DETERMINANTS OF ION SPECIFICITY ON EF-HANDS SITES - CONVERSION OF THE CA2+/MG2+ SITE OF SMOOTH-MUSCLE MYOSIN REGULATORY LIGHT-CHAIN INTO A CA2+-SPECIFIC SITE

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 Ca2+/Mg2+ site present in chicken smooth muscle myosin regulatory light chain (RLC) into a Ca2+-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 Mg2+ binding) or two (for Ca2+ 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 Ca2+ regulation to the actin-activated myosin ATPase demonstrating that in these hybrid myosins, the regulatory function of the Ca2+-specific site (present on the essential light chain) does not the occupancy of the Ca2+/Mg2+ site (present on the regulatory light chain).