Maximal Activation of Skeletal Muscle Thin Filaments Requires Both Rigor Myosin S1 and Calcium

The regulation by calcium and rigor-bound myosin-S1 of the rate of acceleration of 2′-deoxy-3′-O-(N-methylanthraniloyl)ADP (mdADP) release from myosin-mdADP-Pi by skeletal muscle thin filaments (reconstituted from actin-tropomyosin-troponin) was measured using double mixing stopped-flow fluorescence with the nucleotide substrate 2′-deoxy-3′-O-(N-methylanthraniloyl). The predominant mechanism of regulation is the acceleration of product dissociation by a factor of ∼200 by thin filaments in the fully activated conformation (bound calcium and rigor S1) relative to the inhibited conformation (no bound calcium or rigor S1). In contrast, only 2–3-fold regulation is due to a change in actin affinity such as would be expected by “steric blocking” of the myosin binding site of the thin filament by tropomyosin. The binding of one ligand (either calcium or rigor-S1) produces partial activation of the rate of product dissociation, but the binding of both is required to maximally accelerate product dissociation to a rate similar to that obtained with F-actin in the absence of regulatory proteins. The data support an allosteric regulation model in which the binding of either calcium or rigor S1 alone to the thin filament shifts the equilibrium in favor of the active conformation, but full activation requires binding of both ligands. The regulation by calcium and rigor-bound myosin-S1 of the rate of acceleration of 2′-deoxy-3′-O-(N-methylanthraniloyl)ADP (mdADP) release from myosin-mdADP-Pi by skeletal muscle thin filaments (reconstituted from actin-tropomyosin-troponin) was measured using double mixing stopped-flow fluorescence with the nucleotide substrate 2′-deoxy-3′-O-(N-methylanthraniloyl). The predominant mechanism of regulation is the acceleration of product dissociation by a factor of ∼200 by thin filaments in the fully activated conformation (bound calcium and rigor S1) relative to the inhibited conformation (no bound calcium or rigor S1). In contrast, only 2–3-fold regulation is due to a change in actin affinity such as would be expected by “steric blocking” of the myosin binding site of the thin filament by tropomyosin. The binding of one ligand (either calcium or rigor-S1) produces partial activation of the rate of product dissociation, but the binding of both is required to maximally accelerate product dissociation to a rate similar to that obtained with F-actin in the absence of regulatory proteins. The data support an allosteric regulation model in which the binding of either calcium or rigor S1 alone to the thin filament shifts the equilibrium in favor of the active conformation, but full activation requires binding of both ligands. The striated muscle thin filament, a complex of five proteins (actin, tropomyosin, and troponins I, C, and T), contains an adaptable network of interactions that responds to the association with ligands (calcium and myosin) that regulate thin filament activation of myosin ATP hydrolysis. Atomic resolution structures containing the core domains of the troponin complex in the calcium-activated state and the calcium-free state have recently been reported (1Takeda S. Yamashita A. Maeda K. Maeda Y. Nature. 2003; 424: 35-41Crossref PubMed Scopus (642) Google Scholar, 2Vinogradova M.V. Stone D.B. Malanina G.G. Karatzaferi C. Cooke R. Mendelson R.A. Fletterick R.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5038-5043Crossref PubMed Scopus (261) Google Scholar), but there is no high resolution structural information on ligand-induced changes that occur within a complete thin filament. Various schemes have been formulated to account for regulation. Steric blocking in its original form (3Huxley H.E. Cold Spring Harbor Symp. Quant. Biol. 1972; 37: 361-376Crossref Google Scholar) evoked a strict competition in which tropomyosin-troponin prevented myosin binding to actin in the absence of calcium. However, steric blocking was inconsistent with subsequent observations that the effect of calcium upon the affinity of myosin for actin during steady state ATP hydrolysis is small (4Chalovich J.M. Eisenberg E. J. Biol. Chem. 1982; 257: 2432-2437Abstract Full Text PDF PubMed Google Scholar). This problem was addressed by the three-state model (5McKillop D.F.A Geeves M.A. Biophys. J. 1993; 65: 693-701Abstract Full Text PDF PubMed Scopus (663) Google Scholar), in which the thin filament can populate three conformational states with regard to myosin binding, and the modified Hill model (6Chen Y. Yan B. Chalovich J.M. Brenner B. Biophys. J. 2001; 80: 2338-2349Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), in which the thin filament primarily exists in two states. The three-state model includes a blocked state in which the myosin binding site of the thin filament is completely occluded. In the closed state the thin filament can form an initial weakly bound actomyosin complex, but the binding cannot proceed to a tightly bound rigor myosin complex. In the open state the thin filament can bind myosin tightly. An integral feature of this model is that the binding of rigor myosin locks the thin filament in the open state, which fully activates product dissociation whether or not calcium is bound to the thin filament. By analogy it was proposed that myosin-ADP-Pi bound similarly to open and closed states, but acceleration of product dissociation only occurred if the thin filament was in the open conformation. Both the modified Hill and Geeves models (5McKillop D.F.A Geeves M.A. Biophys. J. 1993; 65: 693-701Abstract Full Text PDF PubMed Scopus (663) Google Scholar,6Chen Y. Yan B. Chalovich J.M. Brenner B. Biophys. J. 2001; 80: 2338-2349Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) are based upon data in which the active site of myosin was either empty or contained nonhydrolyzable ligands such as ADP, pyrophosphate, and AMP-PNP. 2The abbreviations used are: AMP-PNPadenosine 5′-(β,γ-imino)triphosphateS1myosin subfragment 1A1the 21-kDa alkali light chain of myosin-S1M-ADP-Pimyosin ADP-inorganic phosphatemant2′,3′-O-(N-methylanthraniloyl)mdATP2′-deoxy-3′-O-(N-methylanthraniloyl)ATPmdADP2′-deoxy-3′-O-(N-methylanthraniloyl)ADPMOPS3-[N-morpholino]propane-sulfonic acidTmtropomyosinTntroponinAMactomyosin. However, these myosin-ligand complexes promote an open switch II conformation of myosin (compared with the closed switch II conformation of M-ADP-Pi) (7Xu S. Offer G. Gu J. White H.D. Yu L.C. Biochemistry. 2003; 42: 390-401Crossref PubMed Scopus (58) Google Scholar, 8Kovacs A. R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. K. Biochemistry. PubMed Scopus Google Scholar) and change the mechanism of myosin binding to this are which that skeletal myosin with ligands that an open switch II conformation not a upon binding to actin J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). that the regulation of the binding of myosin to thin filaments in the open conformation not an of the mechanism of the regulation of thin filament product dissociation from myosin subfragment the 21-kDa alkali light chain of myosin-S1 myosin ADP-inorganic 2′-deoxy-3′-O-(N-methylanthraniloyl)ADP troponin The are to the problem of thin regulation is a fully the mechanism of thin filament it is to the effect of ligand on of the actomyosin hydrolysis an Biochemistry. PubMed Scopus Google Scholar) in with a stopped-flow to the of the rate of acceleration of release by skeletal thin filaments containing one (either calcium or rigor or both bound ligands B. White H.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In these the produces a of and myosin-ADP-Pi in a that is by with thin filaments in the B. White H.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of this with a mechanism in which dissociation a structural that is the by calcium and rigor myosin binding to skeletal thin filaments but a model for the regulation of the filament was not have measured the regulation of the acceleration of the dissociation of the from by calcium and rigor myosin binding to the thin filament and a model for regulation in which both calcium and rigor myosin-S1 are required to fully thin filament acceleration of product are of an that to the of product dissociation that are dissociation using binding In an of the ATP is bound to myosin the of the of the have This is a in is and ATP binding is but of ATP are in and ATP binding is with not completely the of product dissociation be by ATP and are that myosin-S1 the can bind to and the thin filament, and ATP bind to and be to an the effect of calcium binding in the absence of rigor it to that would the of myosin and the of the In the in this have used which the of myosin the of the the was prevented from binding to actomyosin a the by a of with the thin a of product rigor myosin that by product dissociation would be by the is that product dissociation from is as in and the dissociation the rate of The fluorescence of and is bound to the active site of skeletal myosin and is the for and J.M. Biochemistry. PubMed Scopus Google Scholar, Geeves M.A. Biochemistry. PubMed Scopus Google Scholar). The rate of dissociation from upon to the of the of the nucleotide and it was expected that the fluorescence change by the dissociation of from would a to the of the acceleration of product dissociation by thin filaments H.D. B. Biochemistry. PubMed Scopus Google Scholar). have to as a substrate it actomyosin and in muscle to or the substrate ATP E. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar). In is a and complex cannot be to the two and in In this data which that the binding of both calcium and rigor myosin-S1 to the thin filament are required to activation of product release from An allosteric model is proposed that the activation of skeletal thin filaments by calcium and rigor myosin and the effect of rigor myosin-S1 upon the thin filament affinity for calcium and the effect of calcium upon the thin filament affinity for rigor and from was from and to the of Biophys. PubMed Scopus Google Scholar) for and from skeletal was used as a of 1982; PubMed Scopus Google Scholar). was from myosin with by the of and Nature. 257: PubMed Scopus Google Scholar) that of of was used to The was on and to myosin-S1 containing only the light chain thin filaments from and as B. White H.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). state ATP hydrolysis was measured by 1982; PubMed Scopus Google Scholar). thin filaments a calcium of the of steady state ATP hydrolysis of to and used for state that the ATP was using a as H.D. B. Biochemistry. PubMed Scopus Google Scholar). The was by mixing the with the of The of was by high containing of nucleotide was a and with a rate of was with a fluorescence with a was and obtained with a was using a The of hydrolysis was by of the of the and in the fluorescence using a double mixing stopped-flow The light from a was using a of dissociation using an and using a to data of and the rate obtained by the data to one or two using the with the In two of data that for the rate The stopped-flow was with in and and a in that both and to the of thin filaments in the by mixing in with The steady state was in a in a with thin filaments in and or The of the was for S1 and and for thin of the of by myosin-S1 was with and the and the of is in The of the the rate of substrate binding and and the the steady state rate The of the of the and a of the equilibrium of hydrolysis of bound to a of the of the upon the of The of is to and can be used with the of from the of the and to the of to be and to be the equilibrium is for ATP the rate of the hydrolysis in the absence of is for ATP similar of to the on the of of from with a of the fluorescence that is with dissociation in a stopped-flow in which the thin filaments calcium rigor myosin-S1 of S1 and are in the and to for for the to be bound and by the In the the complex is with thin filaments containing to the filaments of calcium and to binding of not bound to myosin-S1 in the and to rigor bound myosin-S1 from the The fluorescence is by a in which of thin a to of the was which a small of thin filaments with regulation not that not used in this The of the rate upon thin filament is by the closed in The of upon thin filament is by a with a of This an from the steady state ATP hydrolysis rate in the absence of The thin filament is similar to the steady state rate measured by in been in dissociation B. White H.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) that the activation of product release calcium and with no rigor myosin-S1 bound to the thin filament be due to an partial activation of the thin filament by a small of rigor S1 that either the or was by product release from bound to the thin This is in these ATP contained with the thin filaments in the that rigor myosin be a rate in which calcium is bound to the thin filament in the the fluorescence change from the dissociation is The initial is by a The of the of the and upon thin filament is in of the and to a of and open data that the rate of product dissociation thin filaments is by calcium binding to the thin filament. The of the to the is that expected from the of the myosin nucleotide and mixing to which that there is mixing with the thin The acceleration of dissociation in the of calcium can be with with as a substrate J. Biol. Chem. Full Text PDF PubMed Google Scholar) and of dissociation with B. White H.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of to the upon the of from been from both steady state and binding J.M. A. Cold Spring Harbor Symp. Quant. Biol. 1972; 37: Google Scholar, Eisenberg E. Biochemistry. PubMed Scopus Google Scholar) that thin filaments containing no bound calcium can be activated by myosin bound in the effect of rigor on the rate of dissociation, myosin-S1 is used in the myosin is for binding to thin filaments either or in the fluorescence is with myosin-S1 in the and of mixing with thin filaments to a of thin filaments with bound rigor myosin-S1 The of the upon thin filament is in In this of the S1 was as to a of rigor S1 to actin the The of the and are and calcium produces an of both the and of the dissociation from thin filaments containing bound rigor myosin The of of the and upon thin filament a of rigor bound S1 thin filament actin is by the and open in The thin filament are and The of the is of the which is within of the measured by This that the is a of dissociation from binding to thin filaments and that the is from a rate of is obtained for the the rate of dissociation from bound to actin both rigor S1 and calcium and from actin is the obtained for dissociation, B. White H.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). This is with the of dissociation of from E. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar). The of thin filament actin with rigor myosin-S1 bound that is required to activation of the thin filament was by the of myosin-S1 to The of the of and of dissociation upon the of rigor myosin-S1 to thin filaments is in data are by a in which activation is obtained with rigor S1 bound actin or a of S1 actin to This with a of S1 actin required to fully the thin filament high calcium measured B. White H.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). that the regulatory is either a or is of both of the thin filament. The of the rate of product release upon rigor myosin-S1 that the rate of rigor myosin-S1 binding to the thin filaments not the rate of thin filament activation high or calcium have used double mixing stopped-flow fluorescence to the regulation of thin filament activation of dissociation from by rigor myosin-S1 and calcium. steady state of substrate and bound to the active site and was with thin filaments of activation by calcium rigor bound myosin of the of dissociation and the actin required to from the data in and is in in with to are for B. White H.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, H.D. B. Biochemistry. PubMed Scopus Google Scholar). The the measured rate of and dissociation is with either a mechanism in which and the dissociation of dissociation or a mechanism in which a conformational change that to actin the rate of dissociation of both However, there are the of and The rate of dissociation by fully activated thin filaments bound calcium and rigor is with the rate of dissociation of from which is in the absence of regulatory proteins in of product dissociation been in nucleotide E. Biophys. J. Full Text Full Text PDF PubMed Scopus Google of product dissociation and by thin filaments and F-actin for the rate of dissociation for the and thin filament or actin in and are in is for the rate of dissociation for the and thin filament or actin measured with ATP as the substrate and using to release B. White H.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The for are the thin filament required to the is the rate of the of product release thin filament the and is the rate with F-actin in the absence of regulatory to a to a was of thin filament to a was of thin filament in a but changes are in the of product release in the absence of rigor myosin-S1 In these ATP was with the thin filaments to rigor The data in calcium by a that is that B. White H.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The binding is to be due to rigor activation of the thin filament by a small of myosin-S1 that not have substrate or bound the The rate of the of dissociation in the of calcium rigor myosin-S1 bound to the thin filaments with the thin filament to a of is that the in the which was to be of the thin filament was either by from ATP that was not bound to the myosin-S1 in the or ATP dissociation from actomyosin of this ATP is by the rate of ATP binding to the ATP binding would have been expected to occur which is similar to the In the the and subsequent hydrolysis of is prevented by the of ATP in the thin calcium and rigor myosin are bound to the thin filament, the of product dissociation in with as substrate with ATP as of from would a fluorescence In ATP from actomyosin ATP to and be to and a However, both a by hydrolysis and product dissociation The measured with both are the within and which that either a is measured or that the three have similar The hydrolysis been to be the of ATP hydrolysis of F-actin in the absence of regulatory proteins H.D. B. Biochemistry. PubMed Scopus Google Scholar). proteins have been to the steady state rate of ATP a as but the of the effect been J.M. A. Cold Spring Harbor Symp. Quant. Biol. 1972; 37: Google Scholar, Eisenberg E. Biochemistry. PubMed Scopus Google Scholar). have similar of product dissociation by actin and fully activated thin but the affinity of for thin filaments is for be expected that of actin or thin filament the rate would be for thin actin the would be steric blocking mechanism in which the myosin binding to thin filaments calcium that the effect of regulation upon the mechanism would be to the affinity of for thin a mechanism was inconsistent with subsequent observations that the effect of calcium upon the affinity of myosin for actin during steady state ATP hydrolysis is small (4Chalovich J.M. Eisenberg E. J. Biol. Chem. 1982; 257: 2432-2437Abstract Full Text PDF PubMed Google Scholar). The in are with this in that there is only a in the affinity of for maximally activated thin filaments (calcium and rigor relative to either activated (calcium or rigor or maximally inhibited thin filaments calcium or rigor The three-state model is based upon equilibrium and of rigor myosin-S1 myosin ligands that open switch II myosin binding to the thin filament. The model the binding of either rigor myosin or myosin with ligands such as ADP, pyrophosphate, and to actin thin by the mechanism that the equilibrium of the is by the bound is that myosin exists in two or and that the conformation is upon the ligands bound to the active the ligands only produces the switch II conformation of the myosin nucleotide binding site that is required to a the myosin to actin J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In there are of the three-state model that are not with the data in the acceleration of product release by thin filaments high calcium but rigor myosin bound is the acceleration by the calcium of the equilibrium the closed and open states, in the three-state model that rigor binding fully product release calcium but as is in rigor binding produces of the rate of product release that with both calcium and rigor bound to the thin filament. are by a structural of the of the of the of troponin in muscle 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In this 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) rigor binding of myosin calcium produces of the change in that is by rigor myosin high calcium. that the three-state model was to the of the of activation of product dissociation upon calcium and rigor S1 and that release or a release was the but not an The model that is with the data in is in in which calcium and rigor myosin-S1 the equilibrium and active of the thin filament. model is similar to of the original model proposed by Hill Eisenberg E. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) in which the thin filament conformation is either active or with regard to the to accelerate product dissociation from (6Chen Y. Yan B. Chalovich J.M. Brenner B. Biophys. J. 2001; 80: 2338-2349Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The data in for the a of the the activation of the ATP hydrolysis and calcium and rigor myosin binding to the thin filament to be The equilibrium the form in the absence of bound calcium and rigor myosin but primarily in the state if only one ligand either calcium or rigor S1) is the equilibrium in favor of the active form only if both calcium and rigor myosin are bound to the thin filament. The of the conformational equilibrium upon the ligands calcium and myosin (no (calcium and calcium are from the of activation as in the of The of the for the equilibrium the and active is for and but only a of be obtained for In the of the equilibrium for ligand (calcium and rigor to the active and of the thin filament and the ligand that can be from are on the The data are with a mechanism in which the affinity of ligand is only by the equilibrium the active and conformation of the filaments and not by binding of the ligand that and This is as there is not an the calcium and myosin on the thin filament and the the would be by the conformation of the thin filament. such a mechanism is a of can be for which is with the of In for the affinity of ligand the of and of the ligand are in that calcium and myosin would be expected to a in the affinity of the ligand for thin are with the measured in the calcium affinity of thin filaments in the of rigor bound myosin-S1 A. Biol. 1972; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of calcium and rigor binding to thin filaments is by the in equilibrium the thin filament from ligand An feature of the model in is that it the small changes in calcium affinity and rigor S1 affinity of thin filaments a of regulation by calcium and S1 The model in that the equilibrium the active and of the thin filaments is the product dissociation the was not would the of active thin and the effect upon the mechanism would be to the affinity of for thin filaments in the inhibited This is by the similar for one (either calcium or rigor S1) or both ligands are bound to the thin filament. However, the small in the affinity of thin filaments in the absence of calcium and bound rigor myosin-S1 be to either the rate of active and thin filament similar to the of product release in the absence of calcium and or a steric blocking as by the of three-state that produces a small change in of the of thin filaments recently be used to the of the of upon calcium A. C. R. J. Biol. 2005; PubMed Scopus Google Scholar). calcium of the thin filament contained in a that would myosin binding, but in the that would partial high calcium the was with in the blocked conformation and in the closed The structural data an in the of thin filament binding to calcium. The calcium of the structural change is in with the in which that calcium binding to the thin filament produces only a small in the of that the change in the the blocked and closed only a small of the regulatory mechanism and that the mechanism of regulation is of the rate of product dissociation The data obtained in and that the association of both calcium and myosin-S1 to skeletal thin filaments is required for activation of the rate of product release from The data can be by an allosteric regulation model in which calcium and rigor S1 binding to the thin filament the equilibrium in favor of an active conformation, but full activation requires binding of both ligands. The predominant mechanism of regulation is acceleration of product dissociation by a factor of ∼200 by thin filaments in the fully activated conformation (bound calcium and rigor S1) relative to the maximally inhibited conformation (no bound calcium or rigor S1). the form of the model in is similar to the closed and open of the three-state model closed and open there are the two In the allosteric model of myosin-S1 and calcium the thin filament by the both the equilibrium of the thin filament from the to the active conformation. activation by rigor myosin-S1 requires no myosin-S1 bound actin B. White H.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and This is in with of fluorescence that a of myosin-S1 actin M.A. Biophys. J. Full Text PDF PubMed Scopus Google Scholar). that the regulatory is the structural on one of a thin filament and actin with a are the actin on both of the filament or actin on one of the filament in both from the site of myosin-S1 In of the for myosin binding to thin filaments it that of myosin-S1 to the myosin-S1 is the actin filament full in of the filament that have been activated by binding of of are by the model in in which calcium and myosin-S1 binding to the thin filament are allosteric of the of the thin filament to product dissociation allosteric of myosin-S1 is with the of S1 binding to thin filaments containing which not a either high or calcium R. Geeves M.A. Biochemistry. PubMed Scopus Google Scholar). the rate of binding a 2–3-fold relative to In the used in and the of the rigor S1 bound to the thin filaments is the of the of S1 binding to thin filaments an equilibrium be that product dissociation from is activated by thin filaments calcium in the absence of rigor to thin filament activation of product release in the of a of myosin to actin that the is a of activation of product release in the absence of either ligand bound to the thin filament. This rate the rate of ATP hydrolysis that is in or muscle H.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, C. C. Biophys. J. Google Scholar). that an regulatory mechanism in muscle to the regulation.

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