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• W3093004359 abstract "Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre– and post–power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport. Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre– and post–power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport. Understanding how motor proteins such as myosin couple structural changes in their ATPase cycle with mechanical force is a fundamental question in biophysics. Indeed, the actomyosin ATPase mechanism is utilized for many different mechanical tasks in cells including muscle contraction, cell division, and organelle transport (1Hartman M.A. Spudich J.A. The myosin superfamily at a glance.J. Cell Sci. 2012; 125 (22566666): 1627-163210.1242/jcs.094300Crossref PubMed Scopus (144) Google Scholar). The large family of myosins, which includes over 20 classes and more than 40 different human genes, is defined by a structurally conserved motor domain and uses a conserved ATPase cycle to convert chemical energy from ATP hydrolysis into mechanical work (2Heissler S.M. Sellers J.R. Various themes of myosin regulation.J. Mol. Biol. 2016; 428 (26827725): 1927-194610.1016/j.jmb.2016.01.022Crossref PubMed Scopus (54) Google Scholar, 3Berg J.S. Powell B.C. Cheney R.E. A millennial myosin census.Mol. Biol. Cell. 2001; 12 (11294886): 780-79410.1091/mbc.12.4.780Crossref PubMed Scopus (602) Google Scholar, 4Robert-Paganin J. Pylypenko O. Kikuti C. Sweeney H.L. Houdusse A. Force generation by myosin motors: a structural perspective.Chem. Rev. 2020; 120 (31689091): 5-3510.1021/acs.chemrev.9b00264Crossref PubMed Scopus (23) Google Scholar). A detailed understanding of the structural and functional aspects of the myosin ATPase pathway will enhance our ability to utilize myosins as drug targets to treat various genetic and pathological diseases (5Trivedi D.V. Nag S. Spudich A. Ruppel K.M. Spudich J.A. The myosin family of mechanoenzymes: from mechanisms to therapeutic approaches.Annu. Rev. Biochem. 2020; 89 (32169021): 667-69310.1146/annurev-biochem-011520-105234Crossref PubMed Scopus (9) Google Scholar). The lever arm hypothesis suggests that myosins generate force by coupling specific conformational changes in the active site to large conformational changes in the actin-binding and lever arm regions (4Robert-Paganin J. Pylypenko O. Kikuti C. Sweeney H.L. Houdusse A. Force generation by myosin motors: a structural perspective.Chem. Rev. 2020; 120 (31689091): 5-3510.1021/acs.chemrev.9b00264Crossref PubMed Scopus (23) Google Scholar, 6Huxley A.F. Mechanics and models of the myosin motor.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2000; 355 (10836496): 433-44010.1098/rstb.2000.0584Crossref PubMed Scopus (86) Google Scholar, 7Holmes K.C. The swinging lever-arm hypothesis of muscle contraction.Curr. Biol. 1997; 7 (9081660): R112-R11810.1016/S0960-9822(06)00051-0Abstract Full Text Full Text PDF PubMed Google Scholar, 8Tyska M.J. Warshaw D.M. The myosin power stroke.Cell Motil. Cytoskeleton. 2002; 51 (11810692): 1-1510.1002/cm.10014Crossref PubMed Scopus (144) Google Scholar). The lever arm domain consists of a single α-helix that extends from the motor and binds a variety of calmodulin or calmodulin-like light chains. When myosin is bound to actin, its cytoskeletal track, the rotation of the lever arm can produce a power stroke that drives actin filament sliding or allows myosin to walk along actin filaments. A key question in the field is how does actin binding trigger the dramatic acceleration in the release of the products of ATP hydrolysis, specifically phosphate release, which can be accelerated over 1000-fold (9Lymn R.W. Taylor E.W. Mechanism of adenosine triphosphate hydrolysis by actomyosin.Biochemistry. 1971; 10 (4258719): 4617-462410.1021/bi00801a004Crossref PubMed Scopus (982) Google Scholar, 10Geeves M.A. The ATPase mechanism of myosin and actomyosin.Biopolymers. 2016; 105 (27061920): 483-49110.1002/bip.22853Crossref PubMed Scopus (30) Google Scholar), and how this is coupled to force production (i.e. lever arm rotation when myosin is bound to its track). There have been many approaches to addressing this question, including X-ray crystallography (4Robert-Paganin J. Pylypenko O. Kikuti C. Sweeney H.L. Houdusse A. Force generation by myosin motors: a structural perspective.Chem. Rev. 2020; 120 (31689091): 5-3510.1021/acs.chemrev.9b00264Crossref PubMed Scopus (23) Google Scholar), solution biochemical analysis (10Geeves M.A. The ATPase mechanism of myosin and actomyosin.Biopolymers. 2016; 105 (27061920): 483-49110.1002/bip.22853Crossref PubMed Scopus (30) Google Scholar), single-molecule mechanical studies (11Batters C. Veigel C. Mechanics and activation of unconventional myosins.Traffic. 2016; 17 (27061900): 860-87110.1111/tra.12400Crossref PubMed Scopus (16) Google Scholar, 12Sun Y. Goldman Y.E. Lever-arm mechanics of processive myosins.Biophys. J. 2011; 101 (21723809): 1-1110.1016/j.bpj.2011.05.026Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 13Ishii Y. Ishijima A. Yanagida T. Single molecule nanomanipulation of biomolecules.Trends Biotechnol. 2001; 19 (11356282): 211-21610.1016/S0167-7799(01)01635-3Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), and muscle mechanics studies (14Lombardi V. Piazzesi G. Reconditi M. Linari M. Lucii L. Stewart A. Sun Y.B. Boesecke P. Narayanan T. Irving T. Irving M. X-ray diffraction studies of the contractile mechanism in single muscle fibres.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2004; 359 (15647164): 1883-189310.1098/rstb.2004.1557Crossref PubMed Scopus (26) Google Scholar, 15Stehle R. Tesi C. Kinetic coupling of phosphate release, force generation and rate-limiting steps in the cross-bridge cycle.J. Muscle Res. Cell Motil. 2017; 38 (28918606): 275-28910.1007/s10974-017-9482-8Crossref PubMed Scopus (10) Google Scholar). The challenge is to integrate all of the results in the literature into a model that describes the sequence of events associated with actin-binding, lever arm rotation (referred to as the power stroke), and force generation (referred to as the working stroke). Structural studies have defined two main conformations of the lever arm, the pre–power stroke state, in which ATP or the products of ATP hydrolysis are tightly bound to the active site, and a post–power stroke state, in which ADP or no nucleotide is bound (16Sweeney H.L. Houdusse A. Structural and functional insights into the myosin motor mechanism.Annu. Rev. Biophys. 2010; 39 (20192767): 539-55710.1146/annurev.biophys.050708.133751Crossref PubMed Scopus (271) Google Scholar). Crystallography results on myosin VI have provided insight into the phosphate release mechanism by demonstrating evidence of the escape route of phosphate through a phosphate tunnel or back door (17Llinas P. Isabet T. Song L. Ropars V. Zong B. Benisty H. Sirigu S. Morris C. Kikuti C. Safer D. Sweeney H.L. Houdusse A. How actin initiates the motor activity of myosin.Dev. Cell. 2015; 33 (25936506): 401-41210.1016/j.devcel.2015.03.025Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Based on the crystallography studies, as well as other results in the literature, this group proposed that myosin binding to actin triggers structural changes in the active site that allow phosphate to be released through the back door. They propose that once phosphate is transitioned from the active site into the tunnel, strong actin binding and lever arm swing can occur and then finally phosphate leaves the tunnel into solution (4Robert-Paganin J. Pylypenko O. Kikuti C. Sweeney H.L. Houdusse A. Force generation by myosin motors: a structural perspective.Chem. Rev. 2020; 120 (31689091): 5-3510.1021/acs.chemrev.9b00264Crossref PubMed Scopus (23) Google Scholar). Therefore, in this model, only when myosin is tightly bound to its track does it trigger the movement of the force-generating lever arm and allow the release of phosphate, a highly irreversible step in the absence of load. Consequently, studies that have directly measured the lever arm rotation by FRET in real time demonstrated that the power stroke occurs before the release of phosphate is observed (18Trivedi D.V. Muretta J.M. Swenson A.M. Davis J.P. Thomas D.D. Yengo C.M. Direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin V.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (26553992): 14593-1459810.1073/pnas.1517566112Crossref PubMed Scopus (29) Google Scholar, 19Muretta J.M. Rohde J.A. Johnsrud D.O. Cornea S. Thomas D.D. Direct real-time detection of the structural and biochemical events in the myosin power stroke.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (26578772): 14272-1427710.1073/pnas.1514859112Crossref PubMed Scopus (53) Google Scholar, 20Rohde J.A. Thomas D.D. Muretta J.M. Heart failure drug changes the mechanoenzymology of the cardiac myosin powerstroke.Proc. Natl. Acad. Sci. U.S.A. 2017; 114 (28223517): E1796-E180410.1073/pnas.1611698114Crossref PubMed Scopus (35) Google Scholar). In addition, recent ultra-fast optical trapping studies on myosin II suggest that the power stroke and observed working stroke occur rapidly after attachment to actin, without any delay that might be caused by an intermediate with phosphate in the tunnel (21Woody M.S. Winkelmann D.A. Capitanio M. Ostap E.M. Goldman Y.E. Single molecule mechanics resolves the earliest events in force generation by cardiac myosin.eLife. 2019; 8 (31526481)e4926610.7554/eLife.49266Crossref PubMed Scopus (20) Google Scholar). Both models suggest that the power stroke and strong actin binding are tightly coupled. There are several key questions that remain to test current models. Foremost is defining structural changes in the active site that are triggered by attachment to actin and required for the power stroke and phosphate release. Carefully chosen point mutations designed to inhibit the lever arm rotation or phosphate release could provide a powerful test of these models. It is also unclear whether all myosins follow the same sequence of events or whether there are variations within the myosin family that are important for tuning myosins for their biological function. We have developed a method of measuring the rotation of the lever arm in myosin V in real time using FRET, which involves labeling myosin V containing a single IQ motif with FlAsH at its N terminus and exchanging QSY-labeled calmodulin at the first IQ motif (18Trivedi D.V. Muretta J.M. Swenson A.M. Davis J.P. Thomas D.D. Yengo C.M. Direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin V.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (26553992): 14593-1459810.1073/pnas.1517566112Crossref PubMed Scopus (29) Google Scholar). We directly measured lever arm rotation in the recovery and power stroke steps of the myosin V ATPase cycle and found that the power stroke occurs in two steps: a rapid step that is faster than phosphate release and a slower step that occurs before ADP release (18Trivedi D.V. Muretta J.M. Swenson A.M. Davis J.P. Thomas D.D. Yengo C.M. Direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin V.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (26553992): 14593-1459810.1073/pnas.1517566112Crossref PubMed Scopus (29) Google Scholar, 22Gunther L.K. Rohde J.A. Tang W. Walton S.D. Unrath W.C. Trivedi D.V. Muretta J.M. Thomas D.D. Yengo C.M. Converter domain mutations in myosin alter structural kinetics and motor function.J. Biol. Chem. 2019; 294 (30518549): 1554-156710.1074/jbc.RA118.006128Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). In the current paper we use a point mutation in the conserved switch I region of the active site to demonstrate the coupling between the actin- and nucleotide-binding regions during the power stroke and phosphate release steps. We also characterized the impact of the mutation in single-molecule mechanics and kinetics in an optical trapping assay. Our results have led to a model suggesting that the transition from a weak-binding state to an initial force-bearing state requires structural changes in the active site and that these actin-induced structural changes are crucial for triggering the power stroke. Thus, generating the myosin power stroke requires priming the key structural elements for force generation; actin-binding region, active site, and lever arm. The switch I region of the active site, which shares homology with other P-loop ATPases including kinesin and G-proteins, consists of a consensus motif (NXXSSR) (Fig. 1A) (23Kull F.J. Vale R.D. Fletterick R.J. The case for a common ancestor: kinesin and myosin motor proteins and G proteins.J. Muscle Res. Cell Motil. 1998; 19 (10047987): 877-88610.1023/A:1005489907021Crossref PubMed Scopus (138) Google Scholar). Mutating the first serine in this motif was found to reduce but not abolish actin-activated ATPase and in vitro motility in myosin II (24Shimada T. Sasaki N. Ohkura R. Sutoh K. Alanine scanning mutagenesis of the switch I region in the ATPase site of Dictyostelium discoideum myosin II.Biochemistry. 1997; 36 (9369475): 14037-1404310.1021/bi971837iCrossref PubMed Scopus (75) Google Scholar, 25Li X.D. Rhodes T.E. Ikebe R. Kambara T. White H.D. Ikebe M. Effects of mutations in the γ-phosphate binding site of myosin on its motor function.J. Biol. Chem. 1998; 273 (9765269): 27404-2741110.1074/jbc.273.42.27404Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Another study examined the impact of mutating this residue (S217A) in myosin V using steady-state and transient kinetic analysis as well as single-molecule motility (26Forgacs E. Sakamoto T. Cartwright S. Belknap B. Kovács M. Tóth J. Webb M.R. Sellers J.R. White H.D. Switch 1 mutation S217A converts myosin V into a low duty ratio motor.J. Biol. Chem. 2009; 284 (19008235): 2138-214910.1074/jbc.M805530200Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), but optical trapping and structural kinetics were not examined. Thus, we introduced the S217A point mutation into a construct of myosin V contained a single IQ motif (MV), N-terminal tetracysteine tag, and C-terminal Myc and FLAG tags (18Trivedi D.V. Muretta J.M. Swenson A.M. Davis J.P. Thomas D.D. Yengo C.M. Direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin V.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (26553992): 14593-1459810.1073/pnas.1517566112Crossref PubMed Scopus (29) Google Scholar, 22Gunther L.K. Rohde J.A. Tang W. Walton S.D. Unrath W.C. Trivedi D.V. Muretta J.M. Thomas D.D. Yengo C.M. Converter domain mutations in myosin alter structural kinetics and motor function.J. Biol. Chem. 2019; 294 (30518549): 1554-156710.1074/jbc.RA118.006128Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 27Swenson A.M. Trivedi D.V. Rauscher A.A. Wang Y. Takagi Y. Palmer B.M. Málnási-Csizmadia A. Debold E.P. Yengo C.M. Magnesium modulates actin binding and ADP release in myosin motors.J. Biol. Chem. 2014; 289 (25006251): 23977-2399110.1074/jbc.M114.562231Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 28Trivedi D.V. David C. Jacobs D.J. Yengo C.M. Switch II mutants reveal coupling between the nucleotide- and actin-binding regions in myosin V.Biophys. J. 2012; 102 (22713570): 2545-255510.1016/j.bpj.2012.04.025Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 29Trivedi D.V. Muretta J.M. Swenson A.M. Thomas D.D. Yengo C.M. Magnesium impacts myosin V motor activity by altering key conformational changes in the mechanochemical cycle.Biochemistry. 2013; 52 (23725637): 4710-472210.1021/bi4004364Crossref PubMed Scopus (14) Google Scholar). For experiments utilizing FRET to measure lever arm rotation, we labeled MV with the tetracysteine-based dye FlAsH and exchanged QSY-CaM onto the first IQ motif (MV-F.QSY-CaM) (Fig. 1A) (18Trivedi D.V. Muretta J.M. Swenson A.M. Davis J.P. Thomas D.D. Yengo C.M. Direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin V.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (26553992): 14593-1459810.1073/pnas.1517566112Crossref PubMed Scopus (29) Google Scholar, 22Gunther L.K. Rohde J.A. Tang W. Walton S.D. Unrath W.C. Trivedi D.V. Muretta J.M. Thomas D.D. Yengo C.M. Converter domain mutations in myosin alter structural kinetics and motor function.J. Biol. Chem. 2019; 294 (30518549): 1554-156710.1074/jbc.RA118.006128Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). The measurements not involving FRET were done with unlabeled WT and S217A in KMg50 buffer (10 mm imidazole, 50 mm KCl, 1 mm MgCl2, 1 mm EGTA, and 1 mm DTT), whereas the FRET measurements were performed with MV-F.QSY-CaM in KMg50 buffer with 1 mm TCEP instead of DTT. In our previous work we demonstrated that our fluorescence labeling strategy had a slight impact on steady-state ATPase and in vitro motility (<2-fold) because of a reduction in the ADP release rate constant, whereas other steps in the ATPase cycle were unchanged (18Trivedi D.V. Muretta J.M. Swenson A.M. Davis J.P. Thomas D.D. Yengo C.M. Direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin V.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (26553992): 14593-1459810.1073/pnas.1517566112Crossref PubMed Scopus (29) Google Scholar). We examined S217A compared with WT using actin-activated ATPase assays in KMg50 buffer at 25 °C (Fig. 1B) (18Trivedi D.V. Muretta J.M. Swenson A.M. Davis J.P. Thomas D.D. Yengo C.M. Direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin V.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (26553992): 14593-1459810.1073/pnas.1517566112Crossref PubMed Scopus (29) Google Scholar, 22Gunther L.K. Rohde J.A. Tang W. Walton S.D. Unrath W.C. Trivedi D.V. Muretta J.M. Thomas D.D. Yengo C.M. Converter domain mutations in myosin alter structural kinetics and motor function.J. Biol. Chem. 2019; 294 (30518549): 1554-156710.1074/jbc.RA118.006128Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). S217A reduced the maximum rate of actin-activated ATPase (kcat) 3–4-fold and increased the KATPase (the actin concentration at which ATPase is half-maximal) 5-fold (Fig. 1B and Table 1), which was similar to the previous myosin V study (26Forgacs E. Sakamoto T. Cartwright S. Belknap B. Kovács M. Tóth J. Webb M.R. Sellers J.R. White H.D. Switch 1 mutation S217A converts myosin V into a low duty ratio motor.J. Biol. Chem. 2009; 284 (19008235): 2138-214910.1074/jbc.M805530200Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar).Table 1Summary of steady-state and transient kinetic resultsWTS217ASteady-state ATPase values (±S.E.), n = 4aSteady-state ATPase measurements. v0 (s−1)0.05 ± 0.060.01 ± 0.01 kcat (s−1)11.3 ± 0.63.3 ± 0.5bp < 0.01. KATPase (µm)1.8 ± 0.49.6 ± 1.5bp < 0.01.Rate/equilibrium constants (±S.E.) ATP binding/hydrolysis (myosin), n = 3cIntrinsic tryptophan fluorescence.K1Tk+2T (µm−1·s−1)1.8 ± 0.32.9 ± 0.9K0.5 (µm)196 ± 453.2 ± 1.3dp < 0.0001.k+H + k−H (maximum rate, s−1)343 ± 319.3 ± 0.5dp < 0.0001. ATP binding (actomyosin), n = 3ePyrene actin.K´1Tk´+2T (µm−1·s−1)1.7 ± 0.20.4 ± 0.1bp < 0.01.K0.5 (µm)577 ± 172918 ± 224k´+2T (s−1)1003 ± 157404 ± 60 Recovery stroke, n = 3fFRET.k+RCF (fast phase, maximum rate, s−1)479 ± 14906 ± 50bp < 0.01.K0.5 (µm)191 ± 16470 ± 53bp < 0.01.k+RCS (slow phase, maximum rate, s−1)32 ± 317 ± 1bp < 0.01. Pyrene actin binding: M.ADP.Pi (µm−1·s−1), n = 1ePyrene actin.6.7 ± 0.40.6 ± 0.1 Actin-activated phosphate release, n = 1gMDCC-PBP fluorescence.KAssoc × k+Pi(actin concentration dependence, µm−1·s−1)19.4 ± 4.0hThe data are from Ref. 22.0.9 ± 0.1k+Pi (maximum rate, s−1)206 ± 34hThe data are from Ref. 22.N.D. Power stroke, n = 3fFRET.KAssoc × kPWF (actin concentration dependence, µm−1·s−1)19.4 ± 0.90.9 ± 0.1dp < 0.0001.k+PWF (fast phase, maximum rate, s−1)417 ± 95N.D.k+PWS (slow phase, maximum rate, s−1)94 ± 36N.D. Actomyosin ADP Release, n = 5 k´+D (s−1)imantADP fluorescence.25.2 ± 1.846.1 ± 6.0dp < 0.0001.a Steady-state ATPase measurements.b p < 0.01.c Intrinsic tryptophan fluorescence.d p < 0.0001.e Pyrene actin.f FRET.g MDCC-PBP fluorescence.h The data are from Ref. 22Gunther L.K. Rohde J.A. Tang W. Walton S.D. Unrath W.C. Trivedi D.V. Muretta J.M. Thomas D.D. Yengo C.M. Converter domain mutations in myosin alter structural kinetics and motor function.J. Biol. Chem. 2019; 294 (30518549): 1554-156710.1074/jbc.RA118.006128Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar.i mantADP fluorescence. Open table in a new tab We performed transient and structural kinetic analysis to determine how the S217A mutation impacts the key structural and biochemical transitions in the myosin ATPase cycle. The data were analyzed based on the established kinetic scheme (Scheme 1) of the ATPase cycle also utilized in recent manuscripts (18Trivedi D.V. Muretta J.M. Swenson A.M. Davis J.P. Thomas D.D. Yengo C.M. Direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin V.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (26553992): 14593-1459810.1073/pnas.1517566112Crossref PubMed Scopus (29) Google Scholar, 22Gunther L.K. Rohde J.A. Tang W. Walton S.D. Unrath W.C. Trivedi D.V. Muretta J.M. Thomas D.D. Yengo C.M. Converter domain mutations in myosin alter structural kinetics and motor function.J. Biol. Chem. 2019; 294 (30518549): 1554-156710.1074/jbc.RA118.006128Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). In Scheme 1, the rate constants going from left to right are indicated with a positive subscript (e.g. k+i), whereas those going right to left have a negative subscript (e.g. k−i). The rate of ATP binding and hydrolysis in the absence of actin was measured by mixing MV 1IQ with varying concentrations of ATP and monitoring the enhancement in tryptophan fluorescence (Fig. S1). The data were fit to a hyperbolic function, and the maximum rate of tryptophan fluorescence change was determined, which is thought to report the ATP hydrolysis rate constant (k+H+k−H). The second-order binding constant for ATP binding to myosin can be obtained by determining the slope of linear portion of the curve a low ATP concentrations (K1Tk+2T). We found that S217A reduces the maximum rate of ATP hydrolysis 40-fold while slightly enhancing the second-order binding constant for ATP binding to myosin (Fig. S1 and Table 1). ATP binding to myosin triggers movement of the lever arm into the pre–power stroke state (recovery stroke), which is important for priming myosin for force generation. The rate of the recovery stroke was examined by monitoring the FRET signal associated with the FlAsH-QSY donor–acceptor pair upon mixing MV-F:QSY-CaM with varying concentrations of ATP (Fig. 2, A–C). The FlAsH fluorescence increase was best fit to a two-exponential function at most ATP concentrations (Fig. 2C). The amplitude of the fast phase dominated the signal (90% or greater) in both S217A and WT. By fitting the fast and slow rate constants to a hyperbolic function, we determined that S217A increases the maximum rate of the fast recovery stroke rate constant (k+RCF) (Fig. 2A), whereas the slow phase of the recovery stroke (k+RCS) (Fig. 2B) was fairly similar to WT. Because the slower phase of the recovery stroke represents a small fraction of the total signal, we have not attempted to include this transition in our kinetic modeling. Myosin adopts a weak actin-binding conformation upon binding to ATP that dramatically weakens its affinity for actin. Pyrene actin is quenched when myosin is tightly bound to actin, and upon ATP binding there is an increase in pyrene fluorescence that can be used to monitor the rate of ATP binding and maximum rate of transition into the weak binding states (Fig. S2). In S217A the fluorescence transients were best fit to a two-exponential function, and the fast phase was ∼70% of the signal, whereas in WT most transients were single-exponential. We found that S217A decreases the maximum rate of ATP-induced transition into the weak binding states (k´+2T) (2-fold) and second-order rate constant (K´1Tk´+2T) for ATP binding (4-fold) (Fig. S2 and Table 1). The slower phase of the ATP-induced dissociation transients may be associated with an isomerization prior to ATP binding found in other myosins (30Geeves M.A. Perreault-Micale C. Coluccio L.M. Kinetic analyses of a truncated mammalian myosin I suggest a novel isomerization event preceding nucleotide binding.J. Biol. Chem. 2000; 275 (10781577): 21624-2163010.1074/jbc.M000342200Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The power stroke and phosphate release rate constants are accelerated when myosin with the products of ATP hydrolysis in the active site (M.ADP.Pi) binds to actin (Fig. 3). We performed sequential mix experiments to examine the actin-activated power stroke and phosphate release rate constants. We monitored the FRET signal upon mixing MV-F.QSY-CaM with substoichiometric ATP, allowing the reaction to age for 20 s and then mixing with varying concentrations of actin. The FlAsH fluorescence transients used to monitor the change in FRET were best fit by a two-exponential function (Fig. 3C). In WT, the fast and slow power stroke rate constants were hyperbolically dependent on actin concentration as we previously reported (18Trivedi D.V. Muretta J.M. Swenson A.M. Davis J.P. Thomas D.D. Yengo C.M. Direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin V.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (26553992): 14593-1459810.1073/pnas.1517566112Crossref PubMed Scopus (29) Google Scholar, 22Gunther L.K. Rohde J.A. Tang W. Walton S.D. Unrath W.C. Trivedi D.V. Muretta J.M. Thomas D.D. Yengo C.M. Converter domain mutations in myosin alter structural kinetics and motor function.J. Biol. Chem. 2019; 294 (30518549): 1554-156710.1074/jbc.RA118.006128Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar) (Fig. 3A). The fast and slow phases of the FRET transients were linearly dependent on actin concentration in S217A (Fig. 3B). Although the maximum rate of the fast power stroke was not determined for S217A, the actin dependence of the fast power stroke rate constant was found to be 20-fold slower in S217A than WT (KAssoc × kPWF = 0.9 ± 0.1 versus 19.4 ± 0.9 μm−1·s−1, respectively). Similarly, the slow power stroke was 5-fold slower at 30 μm actin in S217A compared with WT. We examined the phosphate release rate constant using unlabeled MV constructs and a sequential mix setup identical to that described above, except that the phosphate-binding protein was pres" @default.
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• W3093004359 date "2020-12-01" @default.
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• W3093004359 title "FRET and optical trapping reveal mechanisms of actin activation of the power stroke and phosphate release in myosin V" @default.
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