Comparing skeletal and cardiac calsequestrin structures and their calcium binding: A proposed mechanism for coupled calcium binding and protein polymerization

Abstract

Calsequestrin, the major calcium storage protein of both cardiac and skeletal muscle, binds and releases large numbers of Ca2+ ions for each contraction and relaxation cycle. Here we show that two crystal structures for skeletal and cardiac calsequestrin are nearly superimposable not only for their subunits but also their front-to-front-type dimers. Ca2+ binding curves were measured using atomic absorption spectroscopy. This method enables highly accurate measurements even for Ca2+ bound to polymerized protein. The binding curves for both skeletal and cardiac calsequestrin were complex, with binding increases that correlated with protein dimerization, tetramerization, and oligomerization. The Ca2+ binding capacities of skeletal and cardiac calsequestrin are directly compared for the first time, with ∼80 Ca2+ ions bound per skeletal calse-questrin and ∼60 Ca2+ ions per cardiac calsequestrin, as compared with net charges for these molecules of ∼80 and ∼69, respectively. Deleting the negatively charged and disordered C-terminal 27 amino acids of cardiac calsequestrin results in a 50% reduction of its calcium binding capacity and a loss of Ca2+-dependent tetramer formation. Based on the crystal structures of rabbit skeletal muscle calsequestrin and canine cardiac calsequestrin, Ca2+ binding capacity data, and previous light-scattering data, a mechanism of Ca2+ binding coupled with polymerization is proposed.