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A Kinesin Mutation That Uncouples Motor Domains and Desensitizes the γ-Phosphate Sensor

2000; Elsevier BV; Volume: 275; Issue: 29 Linguagem: Inglês

10.1074/jbc.m001124200

1083-351X

Katherine M. Brendza, Christopher A. Sontag, William M. Saxton, Susan P. Gilbert,

Mitochondrial Function and Pathology

Conventional kinesin is a processive, microtubule-based motor protein that drives movements of membranous organelles in neurons. Amino acid Thr291 ofDrosophila kinesin heavy chain is identical in all superfamily members and is located in α-helix 5 on the microtubule-binding surface of the catalytic motor domain. Substitution of methionine at Thr291 results in complete loss of function in vivo. In vitro, the T291M mutation disrupts the ATPase cross-bridge cycle of a kinesin motor/neck construct, K401-4 (Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506–31514). The pre-steady-state kinetic analysis presented here shows that ATP binding is weakened significantly, and the rate of ATP hydrolysis is increased. The mutant motor also fails to distinguish ATP from ADP, suggesting that the contacts important for sensing the γ-phosphate have been altered. The results indicate that there is a signaling defect between the motor domains of the T291M dimer. The ATPase cycles of the two motor domains appear to become kinetically uncoupled, causing them to work more independently rather than in the strict, coordinated fashion that is typical of kinesin. Conventional kinesin is a processive, microtubule-based motor protein that drives movements of membranous organelles in neurons. Amino acid Thr291 ofDrosophila kinesin heavy chain is identical in all superfamily members and is located in α-helix 5 on the microtubule-binding surface of the catalytic motor domain. Substitution of methionine at Thr291 results in complete loss of function in vivo. In vitro, the T291M mutation disrupts the ATPase cross-bridge cycle of a kinesin motor/neck construct, K401-4 (Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506–31514). The pre-steady-state kinetic analysis presented here shows that ATP binding is weakened significantly, and the rate of ATP hydrolysis is increased. The mutant motor also fails to distinguish ATP from ADP, suggesting that the contacts important for sensing the γ-phosphate have been altered. The results indicate that there is a signaling defect between the motor domains of the T291M dimer. The ATPase cycles of the two motor domains appear to become kinetically uncoupled, causing them to work more independently rather than in the strict, coordinated fashion that is typical of kinesin. kinesin heavy chain Drosophila kinesin heavy chain gene KHC fragment containing the N-terminal 401 amino acids wild type K401 K401 with T291M amino acid substitution 5′-adenylyl imidodiphosphate microtubule microtubule-K401 complex 2′(3′)-O-(N-methylanthraniloyl)-adenosine 5′-triphosphate 2′(3′)-O-(N-methylanthraniloyl)-adenosine 5′-diphosphate Structural analyses of kinesin (2.Kull F.J. Vale R.D. Fletterick R.J. J. Muscle Res. Cell Motil. 1998; 19: 877-886Crossref PubMed Scopus (140) Google Scholar, 3.Kozielski F. Sack S. Marx A. Thormählen M. Schönbrunn E. Biou V. Thompson A. Mandelkow E.M. Mandelkow E. Cell. 1997; 91: 985-994Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 4.Sack S. Müller A. Marx M. Thormählen M. Mandelkow E.-M. Brady S.T. Mandelkow E. Biochemistry. 1997; 36: 16155-16165Crossref PubMed Scopus (181) Google Scholar, 5.Kull F.J. Sablin E.P. Lau R. Fletterick R.J. Vale R.D. Nature. 1996; 380: 550-555Crossref PubMed Scopus (581) Google Scholar) and family relatives Ncd (6.Sablin E.P. Kull F.J. Cooke R. Vale R.D. Fletterick R.J. Nature. 1996; 380: 555-559Crossref PubMed Scopus (324) Google Scholar,7.Sablin E.P. Case R.B. Dai S.C. Hart C.L. Ruby A. Vale R.D. Fletterick R.J. Nature. 1998; 395: 813-816Crossref PubMed Scopus (185) Google Scholar) and Kar3 (8.Gulick A.M. Song H. Endow S.A. Rayment I. Biochemistry. 1998; 37: 1769-1776Crossref PubMed Scopus (89) Google Scholar) have revealed that the topography of the nucleotide-binding pocket is quite similar to that of myosins and the G proteins (9.Vale R.D. J. Cell Biol. 1996; 135: 291-302Crossref PubMed Scopus (244) Google Scholar, 10.Furch M. Fujita-Becker S. Geeves M.A. Holmes K.C. Manstein D.J. J. Mol. Biol. 1999; 290: 797-809Crossref PubMed Scopus (73) Google Scholar). The results suggest further that these proteins may share a common mechanism to sense the state of the nucleotide bound at the active site and to respond through structural transitions to communicate with protein partners. For dimeric kinesin, coordination of the motor domains is controlled in part by the nucleotide state at the active sites (11.Hackney D.D. Proc. Natl. Acad. Sci. 1994; 91: 6865-6869Crossref PubMed Scopus (307) Google Scholar, 12.Ma Y.Z. Taylor E.W. J. Biol. Chem. 1997; 272: 724-730Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 13.Gilbert S.P. Moyer M.L. Johnson K.A. Biochemistry. 1998; 37: 792-799Crossref PubMed Scopus (154) Google Scholar) and is required for the cyclic interaction with the microtubule lattice for unidirectional, processive movement. Through pre-steady-state kinetic analysis, it has been determined that ATP binding by the microtubule-bound motor domain allows the partner motor domain to bind to the microtubule and quickly release its ADP (12.Ma Y.Z. Taylor E.W. J. Biol. Chem. 1997; 272: 724-730Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 13.Gilbert S.P. Moyer M.L. Johnson K.A. Biochemistry. 1998; 37: 792-799Crossref PubMed Scopus (154) Google Scholar). Other points of coordination are also evident. Thus, the two motor domains of the kinesin heavy chain (KHC)1 dimer are at different stages in their ATPase cycles at any given time, keeping the heads out of phase and allowing for processive movement (reviewed in Refs. 14.Block S.M. J. Cell Biol. 1998; 140: 1281-1284Crossref PubMed Scopus (38) Google Scholar and15.Mandelkow E. Johnson K.A. Trends Biochem. Sci. 1998; 23: 429-433Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Much has been learned about kinesin through structural studies. The first motor domain structural intermediate to be determined was the KHC·ADP intermediate (3.Kozielski F. Sack S. Marx A. Thormählen M. Schönbrunn E. Biou V. Thompson A. Mandelkow E.M. Mandelkow E. Cell. 1997; 91: 985-994Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 4.Sack S. Müller A. Marx M. Thormählen M. Mandelkow E.-M. Brady S.T. Mandelkow E. Biochemistry. 1997; 36: 16155-16165Crossref PubMed Scopus (181) Google Scholar, 5.Kull F.J. Sablin E.P. Lau R. Fletterick R.J. Vale R.D. Nature. 1996; 380: 550-555Crossref PubMed Scopus (581) Google Scholar). Recently, the structures of KHC under conditions that mimic different nucleotide states were solved (16.Müller J. Marx A. Sack S. Song Y.-H. Mandelkow E. Biol. Chem. 1999; 380: 981-982Crossref PubMed Scopus (22) Google Scholar). This work revealed that in the absence of microtubules there is little change in KHC structure regardless of the nucleotide present. Thus, with no existing structures of microtubule·kinesin intermediates, the transition states important for efficient ATP hydrolysis remain enigmatic. Because it is difficult to integrate mechanistic models with the available information, alternative approaches are required to understand the structural requirements for motor coordination and processive movement. Site-directed mutagenesis has already identified structural elements that are important for certain aspects of KHC function. Studies by Woehlke et al. (17.Woehlke G. Ruby A.K. Hart C.L. Ly B. Hom-Booher N. Vale R.D. Cell. 1997; 90: 207-216Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar) highlighted charged surface residues necessary for the interaction of KHC with microtubules. Romberget al. (18.Romberg L. Pierce D.W. Vale R.D. J. Cell Biol. 1998; 140: 1407-1416Crossref PubMed Scopus (139) Google Scholar) showed that the neck region of KHC is important for efficiency but is not essential for processive movement. They proposed a model in which interactions between the β-sheet region of the neck (β9 and β10) and the catalytic domain are disrupted such that the neck region could adopt a more extended conformation to allow head separation during times when both heads are bound to the microtubule. This hypothesis is supported by the cryo-EM diffraction studies of microtubule·kinesin complexes by Hoenger et al.(19.Hoenger A. Sack S. Thormählen M. Marx A. Müller J. Gross H. Mandelkow E. J. Cell Biol. 1998; 141: 419-430Crossref PubMed Scopus (118) Google Scholar). Studies of mutant KHCs may help us understand both the ATPase mechanism of kinesin and associated structural rearrangements that allow for efficient utilization of the energy generated through ATP hydrolysis. Moore et al. (20.Moore J.D. Song H. Endow S.A. EMBO J. 1996; 15: 3306-3314Crossref PubMed Scopus (24) Google Scholar) determined that a point mutation in β6 of Ncd greatly reduces the affinity of the motor for microtubules and reduces the velocity of microtubule-based movement. Song and Endow (21.Song H.B. Endow S.A. Nature. 1998; 396: 587-590Crossref PubMed Scopus (52) Google Scholar) have shown that a point mutation in α4 of both Kar3 and Ncd uncouple the nucleotide and microtubule-binding sites, perhaps due to a break in Loop11-mediated communication between the two sites. Recent studies by Rice et al. (22.Rice S. Lin A.W. Safer D. Hart C.L. Naber N. Carragher B.O. Cain S.M. Pechatnikova E. Wilson-Kubalek E.M. Whittaker M. Pate E. Cooke R. Taylor E.W. Milligan R.A. Vale R.D. Nature. 1999; 402: 778-784Crossref PubMed Scopus (650) Google Scholar) have provided insight into structural changes that may allow for force generation and plus end-directed movement of KHC. They have shown that ATP binding promotes a large conformational change in the kinesin neck linker that is directed toward the plus end of the microtubule. These results with monomeric kinesin K349 emphasize that there are conformational transitions in the microtubule-bound kinesin dimer during ATP turnover that are beyond the view of current crystallography efforts. To gain insight into the mechanisms of kinesin mechanochemistryin vivo, we screened for recessive lethal mutations in the kinesin heavy chain (Khc) that disrupt axonal transport (1.Brendza K.M. Rose D.J. Gilbert S.P. Saxton W.M. J. Biol. Chem. 1999; 274: 31506-31514Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar,23.Saxton W.M. Hicks J. Goldstein L.S.B. Raff E.C. Cell. 1991; 64: 1093-1102Abstract Full Text PDF PubMed Scopus (163) Google Scholar). One of the mutations found, Khc 4, causesin vivo phenotypes that suggest a nearly complete loss of function. Sequencing revealed that there is a threonine to methionine substitution at amino acid position 291, an almost completely conserved residue in the kinesin superfamily. 2Greene, E. A., and Henikoff, S., The kinesin home page. 2000.http://www.blocks.fhcrc.org/∼kinesin/. Thr291 is located in α-helix 5, which lies on the microtubule-binding surface of the motor domain. The steady-state ATPase kinetics showed that this mutation causes defects in both ATP and microtubule binding. The k cat is very similar to wild type, yet there is a 3-fold increase in bothK m, ATP andK 0.5, Mt (1.Brendza K.M. Rose D.J. Gilbert S.P. Saxton W.M. J. Biol. Chem. 1999; 274: 31506-31514Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In an effort to determine more precisely how this mutation alters the ATPase cycle and consequently how Thr291 contributes to KHC mechanochemistry, we have pursued a mechanistic analysis of K401-4. The results provide direct evidence that ATP binding is significantly weakened by the T291M mutation and that the binding pocket has lost the ability to distinguish ATP from ADP. These data suggest that structural arrangements important for sensing the γ-phosphate have been disrupted. Furthermore, the kinetics indicate that the two motor domains of the mutant dimer are defective in their head-head communication. Thus, the T291M substitution allows the motor domains to work more independently rather than in the strictly coordinated fashion that is characteristic of kinesin. [α-32P]ATP (>3000 Ci/mmol) was from NEN Life Science Products; polyethyleneimine-cellulose TLC plates (EM Science of Merck, 20 × 20 cm, plastic-backed) were from VWR Scientific (Bridgeport, NJ); Taxol (Taxus brevifolia) was from Calbiochem-Novabiochem; ATP, GTP, AMP-PNP, and S-Sepharose were from Amersham Pharmacia Biotech (Uppsala, Sweden); Bio-Rad Protein Assay, ovalbumin, IgG, and DEAE-Sephacel were from Bio-Rad. K401-4 was bacterially expressed, purified, and characterized as described previously (1.Brendza K.M. Rose D.J. Gilbert S.P. Saxton W.M. J. Biol. Chem. 1999; 274: 31506-31514Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Six preparations of K401-4 were used in the experiments reported, and each was evaluated to determine the steady state parameters:k cat = 18 ± 0.6 s−1, K m, ATP = 224 ± 4.9 μm, and K 0.5, Mt= 3.6 ± 0.01 μm. All experiments reported were performed in ATPase buffer (50 mm HEPES, pH 7.2, with KOH, 5 mm magnesium acetate, 0.1 mm EGTA, 0.1 mm EDTA, 50 mm potassium acetate) at 25 °C. Experiments were conducted at 42,000 rpm in a Beckman Optima XLA analytical ultracentrifuge equipped with absorbance optics and an An60Ti rotor. ATPase buffer was used at 24.7 °C. Velocity data were analyzed using DCDT+, version 1.12 (25.Philo J.S. Anal. Biochem. 2000; 279: 151-163Crossref PubMed Scopus (239) Google Scholar). The reported weight average sedimentation coefficient values ( s20,w) obtained from DCDT+ are corrected for the solution density and viscosity (26.Correia J.J. Gilbert S.P. Moyer M.L. Johnson K.A. Biochemistry. 1995; 34: 4898-4907Crossref PubMed Scopus (51) Google Scholar) and are calculated by a weighted integration over the entire range of sedimentation coefficients covered by the g(s) distribution. To verify these results, the data were also analyzed with DCDT (27.Stafford III, W.F. Anal. Biochem. 1992; 203: 295-301Crossref PubMed Scopus (523) Google Scholar) and, where appropriate, SVEDBERG, version 6.37 (28.Philo J.S. Biophys. J. 1997; 72: 435-444Abstract Full Text PDF PubMed Scopus (215) Google Scholar). Purified tubulin was cold-depolymerized and clarified the morning of each experiment. Microtubules were assembled by the addition of taxol to 20 μm. The microtubules were collected by centrifugation, and the microtubule pellet was resuspended in ATPase buffer plus 20 μm taxol to stabilize the microtubules (29.Gilbert S.P. Johnson K.A. Biochemistry. 1993; 32: 4677-4684Crossref PubMed Scopus (77) Google Scholar). For all of the experiments in which microtubules were present, 20 μm taxol was included to maintain the polymer state. The pre-steady-state kinetics of mantATP binding, mantADP release, K401 binding to microtubules, and detachment of K401 from microtubules were measured using a KinTek StopFlow Instrument (SF-2001, KinTek Corp., Austin, TX) at 25 °C in ATPase buffer. N-methylanthraniloyl fluorescence (mantATP and mantADP) was excited at 360 nm (mercury arc lamp) and detected after being passed through a 400-nm cut-off filter. mantATP binding data in Fig. 1 B were fit to the equation, kobs=k+1[mantATP]+k−1Equation 1 where k obs is the rate constant obtained from the exponential phase of the fluorescence change,k +1 defines the second order rate constant for mantATP binding, and k −1 corresponds to the observed rate constant of mantATP release as determined by they intercept. The dissociation kinetics of Mt·K401 complex and association kinetics of K401 with microtubules were determined by the change in turbidity monitored at 340 nm. All concentrations reported are final after mixing. For Fig. 3 B, the observed rate constants of microtubule association were fit to the equation, kobs=k+5[tubulin]+k−5Equation 2 where k obs is the rate constant obtained from the fast, exponential phase, k +5 defines the second order rate constant for microtubule association, andk −5 corresponds to the observed rate constant of motor dissociation as determined by the y intercept.Figure 3Kinetics of microtubule binding. 4 μm K401-4·ADP was rapidly mixed with varying concentrations of microtubules (2.5–12 μm), and turbidity was monitored in the stopped-flow apparatus. A, a representative stopped-flow record at 7 μm microtubules, which is the average of seven traces. The smooth line is the best fit of the data to a single exponential plus a linear term. The rate constant of the initial exponential phase is 70 ± 1.6 s−1. The second phase at a rate of 0.07 ± 0.001 s−1 is too slow to be considered on the pathway and is attributed to a conformational change after microtubule association. B, the rate constant from the initial fast phase of each transient was plotted as a function of microtubule concentration. The data were fit to Equation 2, the slope providing the apparent second order rate constant for microtubule association, k +5 = 8.2 ± 0.5 μm−1s−1, and the y intercept providingk −5 = 14.3 ± 3.7 s−1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The pre-steady-state experiments to determine the rate constant of ATP hydrolysis were performed with a rapid chemical quenched-flow instrument (KinTek Corp., Austin, TX) at 25 °C in ATPase buffer as described previously (30.Gilbert S.P. Johnson K.A. Biochemistry. 1994; 33: 1951-1960Crossref PubMed Scopus (86) Google Scholar). For each time point, a preformed Mt·K401 complex (10 μm K401, 25 μm tubulin, 20 μm taxol; final concentrations after mixing) was reacted with [α-32P]ATP for times ranging from 5 to 400 ms. The reaction mixture was then quenched with 4 n HCl and expelled from the instrument. Chloroform (100 μl) was added immediately to the reaction mix to denature the protein, followed by neutralization (pH 7–7.8) by the addition of 2 m Tris, 3m NaOH. The acid quench stops the reaction and denatures the protein; therefore, the product formed is the sum of three intermediates:K·[α-32P]ADP·Pi,K·[α-32P]ADP, and [α-32P]ADP released from the active site. The concentration of product ([α-32P]ADP) was plotted as a function of time, and the data were fit to the burst equation, Product=A∗[1−exp(−kbt)]+ksstEquation 3 where A is the amplitude of the burst, representing the formation of [α-32P]ADP·Pi at the active site; k b is the rate constant of the pre-steady-state burst; k ss is the rate constant of the linear phase, corresponding to steady-state turnover; andt is the time in seconds. All molecular modeling was performed on a Silicon Graphics workstation using UCSF MidasPlus Molecular Interactive Display and Simulation software (Computer Graphics Laboratory, University of California, San Francisco, CA). To explore the apparent weak binding of ATP by K401-4 revealed by the steady-stateK m, ATP (1.Brendza K.M. Rose D.J. Gilbert S.P. Saxton W.M. J. Biol. Chem. 1999; 274: 31506-31514Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), we began the pre-steady-state kinetic analysis by measuring the kinetics of ATP binding and ATP hydrolysis (Figs. 1 and2). For the ATP binding studies, the fluorescent ATP analog mantATP was used (Fig. 1). This analog has been used in previous characterizations of kinesin motors and has been shown experimentally to be a good ATP analog because of the fluorescence enhancement and similarity to ATP (31.Sadhu A. Taylor E.W. J. Biol. Chem. 1992; 267: 11352-11359Abstract Full Text PDF PubMed Google Scholar, 32.Gilbert S.P. Webb M.R. Brune M. Johnson K.A. Nature. 1995; 373: 671-676Crossref PubMed Scopus (248) Google Scholar). For K401-wt, the microtubule-activated steady-state k cat reported was 19 s−1 for both ATP and mantATP with the K m, ATP = 62 μm andK m, mantATP = 150 μm(32.Gilbert S.P. Webb M.R. Brune M. Johnson K.A. Nature. 1995; 373: 671-676Crossref PubMed Scopus (248) Google Scholar). The preformed Mt·K401-4 complex (30 μmmicrotubules, 10 μm K401-4) was rapidly mixed with varying concentrations of mantATP in the stopped-flow apparatus, and the change in fluorescence was monitored. A representative stopped-flow record is shown in Fig. 1 A. Binding of mantATP causes a biphasic fluorescence transient with a rapid exponential increase in fluorescence (associated with mantATP binding) followed by a significantly slower exponential decrease in fluorescence. The observed decrease in fluorescence is independent of substrate concentration (k obs = 3.1–3.8 s−1) and is not fast enough to be attributed to ATP hydrolysis. This biphasic fluorescence transient is characteristic of kinesin and has been observed by others (34.Moyer M.L. Gilbert S.P. Johnson K.A. Biochemistry. 1998; 37: 800-813Crossref PubMed Scopus (127) Google Scholar, 36.Ma Y.Z. Taylor E.W. Biochemistry. 1995; 34: 13242-13251Crossref PubMed Scopus (103) Google Scholar). The smooth line is the best fit of the data to a double exponential, providing k obsof the initial, fast reaction at 135 s−1. The rate of the fast exponential phase increased as a function of ATP concentration, and the data were fit to Equation 1 (Fig.1 B). The slope of the line provided the second order rate constant for mantATP binding, k +1 = 1 μm−1s−1, which is similar to the observed rate constant for K401-wt at 2 μm−1s−1 (30.Gilbert S.P. Johnson K.A. Biochemistry. 1994; 33: 1951-1960Crossref PubMed Scopus (86) Google Scholar, 34.Moyer M.L. Gilbert S.P. Johnson K.A. Biochemistry. 1998; 37: 800-813Crossref PubMed Scopus (127) Google Scholar). The y intercept predicts an off rate for mantATP with k −1 = 157 s−1. We next measured the kinetics of ATP hydrolysis for K401-4 through a series of acid quench experiments (Fig. 2). The preformed Mt·K401-4 complex (25 μm microtubules, 10 μm K401-4) was rapidly mixed with [α-32P]MgATP in the chemical quench instrument. Fig. 2 A shows the time course of ATP hydrolysis by K401-4 at six different ATP concentrations. Each transient was biphasic with an initial exponential rate of ADP·Piformation (the burst), followed by a slower rate of product formation (the linear phase) corresponding to steady-state turnover. The exponential burst of product formation at the active site indicates that a step after ATP hydrolysis is rate-limiting for K401-4 as observed for K401-wt (30.Gilbert S.P. Johnson K.A. Biochemistry. 1994; 33: 1951-1960Crossref PubMed Scopus (86) Google Scholar). At high ATP concentrations, ATP binding becomes faster than ATP hydrolysis. Because substrate binding is no longer limiting, the maximum rate constant for the exponential burst phase is the rate constant of ATP hydrolysis. The rate constant for ATP hydrolysis (k +2 = 257 s−1) was determined by plotting the burst rates as a function of ATP concentration (Fig. 2 B) and fitting the data to a hyperbola. Note that the K401-4 rate constant (257 s−1) is significantly faster than the 100 s−1 rate constant observed for dimeric K401-wt ATP hydrolysis (30.Gilbert S.P. Johnson K.A. Biochemistry. 1994; 33: 1951-1960Crossref PubMed Scopus (86) Google Scholar). It is interesting that a monomeric KHC motor domain (K341) also shows a rapid rate of ATP hydrolysis at >300 s−1 (35.Moyer M.L. Gilbert S.P. Johnson K.A. Biochemistry. 1996; 35: 6321-6329Crossref PubMed Scopus (30) Google Scholar). For K401-4, theK d,ATP is 236 μm (Fig.2 B), which is equivalent to the steady-stateK m, ATP at 236 μm (1.Brendza K.M. Rose D.J. Gilbert S.P. Saxton W.M. J. Biol. Chem. 1999; 274: 31506-31514Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Comparison of the K d, ATP for K401-wt at 60 μm (30.Gilbert S.P. Johnson K.A. Biochemistry. 1994; 33: 1951-1960Crossref PubMed Scopus (86) Google Scholar) with theK d, ATP for K401-4 indicates that the mutant motor binds ATP much more weakly. The acid quench experiments provide direct evidence that the T291M mutation significantly weakens ATP binding and increases the rate of ATP hydrolysis. We next looked at the rate of K401-4·ADP binding to the microtubule (Fig.3) to determine if a microtubule binding defect was indeed contributing to the abnormally highK 0.5, Mt measured through steady-state analysis (1.Brendza K.M. Rose D.J. Gilbert S.P. Saxton W.M. J. Biol. Chem. 1999; 274: 31506-31514Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The rate of formation of the Mt·K401-4 complex was monitored by turbidity measurements in the stopped-flow instrument. K401-4·ADP (4 μm) was rapidly mixed with taxol-stabilized microtubules (7 μm), and the change in turbidity was recorded. In this experiment, an increase in turbidity was interpreted as binding of K401 to the microtubule. A representative stopped-flow record is shown in Fig. 3 A. The solid line is the best fit of the data to a single exponential function and a linear term, providing the k obs of the fast exponential phase at 70 s−1. Fig. 3 B shows that the observed rates of microtubule association increased linearly as a function of microtubule concentration. These data were fit to Equation2, the slope of which provides the apparent second order rate constant for binding k +5 = 8 μm−1s−1; this constant is somewhat less than K401-wt measured at 11–15 μm−1s−1 (15.Mandelkow E. Johnson K.A. Trends Biochem. Sci. 1998; 23: 429-433Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 32.Gilbert S.P. Webb M.R. Brune M. Johnson K.A. Nature. 1995; 373: 671-676Crossref PubMed Scopus (248) Google Scholar). The y intercept predicts a significant off-rate (k −5 = 14 s−1), which was not seen for K401-wt (32.Gilbert S.P. Webb M.R. Brune M. Johnson K.A. Nature. 1995; 373: 671-676Crossref PubMed Scopus (248) Google Scholar). Therefore, the T291M mutation appears to weaken the binding between the KHC motor domains and the microtubule. We also measured the kinetics of ADP release from the Mt·K401-4·ADP intermediate (Fig.4) using mantADP. K401-4 was incubated with mantADP at a ratio of 4:1 to allow for exchange of ADP resident at the active site with mantADP. A preformed K401-4·mantADP complex (3 μm K401-4, 12 μm mantADP) was rapidly mixed with microtubules plus MgATP in the stopped-flow apparatus. As mantADP was released from the more hydrophobic environment of the active site into the aqueous buffer, its fluorescence was quenched. The MgATP (1 mm) present in solution blocked the subsequent rebinding of any mantADP to K401. Fig. 4 A shows a representative stopped-flow record. The solid line is the fit of the data to a single exponential function and a linear term, providing the k obs of the fast exponential reaction at 31 s−1. Fig. 4 B shows that the exponential rate constant associated with the fluorescence change upon mantADP release increased with increasing microtubule concentration. The fit of the data to a hyperbola provides a maximum rate constant of mantADP release, k +6 = 234 s−1 with a K 0.5, Mt at 34 μm. The rate constant of mantADP release is consistent with the fast ADP dissociation kinetics reported for K401-wt at 200–300 s−1 (32.Gilbert S.P. Webb M.R. Brune M. Johnson K.A. Nature. 1995; 373: 671-676Crossref PubMed Scopus (248) Google Scholar), yet theK 0.5, Mt for K401-4 is larger than that for K401-wt (15 μm). These results are consistent with the interpretation that the T291M mutant protein requires a higher concentration of microtubules both for half-maximal activation of steady-state turnover at 3.6 versus 1 μm for K401-wt (1.Brendza K.M. Rose D.J. Gilbert S.P. Saxton W.M. J. Biol. Chem. 1999; 274: 31506-31514Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and for mantADP release (34 versus 15 μm for K401-wt). The effects of the T291M mutation on ATP-promoted detachment of K401·ADP from the microtubule were examined by following changes in turbidity (Fig. 5). The preformed Mt·K401-4 complex (2.9 μm microtubules, 3 μm K401-4) was rapidly mixed with MgATP plus 100 mm KCl in the stopped-flow apparatus. In this experiment, a decrease in turbidity was interpreted as release of the motor from the microtubule. The addition of 100 mm KCl weakens rebinding of the motor to the microtubule after detachment to allow for accurate measurement of the dissociation kinetics (32.Gilbert S.P. Webb M.R. Brune M. Johnson K.A. Nature. 1995; 373: 671-676Crossref PubMed Scopus (248) Google Scholar). Fig. 5 A shows a representative stopped-flow record. The solid line is the best fit of the data to a double exponential, providing a k obs of the initial fast exponential phase at 49 s−1. Thek obs increased as a function of ATP concentration, and the fit of the data to a hyperbola provides a maximum rate constant of detachment, k +3 = 60 s−1 with a K 0.5, ATPof 280 μm (Fig. 5 B). ThisK 0.5, ATP is in agreement with both theK m, ATP at steady-state conditions (1.Brendza K.M. Rose D.J. Gilbert S.P. Saxton W.M. J. Biol. Chem. 1999; 274: 31506-31514Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and the K d,ATP determined by acid quench experiments (Fig. 2 B), reinforcing the interpretation that K401-4 binds ATP weakly. It is striking that for both K401-wt and a dimeric human KHC construct K379 (36.Ma Y.Z. Taylor E.W. Biochemistry. 1995; 34: 13242-13251Crossref PubMed Scopus (103) Google Scholar), this ATP-promoted dissociation step was observed at rates of 12–14 s−1, substantially slower than the 60 s−1 observed for K401-4. The motor domains of wild type dimeric KHC are coupled at this step such that ATP binding by the microtubule-attached motor domain stimulates microtubule binding of the partner motor domain and rapid release of its ADP (11.Hackney D.D. Proc. Natl. Acad. Sci. 1994; 91: 6865-6869Crossref PubMed Scopus (307) Google Scholar, 12.Ma Y.Z. Taylor E.W. J. Biol. Chem. 1997; 272: 724-730Abstract Full Text Full Text PDF PubMed Scopus (166)