Regulation of scallop myosin by the regulatory light chain depends on a single glycine residue.
Specific Ca2+ binding and Ca2+ activation of ATPase activity in scallop myosin require a regulatory light chain (RLC) from regulated (molluscan or vertebrate smooth) myosin; hybrids containing vertebrate skeletal RLCs do not bind Ca2+ and their ATPase activity is inhibited. Chimeras between scallop and chicken skeletal RLCs restore Ca2+ sensitivity to RLC-free myosin provided that residues 81-117 are derived from scallop. Six mutants (R90M, A94K, D98P, N105K, M116Q, and G117C) were generated by replacing amino acids of the scallop RLC with the corresponding skeletal RLC residues in positions conserved in either regulated or nonregulated myosins. Ca2+ binding was abolished by a G117C and a G117A mutation; however, these mutants have a decreased affinity for the heavy chain. None of the other mutations affected RLC function. Replacement of the respective cysteine with glycine in the skeletal RLC has markedly changed the regulatory properties of the molecule. The single cysteine to glycine mutation conferred to this light chain the ability to restore Ca2+ binding and regulated ATPase activity, although Ca2+ activation of the actin-activated ATPase was lower than with scallop RLC. The presence of amino acids other than glycine at this position in vertebrate skeletal myosin RLCs may explain why these are not fully functional in the scallop system. The results are in agreement with x-ray crystallography data showing the central role of G117 in stabilizing the Ca(2+)-binding site of scallop myosin.
Jancso
A
A
Brandeis University, Department of Biology, Waltham, MA 02254.
Szent-Györgyi
A G
AG
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D005998:Glycine; D008969:Molecular Sequence Data; D008974:Mollusca; D009119:Muscle Contraction; D009218:Myosins / Q000737:chemistry* / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D000199:Actins; D011993:Recombinant Fusion Proteins; D009218:Myosins; D002118:Calcium; D005998:Glycine