Mechanism of Blebbistatin Inhibition of Myosin II

Why is this work in the frame?

A frame that forgets how it found something cannot be audited. These are the routes that admitted this work.

No Canadian affiliation. An affiliation-only frame — the usual design — would never have seen this work. It is one of the works that make the case for inverting the frame.

Abstract

Blebbistatin is a recently discovered small molecule inhibitor showing high affinity and selectivity toward myosin II. Here we report a detailed investigation of its mechanism of inhibition. Blebbistatin does not compete with nucleotide binding to the skeletal muscle myosin subfragment-1. The inhibitor preferentially binds to the ATPase intermediate with ADP and phosphate bound at the active site, and it slows down phosphate release. Blebbistatin interferes neither with binding of myosin to actin nor with ATP-induced actomyosin dissociation. Instead, it blocks the myosin heads in a products complex with low actin affinity. Blind docking molecular simulations indicate that the productive blebbistatin-binding site of the myosin head is within the aqueous cavity between the nucleotide pocket and the cleft of the actin-binding interface. The property that blebbistatin blocks myosin II in an actin-detached state makes the compound useful both in muscle physiology and in exploring the cellular function of cytoplasmic myosin II isoforms, whereas the stabilization of a specific myosin intermediate confers a great potential in structural studies. Blebbistatin is a recently discovered small molecule inhibitor showing high affinity and selectivity toward myosin II. Here we report a detailed investigation of its mechanism of inhibition. Blebbistatin does not compete with nucleotide binding to the skeletal muscle myosin subfragment-1. The inhibitor preferentially binds to the ATPase intermediate with ADP and phosphate bound at the active site, and it slows down phosphate release. Blebbistatin interferes neither with binding of myosin to actin nor with ATP-induced actomyosin dissociation. Instead, it blocks the myosin heads in a products complex with low actin affinity. Blind docking molecular simulations indicate that the productive blebbistatin-binding site of the myosin head is within the aqueous cavity between the nucleotide pocket and the cleft of the actin-binding interface. The property that blebbistatin blocks myosin II in an actin-detached state makes the compound useful both in muscle physiology and in exploring the cellular function of cytoplasmic myosin II isoforms, whereas the stabilization of a specific myosin intermediate confers a great potential in structural studies. Myosin IIs are ATP-driven molecular motors forming an essential part of the motile machinery of most eukaryotic cell types examined. Among other functions, they serve such diverse and vital functions as muscle contraction, cytokinesis, cortical tension maintenance, and neurite outgrowth and retraction (1Brown J. Bridgman P.C. J. Histochem. Cytochem. 2003; 51: 421-428Crossref PubMed Scopus (63) Google Scholar, 2Sellers J.R. Myosins. Oxford University Press, New York1999Google Scholar, 3Yumura S. Uyeda T.Q. Int. Rev. Cytol. 2003; 224: 173-225Crossref PubMed Scopus (42) Google Scholar, 4Wylie S.R. Wu P.J. Patel H. Chantler P.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12967-12972Crossref PubMed Scopus (87) Google Scholar, 5Wylie S.R. Chantler P.D. Nat. Cell Biol. 2001; 3: 88-92Crossref PubMed Scopus (80) Google Scholar, 6Wylie S.R. Chantler P.D. Mol. Biol. Cell. 2003; 14: 4654-4666Crossref PubMed Scopus (71) Google Scholar). In studies of myosin II, the use of enzyme inhibitors can be a powerful approach, provided that selective and high affinity compounds are available that do not interfere with other cellular processes. The importance of the latter aspect is emphasized by a recent study (7Ostap E.M. J. Muscle Res. Cell Motil. 2002; 23: 305-308Crossref PubMed Scopus (117) Google Scholar) that showed that 2,3-butanedione-monoxime, a compound widely used to inhibit myosin II, is not specific and probably affects the function of a wide range of proteins. Blebbistatin was recently discovered as a small molecule inhibitor of muscle and non-muscle myosin II (8Straight A.F. Cheung A. Limouze J. Chen I. Westwood N.J. Sellers J.R. Mitchison T.J. Science. 2003; 299: 1743-1747Crossref PubMed Scopus (1107) Google Scholar). The compound is permeable to cell membranes. It is a potent inhibitor of skeletal muscle and non-muscle myosin II isoforms, although it has little or no effect on smooth muscle myosin II and myosins from classes I, V, and X (9Limouze J. Straight A.F. Mitchison T.J. Sellers J.R. J. Muscle Res. Cell Motil. 2004; (in press)PubMed Google Scholar). Because of its selectivity and high affinity for several class II myosins, blebbistatin has the potential to become a popular tool in the fields of cell motility and muscle physiology. For the interpretation of the cellular effects caused by blebbistatin, a detailed understanding is essential of its effects on the myosin II ATPase and on the interaction of the myosin head with actin and substrate. We undertook an in-depth characterization of the effect of blebbistatin on the functional properties of rabbit skeletal muscle myosin II, and we performed blind docking simulations on various atomic structures of the myosin head to determine the binding site and the structural basis of isoform specificity of the inhibitor. We find that blebbistatin exerts its inhibitory effect by binding to the myosin-ADP-Pi complex with high affinity and interfering with the phosphate release process. Thus, the inhibitor blocks myosin in an actin-detached state, and therefore it prevents rigid actomyosin cross-linking, which is a great advantage in in vivo applications. By using molecular simulations, we identify the aqueous cavity between the nucleotide- and actin-binding sites of the myosin head as the productive binding site for blebbistatin. All myosin IIs characterized to date follow a common basic enzymatic mechanism (10Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar). Therefore, the mode of inhibition of other muscle and non-muscle myosin II isoforms is expected to be similar to those described herein. Materials—Rabbit fast skeletal muscle myosin and subfragment-1 (S1) 1The abbreviations used are: S1, subfragment-1; mant, 2′(3)-O-(N-methylanthraniloyl); MOPS, 4-morpholinepropanesulfonic acid. were prepared as described in Refs. 11Margossian S.S. Lowey S. Methods Enzymol. 1982; 85: 55-71Crossref PubMed Scopus (817) Google Scholar and 12Okamoto Y. Sekine T. J. Biochem. (Tokyo). 1985; 98: 1143-1145Crossref PubMed Scopus (111) Google Scholar, respectively. Actin was prepared as in Ref. 13Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar and pyrene-labeled as in Ref 14Cooper J.A. Walker S.B. Pollard T.D. J. Muscle Res. Cell Motil. 1983; 4: 253-262Crossref PubMed Scopus (367) Google Scholar. Blebbistatin was either generously provided by Drs. Aaron F. Straight and Timothy J. Mitchison (Harvard Medical School) or purchased from Toronto Research Chemicals. Contrasting with previous observations (8Straight A.F. Cheung A. Limouze J. Chen I. Westwood N.J. Sellers J.R. Mitchison T.J. Science. 2003; 299: 1743-1747Crossref PubMed Scopus (1107) Google Scholar), we found that the K½ values and maximal extents of inhibition by the two stereoisomers of blebbistatin are very similar (data not shown). Therefore, we used an unresolved blebbistatin mixture in all experiments. Conditions—If not stated otherwise, experiments were carried out at 25 °C. For experiments not involving actin plus those in Fig. 5, the following assay buffer was used: 20 mm MOPS (pH 7.0), 5 mm MgCl2, 100 mm KCl, 0.1 mm EGTA. For experiments involving actin except those in Fig. 5, the following assay buffer was used: 4 mm MOPS (pH 7.0), 2 mm MgCl2, 0.1 mm EGTA. Mg2+ concentration was always kept higher than total nucleotide concentration. Steady-state ATPase Assay—MgATPase activities were measured by an NADH-linked assay as described previously (15Wang F. Kovacs M. Hu A. Limouze J. Harvey E.V. Sellers J.R. J. Biol. Chem. 2003; 278: 27439-27448Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) in 1 mm ATP. Data were corrected for background ATPase activity of actin. Stopped-flow Experiments—These experiments were carried out in a KinTek SF-2001 instrument. Fluorescence excitation/emission setups were the following: tryptophan, 295/347-nm bandpass (50 nm bandwidth); mant and pyrene, 365 nm/400 nm long pass. Light scattering was measured at 340 nm. Actin filaments were stabilized by addition of a 1.5-fold molar excess of phalloidin. If not stated otherwise, postmixing concentrations are as indicated throughout the text. Exceptions are the stopped-flow-based equilibrium titrations, where preincubation concentrations are relevant in the analysis of signal change amplitudes, whereas the kobs values will reflect post-mix concentrations. Volume ratios in stopped-flow mixtures were 1:1 in all experiments. Quenched-flow Experiments—These experiments were performed in a KinTek RQF-3 apparatus by using [γ-32P]ATP as described earlier (15Wang F. Kovacs M. Hu A. Limouze J. Harvey E.V. Sellers J.R. J. Biol. Chem. 2003; 278: 27439-27448Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Reagent concentrations after mixing are indicated. Acto-S1 Cosedimentation—50–100-μl samples were ultracentrifuged at 100,000 rpm in a Beckman TLA-100 rotor for 15 min at 4 °C, and the supernatants and pellets were analyzed by 4–20% SDS-PAGE. Relative amounts of proteins in electrophoretic bands were determined by densitometry using the Kodak ID 3.5 software. Data Analysis—Reported means and standard errors are those of two to six rounds of experiment. Fitting of the data sets was done using the KinTek software and OriginLab 7.0 (Microcal Corp.). Computational Docking—Throughout the search for putative binding sites of (R)-(+)- and (S)-(–)-blebbistatin on the myosin head was performed by using our previously published blind docking approach (16Hetenyi C. van der Spoel D. Protein Sci. 2002; 11: 1729-1737Crossref PubMed Scopus (370) Google Scholar) with the AutoDock 3.0 program package (17Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9042) Google Scholar). Atomic structures for head domains of nucleotide-free skeletal muscle myosin (2MYS), ADP·BeFx-bound smooth muscle myosin (1BR4), Dictyostelium myosin II with ATP (1FMW) or ADP·AlF4 (1MND) bound, and nucleotide-free myosin V (1OE9) of the Protein Data Bank were applied as target molecules during the calculations. For each protein and enantiomer of blebbistatin, 100 docking runs were evaluated. Representative groups (i.e. the binding patterns) were collected and ordered based on the calculated binding free energy of the complexes (16Hetenyi C. van der Spoel D. Protein Sci. 2002; 11: 1729-1737Crossref PubMed Scopus (370) Google Scholar). Steady-state Inhibition—In the experiments described, we used a soluble head fragment of skeletal myosin (S1) that contains the actin- and nucleotide-binding sites and retains the enzymatic properties of the full-length molecule (10Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar). “S1” refers to rabbit skeletal muscle S1 throughout this article. The basal MgATPase activity of S1 measured in the absence of actin (0.08 ± 0.01 s–1 in the absence of blebbistatin) was inhibited by blebbistatin to a large extent (92 ± 4% maximal inhibition) with inhibition at ± blebbistatin for a of determined in this The ATPase activity ± was inhibited with inhibition at ± 0.1 blebbistatin actin the low inhibited basal S1 ATPase ± in blebbistatin to a of ± s–1 of the The inhibitor not a effect on the (i.e. the actin concentration at Fig. and of blebbistatin on skeletal muscle S1 indicated ± ± for blebbistatin ± inhibition ± ± ± ± ± for blebbistatin ± inhibition ± of S1 in ATP (50 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± no ± ± binding to ± ± ± ± indicated NADH-linked Acto-S1 in a low blebbistatin concentrations where the binding equilibrium between enzyme and inhibitor is with enzymatic an in activity be the was inhibition. Fig. that at low blebbistatin the of the of the ATPase was by a between and s–1 in the blebbistatin concentration range of and indicated that the blebbistatin from S1 during ATP is s–1 and the is in the of 0.01 s–1 (data not shown). values a of blebbistatin binding to S1 1 to the inhibition The of kobs on blebbistatin concentration not be determined the of the and were It is that in experiments several ATP the inhibition ATPase activity was 20 s–1 the Blebbistatin to S1 ATPase of the myosin head during ATP and release in its that to the ATPase F. R.S. H. Biochem. J. PubMed Scopus Google Scholar, Biochem. J. PubMed Scopus Google Scholar, R. Biol. Chem. 1999; PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar, A. D.S. Kovacs M. M.A. 2001; PubMed Scopus Google Scholar, H. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, 1982; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). S1 was with excess ATP in the absence of blebbistatin, an in S1 that the binding of ATP was by a state with a the ATP blebbistatin, the was by a to a that is than that of The was a and its kobs was on blebbistatin concentration with a of ± s–1 the of blebbistatin binding to S1 during ATP and an of ± s–1 Fig. S1 a on binding to S1 was with excess in the an was by a with high mant blebbistatin was in both the was by a kobs was similar to that of the at the blebbistatin concentration Fig. and the of the two The in Fig. 2 that blebbistatin will to a S1 ATPase intermediate with high affinity ± to the concentration for and its binding is by a in both and mant The affinity of blebbistatin to is of Blebbistatin is neither the inhibitor nor S1 showed an signal change on mixing the two with S1, blebbistatin caused a in the ATP binding of S1 that ATP binding is a fast this effect is caused by a interaction between S1 and the inhibitor. We determined the affinity of blebbistatin for by an the on mixing ATP with S1 with concentrations of blebbistatin. In the absence of blebbistatin, a with a kobs of s–1 was at 100 ATP concentration S1 was with blebbistatin, were with and The of the with blebbistatin and the was a with a kobs 100 blebbistatin the two kobs values of the were by a of the of the blebbistatin concentration to a and showed a of 25 ± 2 for blebbistatin binding to The ADP affinity of S1 was measured by S1 with various amounts of ADP in the absence and of 100 blebbistatin and mixing with 100 ATP concentrations not shown). The ± s–1 in the absence and ± s–1 in the of blebbistatin) of the the of S1 that was the Here ADP the of ATP binding and therefore that of the The fast s–1 in the absence and s–1 in the of 100 blebbistatin) the S1 that not bound nucleotide and ATP. The of the showed a on ADP and very similar values were determined in the absence and of blebbistatin ± and ± Fig. The ADP affinity of S1 in the absence and of blebbistatin was in a similar stopped-flow where 0.1 S1 was with various ADP concentrations and with 20 and the signal of the mant nucleotide was to the were a of S1 for ADP of ± in the absence and ± in the of blebbistatin not shown). of Blebbistatin on ATP the effect of blebbistatin on the ATP we performed experiments using ATP on its 5 S1 was with a excess of an in was that was by a The of the ± 0.01 was with the basal ATPase activity of S1 measured by the assay The of the was ± of of In the of blebbistatin (in all the 20 was to ± of of S1, whereas the was inhibited by In S1 was with 1 ATP in the absence and of blebbistatin (in all Here the of the fast of ATP is by nucleotide whereas the release. to the showed similar values to the experiments. Because the ATP is by the release of the of the as in and the of the fast in the will the equilibrium of the ATP on the enzyme as where is the The and equilibrium in the absence of blebbistatin was than the values available Biochem. J. PubMed Scopus Google Scholar), which the of this were performed at whereas earlier experiments were done at The was in our experiments. The data are from six experiments performed on S1 Data with prepared and previously were All other determined with our S1 were in with of Blebbistatin on the Actin of in that on binding to S1 was used to follow the actin binding of in Fig. the of blebbistatin does not a change in the binding ± s–1 in the absence and 3.0 ± s–1 in the of blebbistatin in all The actin binding affinity (i.e. that in the absence of of S1 was by a stopped-flow-based where 0.1 was with various amounts of S1 and with excess ATP to Thus, the total of the reflect the concentration of the of S1 mixing with ATP. of the of the on S1 concentration M.A. J. Muscle Res. Cell Motil. PubMed Scopus Google Scholar) showed that the of blebbistatin (in all no effect on the actin affinity of S1 Fig. The of ATP-induced was measured by the on mixing with concentrations of ATP. Fig. that the of blebbistatin (in all not change the binding of ATP to ± 0.1 s–1 in the absence and ± s–1 in the of blebbistatin in all The of effect of the of blebbistatin in the experiments of Fig. 5, and that either the properties of S1 are with S1 that has a very low blebbistatin and little of the complexes has bound blebbistatin of and S1 during Steady-state ATP scattering and performed by mixing 2 S1 plus 5 actin with 20 ATP in the absence of blebbistatin, the of was by a state with a signal that for expected for a excess of S1 by a of the signal change as all S1 to actin ATP was The and scattering with the that an in and a in In the of blebbistatin (in all the was similar no of the signal was in that in blebbistatin, S1 is not bound to actin during ATP to the the inhibitor is not Steady-state ATP was not in the in the of blebbistatin Fig. and In S1 was found in the supernatants of the whereas it with actin in the absence of nucleotide of blebbistatin not an effect on addition of mm ATP to most of the S1 was found in the supernatants both in the absence and of blebbistatin, showing that the inhibitor does not the of and S1 during ATP in the of blebbistatin, a of S1 was and measured has an at (8Straight A.F. Cheung A. Limouze J. Chen I. Westwood N.J. Sellers J.R. Mitchison T.J. Science. 2003; 299: 1743-1747Crossref PubMed Scopus (1107) Google Scholar), all the blebbistatin was in the supernatants of all that S1 not blebbistatin (data not shown). Blind of Blebbistatin on the Myosin performed blind docking simulations of blebbistatin on various myosin atomic structures muscle smooth muscle Dictyostelium myosin II in the and and myosin to the structural basis of the interaction of S1 with blebbistatin. Computational of the binding sites interaction for each were based on determined K½ values (9Limouze J. Straight A.F. Mitchison T.J. Sellers J.R. J. Muscle Res. Cell Motil. 2004; (in press)PubMed Google Scholar). muscle myosin and Dictyostelium myosin II are inhibited inhibition at and blebbistatin, whereas myosin V and smooth muscle myosin are not or very inhibited by blebbistatin. this it was to functional and binding of the binding of skeletal muscle myosin and Dictyostelium myosin II were the of the binding of Dictyostelium myosin II and those of myosins were as inhibitory binding The of the binding were within the aqueous cavity between the actin binding cleft and the nucleotide-binding pocket It was that this was a inhibitory binding site of blebbistatin. The binding both in the and of Dictyostelium myosin II. The binding of blebbistatin in the aqueous cavity of the state has the interaction with the protein the 100 on the calculated blebbistatin binding to this site is to the state than to the of the binding of the (R)-(+)- and of blebbistatin showed no in the of all on the of the we a mechanism for blebbistatin inhibition of basal ATPase activity of S1 as in Blebbistatin binds to 25 and does not interfere with the nucleotide binding process. It has that ADP S1 a that is the state based on the of the II in the active site of S1 (10Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar). Because blebbistatin does not the ADP affinity of S1 Fig. it that the state complex has the affinity for blebbistatin as The affinity of blebbistatin for both and S1 in the is than the K½ and the affinity of blebbistatin for S1 during ATP and Thus, it can be that blebbistatin binds preferentially to a S1 It has that ATP an state complex will be (10Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar) that the state in which ATP (10Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar, A. D.S. Kovacs M. M.A. 2001; PubMed Scopus Google Scholar, C. J. Biochem. J. 2001; PubMed Scopus Google Scholar, A.J. R. I. PubMed Scopus Google Scholar). If blebbistatin binds to the state than to the it from that the equilibrium of ATP will be by the inhibitor. this was in the experiments of Fig. 4 Thus, as is the state will blebbistatin with a of a similar to that in the of the of II is to the release of phosphate (10Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar, A. D.S. Kovacs M. M.A. 2001; PubMed Scopus Google Scholar, C. J. Biochem. J. 2001; PubMed Scopus Google Scholar). Thus, the stabilization of the state by blebbistatin will be by an inhibition of phosphate release an effect on the the that is in the ATPase in the absence of the inhibitor. In this the inhibitory mechanism of blebbistatin is similar to that of a specific inhibitor of fast skeletal myosin II M.A. E.M. 2003; PubMed Scopus Google Scholar). Blebbistatin slows down the ATPase of S1 from s–1 to The inhibited will be determined by the of the complex either in the of phosphate release or blebbistatin from inhibition of the ATPase by blebbistatin that of the basal the extent of maximal not blebbistatin, and in its The is not by the that actomyosin interaction is not by blebbistatin. Acto-S1 binding and ATP-induced are not by the of blebbistatin, probably of the very low blebbistatin affinity of Thus, the between and actin-binding sites and the between the of two were not a of structural Blebbistatin does not inhibit binding of nucleotide to it has for S1 and to in the Blebbistatin has no effect on several myosins (9Limouze J. Straight A.F. Mitchison T.J. Sellers J.R. J. Muscle Res. Cell Motil. 2004; (in press)PubMed Google Scholar), although the of the structural the site is throughout the myosin J.R. Myosins. Oxford University Press, New York1999Google Scholar). Thus, it is that the inhibitor does not the nucleotide pocket of myosin II specific in the of that blebbistatin and actin binding to S1 All of are in with the docking that the productive blebbistatin-binding site is within the aqueous cavity the of the actin binding and to the binding site be to extent in the and of S1, of the II a state of the actin cleft in an blebbistatin affinity. indicate that blebbistatin binding in the cavity is in the II state than in the state, which is in with the binding data for state and for state, The binding site is in atomic structures of myosin isoforms that are not inhibited by blebbistatin muscle myosin and myosin V (9Limouze J. Straight A.F. Mitchison T.J. Sellers J.R. J. Muscle Res. Cell Motil. 2004; (in press)PubMed Google In blebbistatin can be as based on the types of enzyme it binds to a enzyme intermediate with it does not compete with for binding sites on the and it the Blebbistatin blocks myosin II in an actin-detached Thus, it is a inhibitor the great advantage that it does not effects from rigid actomyosin in cell applications. with its isoform blebbistatin a useful compound in the investigation of all of myosin II We Drs. Aaron F. Straight and Timothy J. Mitchison for and F. for D. for on the and Drs. S. and for

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.

The record

Venue

Journal of Biological Chemistry

Topic

Cardiomyopathy and Myosin Studies

Field

Medicine

Canadian institutions

—

Funders

Wellcome TrustHoward Hughes Medical Institute

Keywords

MyosinActinMyosin ATPaseMyosin headATPaseMeromyosinBiophysicsMyosin light-chain kinaseBiochemistryBinding siteAdenosine triphosphateChemistryBiologyEnzyme

Has abstract in OpenAlex

yes

API

/api/v1/works/W2009582775