The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca2+ binding site, had dramatically altered Ca 2+ binding properties. The KCa value for E22K was decreased by ∼17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca2+. Interestingly, Ca2+ binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the α-helical content of phosphorylated R58Q greatly increased with Ca2+ binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower KCa than wild-type light chain, whereas phosphorylation of this mutant increased the Ca2+ affinity 6-fold. Whereas phosphorylation of wild-type light chain decreased its Ca2+ affinity, the opposite was true for A13T. The α-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca2+ binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.
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