-contraction machinery, suggesting that they may contribute to hold excitation-contraction coupling proteins to the sites where the j-SR is being organized.

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.