Selective Perturbation of the Myosin Recovery Stroke by Point Mutations at the Base of the Lever Arm Affects ATP Hydrolysis and Phosphate Release

After ATP binding the myosin head undergoes a large structural rearrangement called the recovery stroke. This transition brings catalytic residues into place to enable ATP hydrolysis, and at the same time it causes a swing of the myosin lever arm into a primed state, which is a prerequisite for the power stroke. By introducing point mutations into a subdomain interface at the base of the myosin lever arm at positions Lys84 and Arg704, we caused modulatory changes in the equilibrium constant of the recovery stroke, which we could accurately resolve using the fluorescence signal of single tryptophan Dictyostelium myosin II constructs. Our results shed light on a novel role of the recovery stroke: fine-tuning of this reversible equilibrium influences the functional properties of myosin through controlling the effective rates of ATP hydrolysis and phosphate release. After ATP binding the myosin head undergoes a large structural rearrangement called the recovery stroke. This transition brings catalytic residues into place to enable ATP hydrolysis, and at the same time it causes a swing of the myosin lever arm into a primed state, which is a prerequisite for the power stroke. By introducing point mutations into a subdomain interface at the base of the myosin lever arm at positions Lys84 and Arg704, we caused modulatory changes in the equilibrium constant of the recovery stroke, which we could accurately resolve using the fluorescence signal of single tryptophan Dictyostelium myosin II constructs. Our results shed light on a novel role of the recovery stroke: fine-tuning of this reversible equilibrium influences the functional properties of myosin through controlling the effective rates of ATP hydrolysis and phosphate release. Various steps of the myosin mechanochemical cycle are linked to large conformational changes of the motor domain, which contains the actin and ATP binding sites as well as the converter region that forms the base of the extended lever arm domain. The converter/lever arm module is thought to amplify the structural changes occurring at the ATPase active site to produce a large working stroke (1Fisher A. J. Smith C. A. Thoden J. B. Smith R. Sutoh K. Holden H. M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (637) Google Scholar, 2Geeves M. A. Holmes K. C. Adv. Protein Chem. 2005; 71: 161-193Crossref PubMed Scopus (306) Google Scholar). Upon interacting with ATP, the motor domain undergoes a crystallographically identified large structural rearrangement before hydrolysis takes place. During this transition, the movement of the switch-2 loop of the active site toward the γ-phosphate of ATP brings catalytically important residues to their active positions. This open-closed transition of switch-2 is coupled to a large rotation of the lever arm (from down to a primed up state), and thus the conformational rearrangement has been termed the recovery stroke, which constitutes the priming of the myosin head in an actin-detached state. Rebinding of myosin to actin in the post-recovery (up) conformation is a prelude to the power stroke and force generation (3Zeng W. Conibear P. B. Dickens J. L. Cowie R. A. Wakelin S. Malnasi-Csizmadia A. Bagshaw C. R. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2004; 359: 1843-1855Crossref PubMed Scopus (45) Google Scholar). Kinetic studies on a Dictyostelium myosin II motor domain construct containing a single ATP-sensitive tryptophan sensor (W501+ located in the relay-converter module) have revealed the correspondence between identified structural states and ATPase intermediates (4Malnasi-Csizmadia A. Woolley R. J. Bagshaw C. R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 5Malnasi-Csizmadia A. Pearson D. S. Kovacs M. Woolley R. J. Geeves M. A. Bagshaw C. R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar). In the model shown in Reaction 1, ATP binding to apo-myosin (Mapo) 3The abbreviation used is: M, myosin; TES, 2-2-hydroxy-1, 1-bis (hydroxymethyl) ethylaminoethanesulfonic acid. (K1k2) is followed by the recovery stroke (K3a), which is reversible and rapid compared with the subsequent hydrolysis step (K3b). Following hydrolysis, the reversal of the recovery stroke (K4) is thought to occur before the actual release of Pi (k5). Reaction 1 and the current study deals with the myosin ATPase in the absence of actin, where lever arm motions are uncoupled from the performance of external work. ADP release (K6K7) occurs practically as a reversal of the ATP binding process. (Reaction 1 ignores possible protein structural and/or dynamic differences between the Mdown·ATP, Mdown·ADP, and Mdown·ADP·Pi states. ) The above mechanism gives rise to the hypothesis that a shift in the equilibrium constant of the recovery stroke (K3a) will have an effect on the apparent rate constant of ATP hydrolysis (because appkH = k3bK3a/ (1+K3a) + k-3b). Furthermore, because Pi release is thought to occur in the switch-2 open (down) conformation, the reversal of the recovery stroke (K4) may influence the steady-state ATPase rate, which is controlled by the effective rate of Pi release. Thus, besides its role in ATP hydrolysis and priming the myosin head, the recovery stroke may be important in fine-tuning the steady-state distribution of myosin molecules in up and down lever arm orientations and, in turn, the strong and weak actin binding states during the contractile cycle. To test this hypothesis, we designed Dictyostelium myosin II motor domain mutants to perturb the recovery stroke selectively. This step is accompanied by a large rotation of the converter region that causes disruption of its interface to the N-terminal subdomain of the myosin head (Fig. 1, A and B). One good candidate for a directed change in the interaction pattern of this interface is the Lys84 (N-terminal subdomain) -Arg704 (converter) residue pair. In the down (pre-recovery) conformation the two side chains, unusually, run almost parallel to each other and their positive charges are separated by only ∼0. 5 nm (Fig. 1A). In the up (post-recovery) structure this complex is disrupted (Fig. 1B), implying that the introduction of site-directed substitutions in these positions will possibly affect the recovery stroke equilibrium constant. We constructed and characterized the K84M point mutant in which the positive charge of Lys84 was removed and the side chain replaced by a roughly isosteric one and R704E in which we intended to convert the originally repulsive Lys84-Arg704 interaction into a salt bridge. We introduced these point mutations into the W501+ single tryptophan construct, which has been shown to enable very sensitive resolution of the recovery stroke, while retaining essentially identical kinetic properties to the wild-type enzyme (4Malnasi-Csizmadia A. Woolley R. J. Bagshaw C. R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 5Malnasi-Csizmadia A. Pearson D. S. Kovacs M. Woolley R. J. Geeves M. A. Bagshaw C. R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar). In later sections we will refer to the W501+ control as “wild-type” for simplicity. Protein Expression and Purification—All constructs used in this study are derivatives of the M761 Dictyostelium myosin II motor domain (6Kurzawa S. E. Manstein D. J. Geeves M. A. Biochemistry. 1997; 36: 317-323Crossref PubMed Scopus (76) Google Scholar). Construction of the plasmid for the W501+ motor domain containing the point mutations W36F, W432F, and W584F was described earlier (4Malnasi-Csizmadia A. Woolley R. J. Bagshaw C. R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar). The W501+ construct was further mutagenized to yield the K84M and R704E mutants as described previously (7Kovacs M. Toth J. Malnasi-Csizmadia A. Bagshaw C. R. Nyitray L. J. Muscle Res. Cell Motil. 2004; 25: 95-102Crossref PubMed Scopus (2) Google Scholar). The pDXA-3H expression vector was used for constitutive expression of the motor domains (8Manstein D. J. Hunt D. M. J. Muscle Res. Cell Motil. 1995; 16: 325-332Crossref PubMed Scopus (72) Google Scholar). Dictyostelium AX3 cells were transformed and cultured and the recombinant proteins were prepared as described previously (4Malnasi-Csizmadia A. Woolley R. J. Bagshaw C. R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar). Protein concentrations were determined using Bradford reagent (Sigma). Purity of the preparations (>95%) was checked by 9% SDS-PAGE. Nucleotides and Nucleotide Analog Complexes—ATP (special quality, vanadate-free) was from Roche Applied Science. Other nucleotides were purchased from Sigma. The M·ADP·BeFx complex was prepared by incubation of 5–20 μm motor domain, 50 μm ADP, 3 mm NaF, 50 μm BeCl2 for 30 min. The M·ADP·AlF4 complex was made similarly but with 50 μm AlCl3 instead of BeCl2 and incubation for at least 2 h. Kinetic and Spectroscopic Measurements—All measurements were carried out in a buffer comprising 20 mm TES, pH 7. 5, 40 mm NaCl, and 2 mm MgCl2 as described previously (4Malnasi-Csizmadia A. Woolley R. J. Bagshaw C. R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 5Malnasi-Csizmadia A. Pearson D. S. Kovacs M. Woolley R. J. Geeves M. A. Bagshaw C. R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar, 9Pearson D. S. Holtermann G. Ellison P. Cremo C. Geeves M. A. Biochem. J. 2002; 366: 643-651Crossref PubMed Scopus (30) Google Scholar). Reaction profiles were analyzed by fitting to exponential functions using Origin v7. 5 (Microcal Software). Molecular Dynamics Simulations—Protein Data Bank structure 1FMW and its single point mutants were used as inputs in the modeling studies. Energy minimization and subsequent 5-ns molecular dynamics equilibration were performed with the GROMACS program package (10Lindahl E. Hess B. van der Spoel D. J. Mol. Model. 2001; 7: 306-317Crossref Google Scholar). 17800 explicit water molecules surrounded the proteins during calculations. Structural Integrity of the Mutants—Molecular dynamics simulations of the wild-type and mutant proteins in a water box showed that the amino acid substitutions did not cause significant structural changes even in the vicinity of the targeted side chains and that the structures were stable over a 5-ns time scale (Fig. 1, C–E). The charge separation between Lys84 and Glu704 in the R704E mutant was rather similar to that between Lys84 and Arg704 of the wild-type enzyme (0. 5 nm). Also, the side chain of Met84 in the K84M mutant adopted a very similar conformation to the corresponding Lys84 of the wild-type construct and had a similar proximity to Arg704. Steady-state ATPase Activities—The basal (actin-free) steady-state ATPase activity of K84M was ∼1. 5 times, and that of R704E was ∼3 times, higher than that of the wild-type construct (Table 1). In addition to the NADH-linked assay, ATPase activities were confirmed by multiple turnover tryptophan fluorescence (as described in Ref. (4Malnasi-Csizmadia A. Woolley R. J. Bagshaw C. R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar), data not shown). TABLE 1Kinetic and thermodynamic properties of motor domain constructs Rate and equilibrium constants are based on Reaction 1. TWild-typeaW501+ construct (see Introduction) K84MR704E°CSteady-state basal ATPase activity (s–1) 200. 05 ± 0. 010. 08 ± 0. 010. 17 ± 0. 03ATP binding and hydrolysis K1k2 (μm–1 s–1) 201. 10. 941. 1 appkH (s–1) bApparent rate constant of the ATP hydrolysis step (see “Results”) 53. 4 ± 0. 12. 0 ± 0. 031. 8 ± 0. 01 appkH (s–1) bApparent rate constant of the ATP hydrolysis step (see “Results”) 2032 ± 117 ± 0. 212 ± 0. 1Recovery stroke AMPPNP K3acCalculated from tryptophan fluorescence intensities as K3a = (Fx – Fdown) / (Fup – Fx) where Fdown and Fup are the fluorescence intensities in the down (ADP-bound) and up (ADP·AlF4-bound) states, respectively, and Fx is the intensity of the given nucleotide analog complex (Fig. 3, A–C) 100. 13 ± 0. 040. 04 ± 0. 020. 02 ± 0. 00 K3acCalculated from tryptophan fluorescence intensities as K3a = (Fx – Fdown) / (Fup – Fx) where Fdown and Fup are the fluorescence intensities in the down (ADP-bound) and up (ADP·AlF4-bound) states, respectively, and Fx is the intensity of the given nucleotide analog complex (Fig. 3, A–C) 250. 76 ± 0. 300. 28 ± 0. 040. 10 ± 0. 01 ΔH03a (kJ mol–1) dCalculated from the van't Hoff plots of the recovery stroke (ln K3a = –ΔH03a/RT + ΔS03a/R; 3, and ± ± ± (kJ from the van't Hoff plots of the recovery stroke (ln K3a = –ΔH03a/RT + ΔS03a/R; 3, and ± ± ± K3acCalculated from tryptophan fluorescence intensities as K3a = (Fx – Fdown) / (Fup – Fx) where Fdown and Fup are the fluorescence intensities in the down (ADP-bound) and up (ADP·AlF4-bound) states, respectively, and Fx is the intensity of the given nucleotide analog complex (Fig. 3, ± ± ± K3acCalculated from tryptophan fluorescence intensities as K3a = (Fx – Fdown) / (Fup – Fx) where Fdown and Fup are the fluorescence intensities in the down (ADP-bound) and up (ADP·AlF4-bound) states, respectively, and Fx is the intensity of the given nucleotide analog complex (Fig. 3, ± ± ± ΔH03a (kJ mol–1) dCalculated from the van't Hoff plots of the recovery stroke (ln K3a = –ΔH03a/RT + ΔS03a/R; 3, and ± ± ± (kJ from the van't Hoff plots of the recovery stroke (ln K3a = –ΔH03a/RT + ΔS03a/R; 3, and ± ± ± binding and release (μm–1 from the of the of ADP binding on = + from the of the of ADP binding on = + ± ± ± 30 from the of the of ADP binding on = + ± ± ± 20 ADP (Fig. constant of ADP binding = W501+ construct (see rate constant of the ATP hydrolysis step (see from tryptophan fluorescence intensities as K3a = (Fx – Fdown) / (Fup – Fx) where Fdown and Fup are the fluorescence intensities in the down (ADP-bound) and up (ADP·AlF4-bound) states, respectively, and Fx is the intensity of the given nucleotide analog complex (Fig. 3, from the van't Hoff plots of the recovery stroke (ln K3a = –ΔH03a/RT + ΔS03a/R; 3, and from the of the of ADP binding on = + ADP (Fig. constant of ADP binding = in a on Nucleotide earlier showed a on ADP binding from to down ATP binding caused a large fluorescence as the up (4Malnasi-Csizmadia A. Woolley R. J. Bagshaw C. R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 5Malnasi-Csizmadia A. Pearson D. S. Kovacs M. Woolley R. J. Geeves M. A. Bagshaw C. R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar, A. Kovacs M. Woolley R. J. Bagshaw C. R. J. Biol. Chem. 2001; PubMed Scopus Google (Reaction 1). 2 tryptophan fluorescence of the constructs in the absence of and in ADP and The of the caused by ADP was similar in the fluorescence on ATP addition was in R704E than in the other two constructs. The of ADP and ATP binding to were not by the mutations (Fig. We the of the tryptophan fluorescence of the constructs (Fig. 3, The of the of the wild-type construct in the absence of in ADP, and was similar to that of implying that the motor domain with fluorescence intensities in these in the absence of down in ADP, and up in (Fig. A. Pearson D. S. Kovacs M. Woolley R. J. Geeves M. A. Bagshaw C. R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar). The was in ATP and in the ATP and The intensities and in these have been by of the of a reversible equilibrium of the down and up states, the recovery stroke (Fig. A. Pearson D. S. Kovacs M. Woolley R. J. Geeves M. A. Bagshaw C. R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar). In the ADP and (up) states, the K84M and R704E constructs showed similar profiles to of the that the structures of these states are by the mutations (Fig. 3, and the equilibrium constant of the recovery stroke (K3a) could be from fluorescence intensities as K3a = (Fx Fdown) / (Fup Fx) where and Fup are fluorescence intensities in the of the given ADP state), and state), The equilibrium showed that the K84M and even the R704E caused in of the down conformation (Table 1). In ATP the is complex because the apparent equilibrium constant of the recovery stroke is by the hydrolysis step = + Reaction but a shift toward the down in R704E is even in this (Fig. 3, the mutations the equilibrium constant of the recovery stroke (K3a) times, these have only in the of this The with the recovery stroke is of a large = and a (Fig. 3, and in mutants and the of ATP the W501+ motor domain with ATP in the the ATP binding in Reaction is followed by the rapid recovery stroke (K3a) and the subsequent hydrolysis step (4Malnasi-Csizmadia A. Woolley R. J. Bagshaw C. R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 5Malnasi-Csizmadia A. Pearson D. S. Kovacs M. Woolley R. J. Geeves M. A. Bagshaw C. R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar). The of the of the tryptophan fluorescence is by nucleotide binding but the of this will be by the equilibrium constant of the rapid recovery stroke the fluorescence intensity in the down is and in the up is higher than that in the state, the be a a on the of After this is a subsequent fluorescence that the hydrolysis step the over to in the of the fluorescence (Reaction 1). The rate constant of this the apparent rate constant of ATP hydrolysis, will be a of the recovery stroke equilibrium constant and the rate constants of the hydrolysis step = + + Reaction (4Malnasi-Csizmadia A. Woolley R. J. Bagshaw C. R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 5Malnasi-Csizmadia A. Pearson D. S. Kovacs M. Woolley R. J. Geeves M. A. Bagshaw C. R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar). The of the of the on ATP was in of above 1 mm ATP data not The plots had (K1k2) at and at 20 with earlier wild-type data and effect of the mutations on ATP binding (Table (4Malnasi-Csizmadia A. Woolley R. J. Bagshaw C. R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar). a a of an with a in the mutants compared with the wild-type a in the recovery stroke equilibrium constant (K3a) caused by the mutations (Fig. A and B). the of the at 20 ATP A in the was in K84M and even in This the of the data of the the correspondence between the recovery stroke equilibrium constant (K3a) and the apparent rate constant of the hydrolysis step The fluorescence the were in K84M and even in R704E than in the wild-type a in the ATP hydrolysis the to the fluorescence in (Fig. Reaction 1). of the the recovery stroke is than the and subsequent its be only by its using kinetic A. Pearson D. S. Kovacs M. Woolley R. J. Geeves M. A. Bagshaw C. R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar, C. J. Biochem. J. 2001; PubMed Scopus Google Scholar). Following a rapid the motor analog and showed conformational of the reversible and recovery stroke (Fig. The of these tryptophan fluorescence is the of the and rate constants = + and in the and AMPPNP of the motor domain could thus be determined from the of the and the K3a equilibrium constants determined in equilibrium fluorescence measurements (Fig. 3, 1). that the shift toward the down in caused by the mutations is by a in the rate constant Furthermore, in the plots of these rate constants the of were very similar in the of the mutants were from in the wild-type (Fig. and This that the mutations caused a in the of the but the exponential = A where A is the and is the of the recovery constants were from the of were from the + and the K3a from steady-state fluorescence intensities 3 and In of between of the wild-type and mutant constructs were S. E. of measurements with a given nucleotide are in the wild-type of A in as ± ± ± between wild-type and mutants were between wild-type and mutants were ± between wild-type and mutants were between wild-type and mutants were Rate constants were from the of were from the + and the K3a from steady-state fluorescence intensities 3 and In of between of the wild-type and mutant constructs were S. E. of measurements with a given nucleotide are in the wild-type as between wild-type and mutants were in a of ADP and with ADP in the constructs showed a single exponential in tryptophan fluorescence not of the of on ADP = + + showed that the binding step was not by the the rate constant of the subsequent was (Fig. 1, Reaction 1). The rate constants of ADP were determined by in which the motor were with concentrations of ATP in the (Fig. The mutations had effect on the ADP binding equilibrium constant = of the by to perturb the myosin recovery stroke by the repulsive Lys84-Arg704 interaction into a weak in the K84M mutant and a salt in the R704E construct (Fig. 1). mutations are located at the N-terminal which is from the nucleotide binding from the of nucleotide binding to and release from the mutants as well as molecular dynamic the substitutions did not affect the structure of the nucleotide binding site (Fig. 1). the mutations had a effect on the recovery stroke that conformational of the Thus, the mutations charges to have an effect on recovery stroke but did not the structures of the and of this In constructs ADP the same open lever arm and the post-recovery lever arm state, as by intensity (Fig. 3, Other the subdomain interface in the over the repulsive force between Lys84 and Arg704 in the down in to to The wild-type Lys84-Arg704 repulsive interaction only in the down state, and we that this in the wild-type K84M be that the R704E a salt to the same this to have on the properties of this In in the K84M mutant where the salt was the kinetic and thermodynamic on the recovery stroke were in the same as in the R704E The to ATP and Pi the fluorescence profiles of the interaction of the mutants with ATP and their steady-state ATPase we that even a in the equilibrium constant of the rapid and reversible recovery stroke causes significant of ATP hydrolysis, which takes place the recovery stroke (Fig. 1, Reaction 1). Pi which is the step of the basal ATPase cycle and takes place the reversal of the recovery stroke is by the same mutations (Table 1, Reaction 1). This is with earlier in which of Lys84 caused a in the basal steady-state ATPase activity of Dictyostelium myosin II because the during the cycle K. S. A. Biochemistry. PubMed Scopus Google Scholar). of the that the mutations the rate constant of the recovery stroke did not have a effect on the rate constant (Fig. The structural of the effect is the disruption of the subdomain interface containing residues and the recovery stroke that these residues nm from each other (Fig. 1, A and (1Fisher A. J. Smith C. A. Thoden J. B. Smith R. Sutoh K. Holden H. M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (637) Google Scholar). only a interaction be between these side chains, the mutations not affect the rate constant is the mutations targeted a side chain interaction that could be to have an the rather than the apparent of the recovery stroke (Fig. and The mutations in the study affect in the conformation, and the into the to the conformational change and possibly the in the of the The simulations of the of the myosin recovery stroke using as to the of this S. B. D. Holmes K. C. Smith Sci. S. A. 2005; PubMed Scopus Google Scholar). Protein conformational changes that enzyme activity and protein are to these of the in the the effective rate of ATP hydrolysis and Pi the equilibrium constant of the recovery stroke may influence the distribution of steady-state intermediates during the of the actin of these this effect may influence the the of myosin states during steady-state ATPase The is a functional for the effective of as myosin II a by myosin a the ATP hydrolysis step is reversible in the apparent equilibrium constant of the hydrolysis of ATP is between and for C. R. Biochem. J. PubMed Scopus Google Scholar, M. A. J. Biol. Chem. PubMed Scopus Google Scholar, M. J. Biol. Chem. 2005; PubMed Scopus Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar, Scholar, Kovacs M. A. J. J. Biol. Chem. PubMed Scopus Google Scholar). the step is coupled to the recovery stroke A. Pearson D. S. Kovacs M. Woolley R. J. Geeves M. A. Bagshaw C. R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar, C. J. Biochem. J. 2001; PubMed Scopus Google Scholar), the recovery stroke equilibrium constant (K3a) is to be a of in of ATP = + K3a + Reaction 1). in turn, influence the by the rate of into the strong binding Arg704 is at least myosin and Lys84 is only II Scholar). Thus, the residues in the current study may even a role in the functional of which hypothesis further