Myosin loop 2 is involved in the formation of a trimeric complex of twitchin, actin, and myosin.
Molluscan smooth muscles exhibit a low energy cost contraction called catch. Catch is regulated by twitchin phosphorylation and dephosphorylation. Recently, we found that the D2 fragment of twitchin containing the D2 site (Ser-4316) and flanking immunoglobulin motifs (TWD2-S) formed a heterotrimeric complex with myosin and with actin in the region that interacts with myosin loop 2 (Funabara, D., Hamamoto, C., Yamamoto, K., Inoue, A., Ueda, M., Osawa, R., Kanoh, S., Hartshorne, D. J., Suzuki, S., and Watabe, S. (2007) J. Exp. Biol. 210, 4399-4410). Here, we show that TWD2-S interacts directly with myosin loop 2 in a phosphorylation-sensitive manner. A synthesized peptide, CAQNKEAETTGTHKKRKSSA, based on the myosin loop 2 sequence (loop 2 peptide), competitively inhibited the formation of the trimeric complex. Isothermal titration calorimetry showed that TWD2-S binds to the loop 2 peptide with a K(a) of (2.44 +/- 0.09) x 10(5) m(-1) with two binding sites. The twitchin-binding peptide of actin, AGFAGDDAP, which also inhibited formation of the trimeric complex, bound to TWD2-S with a K(a) of (5.83 +/- 0.05) x 10(4) m(-1) with two binding sites. The affinity of TWD2-S to actin and myosin was slightly decreased with an increase of pH, but this effect could not account for the marked pH dependence of catch in permeabilized fibers. The complex formation also showed a moderate Ca(2+) sensitivity in that in the presence of Ca(2+) complex formation was reduced.
Graduate School of Bioresources, Mie University, Tsu, Mie 514-8507, Japan.
Daisuke
D
Funabara
Rika
R
Osawa
Miki
M
Ueda
Satoshi
S
Kanoh
David J
DJ
Hartshorne
Shugo
S
Watabe
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002151:Calorimetry; D006863:Hydrogen-Ion Concentration; D046912:Multiprotein Complexes / Q000378:metabolism; D009119:Muscle Contraction / Q000502:physiology; D009124:Muscle Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000502:physiology*; D009218:Myosins / Q000378:metabolism*; D049878:Mytilus / Q000378:metabolism*
D000199:Actins; D046912:Multiprotein Complexes; D009124:Muscle Proteins; D009218:Myosins; D002118:Calcium