Determinants of ion specificity on EF-hands sites. Conversion of the Ca2+/Mg2+ site of smooth muscle myosin regulatory light chain into a Ca(2+)-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 Ca(2+)-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 Ca(2+)-specific site (present on the essential light chain) does not depend on the occupancy of the Ca2+/Mg2+ site (present on the regulatory light chain).
da Silva
A C
AC
Departamento de Bioquimica, Universidade de São Paulo, Brazil.
Kendrick-Jones
J
J
Reinach
F C
FC
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship
D000251:Adenosine Triphosphatases; D009218:Myosins; D008274:Magnesium; D002118:Calcium