Chimaeric myosin regulatory light chains: sub-domain switching experiments to analyse the function of the N-terminal EF hand.
The regulatory light chains (RLCs) located on the myosin head, regulate the interaction of myosin with actin in response to either Ca2+ or phosphorylation signals. The RLCs belong to a family of calcium binding proteins and are composed of four "EF hand" ancestral calcium binding motifs (numbered I to IV). To determine the role of the first EF hand (EF hand I) in the regulatory process, chimaeric light chains were constructed by protein engineering, by switching this region between smooth muscle and skeletal muscle myosin RLCs. For example, chimaera G(I)S consisted of EF hand I of the smooth muscle (gizzard) RLC and EF hands II to IV of the skeletal muscle RLC, whereas chimaera S(I)G consisted of EF hand I of the skeletal muscle RLC and EF hands II to IV of the smooth muscle RLC. The chimaeric RLCs were expressed in Escherichia coli using the pLcII expression system, and after isolation and purification their regulatory properties were compared with those of wild-type smooth and skeletal muscle myosin RLCs. The chimaeric RLCs bound to the myosin heads in scallop striated muscle myofibrils from which the endogenous RLCs had been removed ("desensitized" myofibrils) with similar affinities to those of the wild-type smooth and skeletal muscle RLCs. Both chimaeric RLCs were able to regulate the actin-activated Mg(2+)-ATPase activity of scallop myosin: G(I)S inhibited the ATPase in the presence and absence of Ca2+, like the wild-type skeletal muscle RLC, while S(I)G inhibited the myosin ATPase in the absence of Ca2+, and this inhibition was relieved on Ca2+ addition, in the same way as the wild-type smooth muscle RLC. Thus the type of regulation that the RLCs confer on the myosin is determined by the source of EF hands II to IV rather than that of EF hand I.
Messer
N
N
MRC Laboratory of Molecular Biology, Cambridge, U.K.
Kendrick-Jones
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002645:Chickens; D002678:Chimera; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D015870:Gene Expression / Q000187:drug effects; D005895:Gizzard, Avian / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D012758:Shellfish
D004247:DNA; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium