Both cardiac and skeletal calsequestrin (CASQ2 and CASQ1) serve as a major Ca2+ storage/buffer protein in the sarcoplasmic reticulum (SR) by sequestering and releasing large numbers of Ca2+ ions during each muscular contraction and relaxation cycle. CASQ isolated from various species often exists in a phosphorylated form, but phosphorylation’s role is not yet understood. Here, the authors identified two phosphorylation sites, Ser385 and Ser393, for the first time, in human CASQ2 (hCASQ2) by mass-spectroscopy and evaluated the consequences of such phosphorylation. Substitution of these two serines with phosphoserine-mimicking aspartic-acid residues results in a significant increase in helical content, solubility and Ca2+-binding capacity above 6 mM [Ca2+]. However, neither substitution of Ser385 nor Ser393 alone produce any significant changes. Based on the crystal structures of hCASQ2, Ca2+ binding capacity data, turbidity, and light scattering profiles, it was propose that phosphorylation at these two positions produces a disorder-to-order or coil-to-helix transition of the C-terminus, which in turn provides a more stable network of polyanions. Therefore, considering all the previous reports and the new data, the observed dynamic in vivo phosphorylation of CASQ could provide the basis not only for effective regulation of Ca2+ buffering capacity, but also for the junctional SR trafficking mechanism.

•Substrate binding properties of potato AGPase variants were analyzed by ITC.•The heterotetrameric AGPase, SWTLWT, displays two binding sites for ATP.•The homotetrameric enzymes, SS and LS, contain only a single binding site for ATP.•SWTLWT exhibits higher affinity to ATP due to synergistic interaction of LS and SS.•AGPase does not bind glucose 1-phosphate in the presence or absence of ATPγS.Substrate binding properties of the large (LS) and small (SS) subunits of potato tuber ADP-glucose pyrophosphorylase were investigated by using isothermal titration calorimetry. Our results clearly show that the wild type heterotetramer (SWTLWT) possesses two distinct types of ATP binding sites, whereas the homotetrameric LS and SS variant forms only exhibited properties of one of the two binding sites. The wild type enzyme also exhibited significantly increased affinity to this substrate compared to the homotetrameric enzyme forms. No stable binding was evident for the second substrate, glucose-1-phosphate, in the presence or absence of ATPγS suggesting that interaction of glucose-1-phosphate is dependent on hydrolysis of ATP and supports the Theorell–Chance bi bi reaction mechanism.

Mutations of conserved residues of human cardiac calsequestrin (hCSQ2), a high-capacity, low-affinity Ca2+-binding protein in the sarcoplasmic reticulum, have been associated with catecholamine-induced polymorphic ventricular tachycardia (CPVT). In order to understand the molecular mechanism and pathophysiological link between these CPVT-related missense mutations of hCSQ2 and the resulting arrhythmias, we generated three CPVT-causing mutants of hCSQ2 (R33Q, L167H, and D307H) and two non-pathological mutants (T66A and V76M) and investigated the effect of these mutations. In addition, we determined the crystal structure of the corresponding wild-type hCSQ2 to gain insight into the structural effects of those mutations. Our data show clearly that all three CPVT-related mutations lead to significant reduction in Ca2+-binding capacity in spite of the similarity of their secondary structures to that of the wild-type hCSQ2. Light-scattering experiments indicate that the Ca2+-dependent monomer-polymer transitions of the mutants are quite different, confirming that the linear polymerization behavior of CSQ is linked directly to its high-capacity Ca2+ binding. R33Q and D307H mutations result in a monomer that appears to be unable to form a properly oriented dimer. On the other hand, the L167H mutant has a disrupted hydrophobic core in domain II, resulting in high molecular aggregates, which cannot respond to Ca2+. Although one of the non-pathological mutants, T66A, shares characteristics with the wild-type, the other null mutant, V76M, shows significantly altered Ca2+-binding and polymerization behaviors, calling for careful reconsideration of its status.