Ca2+ binding to myosin regulatory light chain affects the conformation of the N-terminus of essential light chain and its binding to actin.
We prepared a new type of skeletal myosin subfragment 1 (S1-MLC1F) containing both, the essential and the regulatory light chains, intact, by exchanging the essential light chains of papain S1 with bacterially expressed longer isoform (MLC1F) of this light chain. We then compared the enzymatic and structural properties of chymotryptic S1, papain S1, and S1-MLC1F in the presence and in the absence of Ca(2+) ions bound to the regulatory light chain. In the presence of Ca(2+), subfragment 1 containing both intact light chains exhibited lower V(max) and lower K(m) for actin activation of S1 ATPase. When S1-MLC1F was cross-linked to actin via the N-terminus of the essential light chain, the yield was much higher when Ca(2+) ions saturated the regulatory light chain. Limited proteolysis of the essential light chain in S1-MLC1F was significantly inhibited in the presence of calcium as compared to chymotryptic S1. We conclude that the effect of binding of Ca(2+) to the regulatory light chain is transmitted to the N-terminal extension of the longer isoform of the essential light chain. The resulting structure of the N-terminus is less susceptible to proteolytic digestion, binds tighter to actin, and has an inhibitory effect on actin-activated myosin ATPase. This new conformation of the N-terminus may be responsible for calcium induced myosin-linked modulation of striated muscle contraction.
Nieznanski
Krzysztof
K
Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Nieznanska
Hanna
H
Skowronek
Krzysztof
K
Kasprzak
Andrzej A
AA
Stepkowski
Dariusz
D
D000199:Actins / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D004789:Enzyme Activation; D046911:Macromolecular Substances; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D018994:Myosin Light Chains / Q000737:chemistry*; D015879:Myosin Subfragments / Q000737:chemistry* / Q000145:classification; D010206:Papain / Q000737:chemistry; D011485:Protein Binding; D051381:Rats
D000199:Actins; D046911:Macromolecular Substances; D018994:Myosin Light Chains; D015879:Myosin Subfragments; D010206:Papain; D002118:Calcium