Determinants of Ion Specificity on EF-hands Sites CONVERSION OF THE Ca/Mg SITE OF SMOOTH MUSCLE MYOSIN REGULATORY LIGHT CHAIN INTO A Ca-SPECIFIC SITE

Abstract Calcium binding proteins mediate a large number of cellular processes. These processes respond to micromolar fluctuations of cytosolic calcium in the presence of a large excess of magnesium. The metal binding sites present in these proteins are either calcium-specific (regulatory sites) or capable of binding both calcium and magnesium (structural sites). Using site-directed mutagenesis we were able to convert the single Ca/Mg site present in chicken smooth muscle myosin regulatory light chain (RLC) into a Ca-specific site. The replacement of the aspartic acid present in the 12th position (-Z coordinating position) of the metal binding loop with a glutamic acid increases calcium affinity and abolishes magnesium binding, rendering the site calcium-specific. To explain this observation, we hypothesize that restrictions on the ability of side chains to change conformation, contributing one (for Mg binding) or two (for Ca binding) coordinations could alter the metal specificity in EF-hands. Other mutations which decrease or abolish calcium binding have also been characterized. When used to substitute the endogenous scallop myosin RLC, these mutants were capable of restoring the Ca regulation to the actin-activated myosin ATPase demonstrating that in these hybrid myosins, the regulatory function of the Ca-specific site (present on the essential light chain) does not depend on the occupancy of the Ca/Mg site (present on the regulatory light chain).