The DNase-I Binding Loop of Actin May Play a Role in the Regulation of Actin-Myosin Interaction by Tropomyosin/Troponin

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 Ca2+ regulation of myosin interaction, in the presence of the tropomyosin-troponin complex, with actin cleaved by subtilisin between Met47 and Gly48. We have compared the effects of actin cleavage by subtilisin and by protease ECP32, between Gly42 and Val43, 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. 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 Ca2+ regulation of myosin interaction, in the presence of the tropomyosin-troponin complex, with actin cleaved by subtilisin between Met47 and Gly48. We have compared the effects of actin cleavage by subtilisin and by protease ECP32, between Gly42 and Val43, 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. It is now generally accepted that the regulation of vertebrate striated muscle contraction involves cooperative and allosteric interactions between actin, myosin, tropomyosin (Tm), 1The abbreviations used are: Tm, tropomyosin; Tn, troponin; S1, myosin subfragment 1; ECP, proteinase ECP32 from E. coli A2 strain; MOPS, 4-morpholinepropanesulfonic acid. 1The abbreviations used are: Tm, tropomyosin; Tn, troponin; S1, myosin subfragment 1; ECP, proteinase ECP32 from E. coli A2 strain; MOPS, 4-morpholinepropanesulfonic acid. and troponin (Tn). Positive cooperativity of actin-myosin interactions in the presence of regulatory proteins is indicated by sigmoidal profiles of both the binding of the myosin heads to the thin filament (1Greene L.E. Eisenberg E. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2616-2620Google Scholar) and the actin-activated ATPase plotted as a function of myosin concentration (2Lehrer S.S. Morris E.P. J. Biol. Chem. 1982; 257: 8073-8080Google Scholar). This behavior, for which the binding of Tm to actin is a necessary and sufficient requirement, has led to a modification of the classic steric blocking hypothesis of the regulation of striated muscle contraction. Based on equilibrium binding studies in solution, Hill et al. (3Hill T.L. Eisenberg E. Greene L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3186-3190Google Scholar) suggested that seven actin monomers together with one molecule of Tm and one of Tn form a functional unit that exists in equilibrium between two states, inactivated and activated; the latter having higher binding affinity for Ca2+ and for myosin or its subfragment 1 (S1). Subsequent kinetic studies have led McKillop and Geeves to a three-state model (4McKillop D.F.A. Geeves M.A. Biophys. J. 1993; 65: 693-701Google Scholar) in which the kinetics of S1 binding to the regulated actin filament is closely coupled to the two-step mechanism of S1 binding to actin alone (5Trybus K.M. Taylor E.W. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7209-7213Google Scholar, 6Geeves M.A. Goody R.S. Gutfreund H. J. Muscle Res. Cell Motil. 1984; 5: 351-361Google Scholar). In this model, removal of calcium converts the actin-Tm-Tn units to a state unable to bind S1 (blocked state). Actin-Tm as well as actin-Tm-Tn with Ca2+ bound to troponin are in a state that permits weak binding of S1 (closed state, no acceleration of ATPase rate) in equilibrium with a third, fully active state (open state) that allows isomerization of the weakly bound S1 to the strongly bound, rigor-like state. The ratio of open/closed thin filament cooperative units depends on the fraction of actin sites occupied by S1, because occupancy of the actin filament with strongly bound S1 stabilizes the open state. These three biochemical states can be related to three distinct positions of the Tm-Tn complex on the actin filament (7Holmes K.C. Biophys. J. 1995; 68: 2s-7sGoogle Scholar, 8Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Google Scholar, 9Craig R. Lehman W. J. Mol. Biol. 2001; 311: 1027-1036Google Scholar). One should emphasize that states analogous to the blocked, closed, and open state are also 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 Ca2+ postulated in the McKillop and Geeves model is a matter of dispute (10Chen Y.-D. Yan B. Chalovich J.M. Brenner B. Biophys. J. 2001; 80: 2338-2349Google Scholar, 11Geeves M.A. S.S. Biophys. J. Scholar, J.M. Yan B. Brenner B. Y.-D. Biophys. J. Scholar, J.M. Chalovich J.M. Biophys. J. Scholar). Both that the cooperativity of Tm and S1 binding to actin on the of interactions between Tm contacts between Tm are also the in the more that to the role of of Tm in of the cooperative unit S.S. Geeves M.A. 1997; Scholar, R. S.S. Geeves M.A. Scholar, R. Geeves M.A. Biophys. J. Scholar). Tm with interactions bind to actin, that the cooperativity involves changes the actin R. Geeves M.A. Biophys. J. Scholar, R. E. W. J. Muscle Res. Cell Motil. Scholar, J. Biol. Chem. Scholar, Hill L.E. J. Biol. Chem. Scholar, J. Biol. Chem. 1993; Scholar, J. Scholar). and in actin has been in the more of cooperative myosin binding and activation of muscle contraction J. Biol. Chem. Scholar, J. E. J. Biol. Chem. 2001; Scholar). lines of evidence point to a possible role of subdomain of actin in transmission of cooperative changes between actin in thin This and in its binding loop are the of the actin filament structure. strongly the interprotomer interactions the filament A. Biol. 1995; 5: Scholar, E.P. Proc. Natl. Acad. Sci. U. S. A. with participation of loop 38–52 in interactions K.C. W. W. and E. W. E. J. Mol. Biol. and of actin myosin in the in by interprotomer of actin between revealed the of of the subdomain 1 for of and with myosin E. E. A. A. E. Scholar, E. E. A. E. Scholar). In of actin is also by subtilisin cleavage loop 38–52 E. J. Cell Biol. Scholar). subdomain of actin were to the affinity of this actin for both Tm and myosin S1 and thin filament regulation by Ca2+ Tm in blocking the myosin binding sites in subdomain 1 Craig R. Lehman W. J. Biol. Chem. Scholar). of revealed a of loop 38–52 on Tm binding to actin K.C. J. Mol. Biol. 1995; and possible of subdomain actin to changes in from muscle in to Ca2+ activation of thin filament has been J.M. Morris E.P. J. Scholar). the studies on both muscle actin and actin with on or indicated that the blocked, closed, and open regulatory states no subdomain E. A. E. J. Biol. Chem. 2001; Scholar). of effects of the regulatory proteins on the affinity for myosin S1 and in of actin has also led to the that subdomain conformation not an active role in the regulation of interactions E. E. J. Biol. Chem. Scholar). We have that cleavage of the binding loop of actin between Gly42 and with protease ECP32 from coli A2 has a effect on the interprotomer interactions in subtilisin cleavage between Met47 and has not H. Biophys. J. Scholar). This to the effects of modifications on the cooperativity of myosin The results of this the that modification of the C-terminal of loop 38–52 by subtilisin cleavage not the regulation of actin-myosin In modification of the N-terminal part of this loop changes in Tm binding to actin, its effect on acto-S1 ATPase at low S1 concentrations, and the of the ATPase by the S1 binding. This is the that changes at the of the binding loop a role in the regulation of muscle contraction. of this has been J. J. H. Biophys. J. Scholar). protease ECP32 from E. coli A2 was by A. of from MOPS, were from was from was from and was from was from and to S. J. Biol. Chem. It was in the form of in and of actin with at was as J. H. J. 1993; Scholar) with an of of on a cleaved between and with subtilisin was as by et al. E. Scholar) that the was at actin of at an to actin ratio of The cleaved actin was to one and was in for to cleaved between and was by a cleavage with partially ECP32 at Scholar). of the of the of were for of the by the of actin cleavage with the of The cleaved actin was used The were form by with at and with from muscle and its S1 were as in S.S. S. 1982; S1 and was by on Scholar). of S1 were by at for muscle and troponin were as in 1982; and 1982; Scholar, of Tm with at was as by and S.S. Scholar). The ratio of to Tm in used this was in the of indicating that a fraction of the was of was by to The of proteins were by the and at and for at and for S1, at and for Tm, and at and for The concentration of Tm was by the of Scholar). of for of the of the enhancement of actin by both ECP and subtilisin cleavage J. H. J. 1993; Scholar, E. was necessary to the for the of of the actin and were and with for The were with a of and in a with tropomyosin at a ratio to actin. the were with the to actin in the from 1 to and the of was and at of the in the between the and to the actin the were to be for and 4 for actin both in the presence and absence of ATPase of S1 and acto-S1 was in and or at The was by to concentration and with The was to J. Biol. Chem. and were in a in the ratio of were at and the was at at of was at at of at was at the of F-actins by Limited with by of with were with subtilisin at an ratio of at different of the were and the was with The were by on to The of actin were from with of of on changes in binding by modifications of actin not be by of the binding of to as because the of results in of its by J. H. J. 1993; Scholar). It is a to from of in that low concentrations, by the in the presence of phalloidin, a to of actin and This is to actin as be binding changes not in the filament but also in of modified to the effects of the modifications of actin on interaction with Tm, to changes in the of in both and the of actin to the of at and and the of at of to of Tm S.S. Scholar). of with F-actins that the in the of Tm was similar for the three as in of the with actin higher concentration than the with actin that was at of Tm, indicating that cleavage at Gly42 Tm to binding. The for actin closely that for of Tm to by a in the of 1 and unmodified or at the indicated on the from one of are The of and F-actins are for the presence of the with an that Tm solution, with Tm in actin, is from by Tm binding. The of actin was at at at The in the absence of actin and at with were to and to concentration of as in the The the were the Hill that the of actin to Tm binding is and the is to the fraction of of bound Tm the at to Tm ratio can be as a between concentration of and bound from the the of the concentration of the were by the to a Hill in the form in fraction of actin binding to Tm of actin binding sites on Tm and is the Hill The for actin was with and of The of for actin from from is an because the binding of the two proteins of the this is in with the in different R. E. W. J. Muscle Res. Cell Motil. Scholar, J. Biol. Chem. Scholar, Eisenberg E. J. Biol. Chem. Scholar, J. Biol. Chem. Scholar, J. Biol. Chem. Scholar). It is to be that the cooperativity of Tm to actin actin is in is not Hill to or to in of in by ATPase S1 concentration of actin-activated S1 ATPase at a The enhancement of actin concentration for by subtilisin and ECP cleavage and was for by the concentration of the modified One can that at the low and S1 used in from the of acto-S1 ATPase on in the absence of regulatory proteins was with both and the modified In with an enhancement of for this subtilisin cleavage of actin E. activation of S1 by actin was an of to actin. similar in actin-activated ATPase was by ECP of the two modifications of actin on the S1 concentration of acto-S1 ATPase in the presence of the regulatory proteins were cleavage no on the of changes in the ATPase with S1 ECP-modified actin, the from inhibition of the ATPase to that with alone at low S1 to activation at S1 S.S. Morris E.P. J. Biol. Chem. 1982; 257: 8073-8080Google and at than with and actin. This not be by possible in the concentration of ECP-modified with S1 from of the present in the that were the a of the from the on be also with actin no activation of S1 ATPase by actin was at its 4 the concentration for of this actin of the in cooperativity of the of ECP-modified actin to of or of on changes in the of acto-S1 ATPase for actin. The of myosin at the indicated on the were in the presence of actin at the concentration of alone or with Tm that been in the absence or in the presence of of are in the to The ATPase of S1 alone has been from In the inhibition of acto-S1 ATPase by Tm at low in a and low S1 to actin ratio of was as a function of Tm In with Eisenberg E. Scholar, L.E. Eisenberg E. 1984; with actin the at the ratio of one Tm seven actin to of the of results were with actin. actin, the effect of Tm was reduced to of the ATPase with actin alone not The cooperativity of actin-myosin interaction is also in the higher of the regulation of ECP-modified by Ca2+ in the presence of Tm-Tn at S1 the concentration of actin. in at S1, the of regulation as in was to be for actin, for actin, and for actin for The to the for that the between and actin is that between and actin is not of on the of with Tm and with 4 the effects of phalloidin, at a actin, on the actin-activated ATPase of S1 as a function of S1 concentration at a concentration of actin. The were the as in for actin, which was used in two different 4 to the for this actin of and which in the absence of should 4 the concentration to that in with and actin. was actin at the concentration of 4 not the S1 ATPase was the effects of on modified actin were than be from enhancement by this of the 4 the acto-S1 ATPase were higher than with actin in the absence of with 4 with and of actin in a enhancement of acto-S1 ATPase effect of was with or actin. the effects of Tm on activation of S1 ATPase by actin in the presence and absence of phalloidin. One can that the from an inhibition to of the ATPase with of S1, in the absence of phalloidin, an inhibition in the of S1 used actin was with phalloidin. It is that binding the effects of ECP cleavage of actin on its interaction with myosin in both the absence and presence of in the effects of on and on actin were to than the inhibition by Tm of acto-S1 ATPase at low S1 to actin and to the S1 concentration for the from inhibition to These results for in the of on that has been used in studies on actin-myosin The effects of on changes in the interaction of actin with S1 to possible of this on the interaction of modified actin with This was by changes in the of with actin at a Tm to actin In with in at this ratio of the two the in the of of was fully with and with the cleaved actin. one can in of a to Tm with actin in a of the to its in the Tm complex with actin. to the latter complex not These that the changes in interaction by ECP cleavage of actin are reversed by phalloidin. of S1 to by in the of studies on regulated actin have led to a that the in the of Tm with a in the between Tm and actin that is with a cooperative from the to open state of the regulated actin of S1 has been to a in the and a in the of the S.S. Scholar). effects were with Tm bound to the modified Our S1 of with and in the absence of a in the by a of and from In with an S.S. of the profiles with actin with changes in S1 binding to actin that the three were bound to seven actin the modified the profiles were to in the actin actin actin. of effects of actin modifications was possible because of with of the changes S1 of the modified in of actin that has to be in a Tm to the of of the changes in on of complex with S1 S1 binding to both and to actin, which is in a of the of at S1 the concentration of the with actin is the of its the of this because of the of from the filament H. Biophys. J. Scholar). In the absence of the of this actin and which is as a of in the of the was also S1 of actin. This effect to the diminished as compared with unmodified actin. the the of actin are by Tm and this effect is cooperative H. Biophys. J. Scholar). Tm is to S1 affinity to actin. One can a binding of S1 to the or with the of the of the to the modifications of actin can be as an of increased cooperativity of the in the state of Tm on the of to a of actin. of the of the has been that cleavage of the binding loop with ECP to with at two sites in subdomain and that are cleaved in but in modified by subtilisin cleavage of the binding loop more closely resembled in its to H. Biophys. J. Scholar). used with subtilisin to effects of the modifications of the binding can be in in unmodified the major of subtilisin cleavage between Met47 and a C-terminal is strongly a of the and in subdomain 4 A. A. E. 1995; Scholar, H. A. U. J. Scholar) results in of the the N-terminal and C-terminal The is a of subtilisin cleavage between and H. A. U. J. Scholar). of that been cleaved between Met47 and a of the N-terminal changes in the with to unmodified were and the of of the were also This with the of ECP-modified of as from the of the and and acceleration of the cleavage between and the C-terminal and its H. A. U. J. Scholar). These not be by the concentration of ECP-modified actin because at subtilisin used in this is to The of monomers with the of is to of and this to for the in of the of cleavage at sites as can be in These results the of to and that modification of the binding loop by its cleavage with ECP to changes in structure. the of S1 to the model of the and biochemical studies have led to a that the myosin binding sites in actin are in its subdomain 1 and at the of 1 and K.C. 1993; Scholar, P. P. E. Biophys. J. 1995; 68: Scholar). of of the binding loop in the weak binding of myosin, suggested by the of the acto-S1 K.C. 1993; the changes in the of a to of actin and in the interaction of this actin with R. Scholar, E. A. E. Biophys. J. Scholar). the of S1 interaction with actin to binding has led to of in of allosteric effects of S1 binding to the sites in actin E. A. E. Biophys. J. Scholar). for signal transmission in the from loop 38–52 to subdomain has been by studies on A. E. Biophys. J. Scholar, S. Scholar). or between loop 38–52 and myosin binding sites in subdomain 1 of actin is also a for the of acto-S1 ATPase this loop is by subtilisin or ECP of communication between the binding loop and myosin binding sites is with the that activation of myosin ATPase actin in its with a from that this loop with 1 and of an the K.C. W. W. Scholar, K.C. J. Mol. Biol. 1993; Scholar, K.C. Biophys. J. 1995; 68: and with biochemical evidence for its interaction with the in subdomain 1 of actin E. W. E. J. Mol. Biol. Scholar, H. A. J. H. J. 1995; Scholar, E. E. Biophys. J. Scholar). the enhancement of the of in with a effect of subtilisin cleavage H. Biophys. J. for participation of the N-terminal part of the binding ECP cleavage but not subtilisin cleavage in the interactions as in the model of et al. K.C. W. W. Scholar). One can that the effect of subtilisin cleavage is to an through its on the conformation of the N-terminal part of the This is to with the in the absence of regulatory the effect of ECP cleavage on acto-S1 ATPase is reduced by binding to actin, the inhibition from subtilisin cleavage is The effect of on the activation of S1 by actin is with an of communication between the N-terminal of loop 38–52 and myosin binding sites because this is to this loop and its contacts with an the filament K.C. J. Mol. Biol. 1993; Scholar). by of interactions in ECP-modified has also been H. Biophys. J. Scholar). the with by H. Biophys. J. Scholar) and subtilisin as a has been that ECP cleavage to a in the conformation of of subdomain in This effect is reversed by binding to the modified H. Biophys. J. Scholar). These point to a possible role of in an of communication between the binding loop and myosin binding sites in subdomain of the of the and form of actin a of the loop to the in conformation of the binding loop by of the from the of its interaction with in subdomain 1 P. R. J. Biol. Chem. Scholar). of that of with the of actin to bind H. J. more evidence for between this of and 38–52 loop on the of subdomain In to cleavage with ECP, the effect of subtilisin cleavage on the conformation of in is H. Biophys. J. and this the that the N- and C-terminal parts of the binding loop with the sites of myosin binding in different It has recently been interaction with myosin in the absence of regulatory actin not from actin in the Ca2+ regulation and the of of its in in the presence of Tm-Tn E. E. J. Biol. Chem. Scholar). The that the of modifications of the actin loop 38–52 for the regulation of actin-myosin interaction on the of the cleavage the between and reduced the effect of Tm Tm-Tn in the absence of on acto-S1 ATPase at low S1 concentrations, and the of the ATPase by S1 binding to actin. The of the effect of Tm to be related to its diminished affinity to ECP-modified because of this actin in a of both the Tm binding of actin and of the effect of Tm on acto-S1 ATPase at low S1 In actin cleaved with subtilisin between and more closely resembled unmodified actin than the ECP-modified actin. These results suggest that the contacts of the N-terminal part of loop which are by ECP cleavage but by subtilisin cleavage H. Biophys. J. a role in both and between different functional states of the thin of of and revealed changes in actin a in of the loop to of a from to in subdomain 4 of actin, and of the binding loop K.C. J. Mol. Biol. 1995; Scholar). It is that changes in the of are The N-terminal of the binding loop and to with the of actin, form a between three K.C. W. W. Scholar, E. W. E. J. Mol. Biol. Scholar, E. E. Biophys. Chem. Scholar). between the N-terminal of the binding loop and the to has been by an of cleavage sites in ECP-modified This be for the regulation of actin-myosin interaction because biochemical R. P. J. Biol. Chem. 1984; K.C. J. Mol. Biol. 1995; and studies Scholar) that and the loop in Tm binding in the state of the thin One can that a of by modification of the N-terminal part of loop the of the Tm the which is for binding of the myosin heads (7Holmes K.C. Biophys. J. 1995; 68: 2s-7sGoogle Scholar, 8Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Google Scholar, 9Craig R. Lehman W. J. Mol. Biol. 2001; 311: 1027-1036Google Scholar). This is one of possible of the of the effect of Tm on acto-S1 ATPase with actin. transmission in the from Tm binding in subdomain 4 to the binding loop through to myosin binding sites in subdomain 1 to the cooperative enhancement of was H. Biophys. J. Tm binding the in indicating stabilization of the interprotomer contacts that are by this and this effect of Tm is These are in with the that changes in the interprotomer interactions of the binding loop to the regulation of the studies on actin indicated that between the three regulatory states of the thin filament no of loop 38–52 E. A. E. J. Biol. Chem. 2001; Scholar). It not to the of changes at the of this loop through are to Tm and myosin binding sites. In this on the from the to open state of the thin Our of the actin-activated ATPase of S1 in the presence of the Tm-Tn complex an that the changes in the equilibrium between the and state also be by the conformation of the N-terminal of actin loop more of this of thin filament regulation be in We of for of protease The of is also