Abstract
Various lines of evidence suggest that communication between tropomyosin and myosin in the regulation of vertebrate-striated muscle contraction involves yet unknown changes in actin conformation. Possible participation of loop 38-52 in this communication has recently been questioned based on unimpaired Ca(2+) regulation of myosin interaction, in the presence of the tropomyosin-troponin complex, with actin cleaved by subtilisin between Met(47) and Gly(48). We have compared the effects of actin cleavage by subtilisin and by protease ECP32, between Gly(42) and Val(43), on its interaction with myosin S1 in the presence and absence of tropomyosin or tropomyosin-troponin. Both individual modifications reduced activation of S1 ATPase by actin to a similar extent. The effect of ECP cleavage, but not of subtilisin cleavage, was partially reversed by stabilization of interprotomer contacts with phalloidin, indicating different pathways of signal transmission from the N- and C-terminal parts of loop 38-52 to myosin binding sites. ECP cleavage diminished the affinity to tropomyosin and reduced its inhibition of acto-S1 ATPase at low S1 concentrations, but increased the tropomyosin-mediated cooperative enhancement of the ATPase by S1 binding to actin. These effects were reversed by phalloidin. Subtilisin-cleaved actin more closely resembled unmodified actin than the ECP-modified actin. Limited proteolysis of the modified and unmodified F-actins revealed an allosteric effect of ECP cleavage on the conformation of the actin subdomain 4 region that is presumably involved in tropomyosin binding. Our results point to a possible role of the N-terminal part of loop 38-52 of actin in communication between tropomyosin and myosin through changes in actin structure.
Highlights
It is generally accepted that the regulation of vertebrate striated muscle contraction involves cooperative and allosteric interactions between actin, myosin, tropomyosin (Tm),1 and troponin (Tn)
One should emphasize that states analogous to the blocked, closed, and open state are present in the Hill model as substates of the two major states, but the incapability of thin filament to bind subfragment 1 (S1) in the absence of Ca2ϩ postulated in the McKillop and Geeves model is a matter of dispute (10 –13)
We have previously shown that proteolytic cleavage of the DNase-I binding loop of actin between Gly42 and Val43 with protease ECP32 from Escherichia coli A2 strain has a profound effect on the interprotomer interactions in F-actin, whereas subtilisin cleavage between Met47 and Gly48 has not [36]
Summary
It is generally accepted that the regulation of vertebrate striated muscle contraction involves cooperative and allosteric interactions between actin, myosin, tropomyosin (Tm), and troponin (Tn). One should emphasize that states analogous to the blocked, closed, and open state are present in the Hill model as substates of the two major states, but the incapability of thin filament to bind S1 in the absence of Ca2ϩ postulated in the McKillop and Geeves model is a matter of dispute (10 –13) Both models assume that the cooperativity of Tm and S1 binding to actin depend on the strength of end-to-end interactions between adjacent Tm molecules. A most recent study of effects of the regulatory proteins on the affinity for myosin S1 and in vitro motility of subtilisin-cleaved actin has led to the conclusion that subdomain 2 conformation does not play an active role in the regulation of actomyosin interactions [35]. A preliminary report of this work has been presented [37]
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