The Use of Site-Directed Mutagenesis to Identify Functional Sites in the Ca2+ ATPase

Our long term research objective has been to understand the mechanism of Ca2+ transport by the Ca2+ ATPase of the sarcoplasmic reticulum and our approach to the solution of this problem has been to investigate structure/function relationships in the enzyme. In recent years, we have used the powerful techniques of cloning, structural prediction and site-directed mutagenesis to gain insight into the roles of specific amino acids in the structure, function and assembly of the protein. These studies have allowed us to evaluate the functional roles of major regions of the Ca2+ ATPase and to define amino acids that are specifically involved in ATP-dependent Ca2+ translocation. We have been able to show that residues in the stalk sector are not involved in Ca2+ binding, but may be involved in regulation of Ca2+ transport, that specific residues within the transmembrane sector are involved with Ca2+ binding and Ca2+ translocation, that residues in the nucleotide binding and hinge domains are involved in ATP binding and in determination of apparent Ca2+ affinity, and that residues throughout the molecule are involved in conformational changes. From these data, we have developed a simple and easily understood hypothesis for the mechanism of ATP-dependent Ca2+ transport.