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• W2099687412 abstract "Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: kcat = 5.5 s-1, K1/2,Mt = 0.7 μm, and Km,ATP = 25 μm; and for Eg5-437: kcat = 2.9 s-1, K1/2,Mt = 4.5 μm, and Km,ATP = 19 μm. 2′(3′)-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 μm-1s-1, followed immediately by ATP hydrolysis at 15 s-1. ATP-dependent Mt·Eg5 dissociation was relatively slow and rate-limiting at 8 s-1 with mantADP release at 40 s-1. Surprisingly, Eg5-367 binds microtubules more effectively (11 μm-1s-1) than Eg5-437 (0.7 μm-1s-1), consistent with the steady-state K1/2,Mt and the mantADP release K1/2,Mt. These results indicate that the ATPase pathway for monomeric Eg5 is more similar to conventional kinesin than the spindle motors Ncd and Kar3, where ADP product release is rate-limiting for steady-state turnover. Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: kcat = 5.5 s-1, K1/2,Mt = 0.7 μm, and Km,ATP = 25 μm; and for Eg5-437: kcat = 2.9 s-1, K1/2,Mt = 4.5 μm, and Km,ATP = 19 μm. 2′(3′)-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 μm-1s-1, followed immediately by ATP hydrolysis at 15 s-1. ATP-dependent Mt·Eg5 dissociation was relatively slow and rate-limiting at 8 s-1 with mantADP release at 40 s-1. Surprisingly, Eg5-367 binds microtubules more effectively (11 μm-1s-1) than Eg5-437 (0.7 μm-1s-1), consistent with the steady-state K1/2,Mt and the mantADP release K1/2,Mt. These results indicate that the ATPase pathway for monomeric Eg5 is more similar to conventional kinesin than the spindle motors Ncd and Kar3, where ADP product release is rate-limiting for steady-state turnover. Eukaryotic cell division requires proper assembly and maintenance of the bipolar spindle, an intricate protein complex composed of a dynamic array of microtubules and microtubule-based motor proteins (reviewed in Refs. 1Inoue S. J. Struct. Biol. 1997; 118: 87-93Crossref PubMed Scopus (21) Google Scholar, 2Compton D.A. Annu. Rev. Biochem. 2000; 69: 95-114Crossref PubMed Scopus (232) Google Scholar, 3Mitchison T.J. Salmon E.D. Nat. 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Hartman G.D. Huber H.E. Kuo L.C. J. Mol. Biol. 2004; 335: 547-554Crossref PubMed Scopus (198) Google Scholar, 36Turner J. Anderson R. Guo J. Beraud C. Fletterick R. Sakowicz R. J. Biol. Chem. 2001; 276: 25496-25502Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Surprisingly, we found that Eg5-437, designed to yield a dimeric motor, is monomeric based on results from sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation as well as analytical gel filtration. The kinetics and equilibrium binding characteristics for Eg5 presented here reveal a unique ATPase mechanism more similar to KinN kinesins, such as conventional kinesin, compared with the well studied KinC spindle motors, Kar3 and Ncd. Experimental Conditions—Experiments were performed at 25 °C in ATPase buffer (20 mm Hepes, pH 7.2, with KOH, 5 mm magnesium acetate, 0.1 mm EDTA, 0.1 mm EGTA, 50 mm potassium acetate, 1 mm dithiothreitol, 5% sucrose). All concentrations reported for experiments are final after mixing. Expression and Purification of Eg5—We have expressed two human Eg5 constructs as described previously (33Maliga Z. Kapoor T.M. Mitchison T.J. Chem. Biol. 2002; 9: 989-996Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Eg5-367 contains the N-terminal 367 amino acids, Met1-Gln367 followed by a His6 tag (molecular mass = 41.7 kDa without MgADP and 42.1 kDa with MgADP), and Eg5-437 contains the N-terminal 437 amino acids, Met1-Thr437, followed by a His6 tag (molecular mass = 49.8 kDa without MgADP and 50.2 kDa with MgADP). Both recombinant Eg5 motors were expressed in the Escherichia coli cell line BL21-CodonPlus(DE3)-RIL (Stratagene, La Jolla, CA) and purified using column chromatography. Briefly, cells were diluted after induction to 1 g/6 ml in lysis buffer (10 mm sodium phosphate buffer, pH 7.2, 20 mm NaCl, 2 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, 2 mm phenylmethylsulfonyl fluoride). The soluble cell lysate was loaded onto a 100-ml S-Sepharose (Sigma-Aldrich Co.) column to select for the Eg5 motor domain. Eg5 was eluted from the S-Sepharose column using a linear NaCl gradient (20-600 mm NaCl). Fractions enriched in Eg5 were pooled and dialyzed against nickel-nitrilotriacetic acid buffer (10 mm sodium phosphate buffer, pH 7.2, 20 mm NaCl, 2 mm MgCl2, 0.1 mm EGTA, 1 mm dithiothreitol, 0.02 mm ATP). The dialysate was loaded onto a 5-ml nickel-nitrilotriacetic acid-agarose (Qiagen, Valencia, CA) column, and Eg5 was eluted using a linear imidazole gradient (0-200 mm imidazole). Fractions enriched in Eg5 were pooled and dialyzed against ATPase buffer plus 200 mm NaCl to remove the imidazole. The dialysate was concentrated by ultrafiltration (Millipore Centriprep 30, Bedford, MA) and further dialyzed against ATPase buffer with 5% sucrose. We determined the Eg5 protein concentration by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc.) with IgG as the protein standard. This purification method yielded 40-150 mg of Eg5 protein per 30 g of E. coli cells at >99% purity. Microtubules for Kinetic Experiments—Microtubules were prepared from bovine brain tubulin, and on the day of each experiment an aliquot of tubulin was thawed, cycled, and stabilized with 20 μm Taxol (paclitaxel, Sigma-Aldrich Co.). Analytical Ultracentrifugation—Sedimentation velocity experiments were conducted at 42,000 rpm, and a sedimentation equilibrium experiment was carried out at 17,000 rpm in a Beckman Optima XLA analytical ultracentrifuge (Beckman Coulter Inc., Fullerton, CA) equipped with absorbance optics and an An60Ti rotor. Velocity and equilibrium data were collected at a wavelength of 235 nm. The conditions for sedimentation velocity experiments included ATPase buffer with and without 50 μm AMP-PNP 1The abbreviations used are: AMP-PNP, adenosine 5′-(β,γ-imino)-triphosphate; Mt, microtubule; mant, 2′(3′)-O-(N-methylanthraniloyl); AXP, ATP or ADP; GST, glutathione S-transferase. (a non-hydrolyzable ATP analog) at 25 °C (Fig. 1A). Velocity data were analyzed by DCDT+ (version 1.15) (74Philo J.S. Anal. Biochem. 2000; 279: 151-163Crossref PubMed Scopus (239) Google Scholar) as described previously (75Correia J.J. Gilbert S.P. Moyer M.L. Johnson K.A. Biochemistry. 1995; 34: 4898-4907Crossref PubMed Scopus (51) Google Scholar) and, where appropriate, SVEDBERG (version 6.39) (76Philo J.S. Biophys. J. 1997; 72: 435-444Abstract Full Text PDF PubMed Scopus (215) Google Scholar) to verify the results. The reported weight average sedimentation coefficient values (s20,w) obtained from DCDT+ were corrected for the solution density and viscosity (75Correia J.J. Gilbert S.P. Moyer M.L. Johnson K.A. Biochemistry. 1995; 34: 4898-4907Crossref PubMed Scopus (51) Google Scholar) and were calculated by a weighted integration over the entire range of sedimentation coefficients covered by the g(s) distribution. Equilibrium data were analyzed by NONLIN (version 1.035) as described previously (75Correia J.J. Gilbert S.P. Moyer M.L. Johnson K.A. Biochemistry. 1995; 34: 4898-4907Crossref PubMed Scopus (51) Google Scholar). Gel Filtration and Stokes Radii Calculations—Purified proteins were resolved by using a Superose-6 HR 10/30 gel filtration column (Amersham Biosciences) equilibrated in ATPase buffer at 25 °C using the System Gold high-pressure liquid chromatography system (Beckman Coulter Inc.) (Fig. 1B). The elution volumes (Ve) of proteins were determined by intrinsic protein fluorescence detection (Jasco FP-2020, Victoria, British Columbia). The Stokes radii (Rs) of Eg5-367, Eg5-437, and Drosophila kinesin heavy chain (K401) were calculated as described previously (77Patel S.S. Hingorani M.M. J. Biol. Chem. 1993; 268: 10668-10675Abstract Full Text PDF PubMed Google Scholar). Briefly, the partition coefficients (Kav) were calculated for four standard proteins of known Stokes radii (Rs): ovalbumin, 3.05 nm; aldolase, 4.81 nm; catalase, 5.22 nm; and ferritin, 6.10 nm. The apparent Stokes radius of each motor was determined from interpolation of a semilog plot of the markers Kavversus the known Rs of the markers (see Table I).Table IPhysical properties of Eg5 motorsProteinPolypeptide molecular massaThe monomer polypeptide with MgADP bound was determined from the amino acid sequence and nucleotide molecular mass (Da).RsbRs is the Stokes radius (nm) determined by analytical gel filtration.s20,wcS20,w is the sedimentation coefficient determined from sedimentation velocity experiments.Calculated molecular massdThe molecular mass (Da) was calculated as described (86).Association stateDaDaEg5-36742,1192.86NDeND, not determined.NDMonomerEg5-43750,2173.373.4948,229MonomerK40145,5314.795.0691,902Dimera The monomer polypeptide with MgADP bound was determined from the amino acid sequence and nucleotide molecular mass (Da).b Rs is the Stokes radius (nm) determined by analytical gel filtration.c S20,w is the sedimentation coefficient determined from sedimentation velocity experiments.d The molecular mass (Da) was calculated as described (86Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1547) Google Scholar).e ND, not determined. Open table in a new tab Phosphocreatine Kinase Coupled Assay—The concentration of active Eg5 sites was determined by performing phosphocreatine kinase coupled assays as previously described (78Gilbert S.P. Mackey A.T. Methods. 2000; 22: 337-354Crossref PubMed Scopus (67) Google Scholar). Briefly, Eg5 (5 μm final concentration) was incubated with trace amounts of [α-32P]ATP for 90 min to convert all radiolabeled ATP to ADP (data not shown). In some experiments, additional non-radiolabeled ADP was then added to yield a final concentration of 5-15 μm [α-32P]ADP. The Eg5·[α-32P]ADP complex was combined with a creatine kinase/phosphocreatine-based ATP regeneration system to convert unbound [α-32P]ADP to [α-32P]ATP. However, ADP tightly bound at the Eg5 active site was inaccessible to the creatine kinase and therefore protected from enzymatic conversion to ATP. This assay measures the Eg5 concentration based on ADP tightly bound at the Eg5 active site. The protected [α-32P]ADP was quantified as a function of time by incubating the Eg5·[α-32P]ADP complex with the regeneration system with or without 2.5 mm unlabeled MgATP (Fig 2A). Each data set was fit to the following single exponential function, [ADP]=A exp(−kofft)+C(Eq. 1) where the active site concentration is the sum of the amplitude (A) and the constant term (C) to extrapolate to zero time (t). In the experiment with excess unlabeled MgATP, koff is the first-order rate constant for ADP release in the absence of microtubules. Steady-State ATPase Kinetics—Eg5 steady-state ATPase activity was determined by following [α-32P]ATP hydrolysis to form [α-32P]ADP·Pi as previously described (78Gilbert S.P. Mackey A.T. Methods. 2000; 22: 337-354Crossref PubMed Scopus (67) Google Scholar). In Fig. 2B, the rate of ATP turnover was plotted as a function of microtubule concentration, and the data were fit to the following quadratic equation,Rate=0.5×kcat×{(E0+K1/2,Mt+Mt0)−[(E0+K1/2,Mt+Mt0)2−(4E0Mt0)]1/2}(Eq. 2) where the Rate is the amount of hydrolysis product formed per second per active site, kcat is the maximum rate constant of product formation at saturating substrate, E0 is the Eg5 site concentration, K1/2,Mt is the tubulin concentration as microtubules needed to provide one-half the maximal velocity, and Mt0 is the microtubule concentration. Mt·Eg5 Cosedimentation Assays—Eg5 at 2 μm was incubated with varying microtubule concentrations (0-6 μm tubulin) in the absence (Fig. 3A) or presence (Fig. 3C) of increasing MgADP concentrations for 30 min, followed by centrifugation in a Beckman Airfuge (Beckman Coulter Inc.) at 30 p.s.i. (100,000 × g) for 30 min as described previously (67Foster K.A. Correia J.J. Gilbert S.P. J. Biol. Chem. 1998; 273: 35307-35318Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The microtubule pellet was resuspended in ATPase buffer to equal the volume of the supernatant. Gel samples for the pellet and supernatant were prepared in 5× Laemmli sample buffer, and the proteins were resolved by SDS-PAGE, followed by staining with Coomassie Brilliant Blue R-250. The gels were analyzed with a Scan Maker X6EL scanner (Microtek, Carson, CA), and the proteins were quantified using National Institutes of Health Image (version 1.62) to determine the fraction of Eg5 in the supernatant and pellet at each microtubule concentration. The fraction of Eg5 that partitioned to the pellet was plotted as a function of microtubule concentration (Fig. 3A), and the data were fit to the following quadratic equation,Mt·E/E0=0.5×{(E0+Kd+Mt0)−[(E0+Kd+Mt0)2−(4E0Mt0)]1/2}(Eq. 3) where Mt·E/E0 is the fraction of Eg5 partitioning with the microtubule pellet, E0 is total Eg5 concentration, Kd is the dissociation constant, and Mt0 is the microtubule concentration. For Fig. 3C, the fraction of Eg5 that partitioned to the pellet was plotted as a function of MgADP concentration. Mt·Eg5·[α-32P]ADP Cosedimentation Assays—Eg5 at 5 μm was incubated with microtubules (6 and 8 μm tubulin for Eg5-367 and Eg5-437, respectively) and with varying concentrations of radiolabeled MgADP (2.5-100 μm) for 30 min as described previously (72Mackey A.T. Gilbert S.P. J. Biol. Chem. 2003; 278: 3527-3535Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The reaction mixture was centrifuged, the supernatant was removed, the unwashed pellet was resuspended in 4 n NaOH, and the volume of the pellet was adjusted with buffer to equal the volume of the supernatant. Control reactions were performed at each microtubule concentration in the absence of Eg5 to determine the amount of radiolabeled nucleotide that was non-specifically trapped in the microtubule pellet. The concentration of [α-32P]ADP for each Mt·Eg5 reaction was corrected for [α-32P]ADP that partitioned with microtubules in the absence of motor. Fig. 3B shows the data fit to the following quadratic equation,Mt·E·ADP=0.5×{(E0+Kd+ADP)−[(E0+Kd+ADP)2−(4E0ADP)]1/2}(Eq. 4) where Mt·E·ADP is the concentration of ADP bound to the Mt·Eg5 complex, E0 is total Eg5, ADP is the total nucleotide present, and Kd is the dissociation constant for ADP. Acid-quench Experiment—The pre-steady-state kinetics of MgATP hydrolysis for Eg5 in the presence of microtubules was defined in this assay. A preformed Mt·Eg5 complex was rapidly mixed with varying concentrations of radiolabeled MgATP plus 100 mm KCl in a quench-flow instrument (Kintek Corp., Austin, TX). We added the additional salt in the ATP syringe to lower steady-state turnover without affecting the pre-steady-state burst of product formation (Fig. 5A). The reaction was incubated (from 10 ms to 1 s) and quenched with formic acid, and radiolabeled product formation was quantified after separating [α-32P]ADP plus Pi from unreacted [α-32P]ATP by thin layer chromatography (78Gilbert S.P. Mackey A.T. Methods. 2000; 22: 337-354Crossref PubMed Scopus (67) Google Scholar). The concentration of [α-32P]ADP was plotted as a function of time (Fig. 5B), and the data were fit to the following burst equation,Product=(A×[1−exp(−kbt)])+ksst(Eq. 5) where A is the amplitude of the initial rapid exponential phase, which corresponds to the formation of [α-32P]ADP·Pi at the Eg5 active site during the first turnover, kb is the rate constant of the exponential burst phase, t is time in seconds, and kss is the rate constant of the linear phase (μm ADP·s-1) corresponding to steady-state turnover. The exponential rate of the pre-steady-state burst (kb) and burst amplitude (A) (Fig. 5C) were plotted as a function of MgATP concentration, and each data set was fit to a hyperbola. Pulse-chase Experiment—This experiment measures the time course of the formation of a kinetically stable Mt·Eg5·ATP intermediate prior to ATP hydrolysis (48Gilbert S.P. Johnson K.A. Biochemistry. 1994; 33: 1951-1960Crossref PubMed Scopus (86) Google Scholar). In these experiments, the time course of ATP turnover (from 10 ms to 1 s) was measured by chasing the Mt·Eg5·[α-32P]ATP intermediate with ∼5 mm MgATP (15 mm MgATP in the quench syringe) for 4 s (8-10 half-lives) in the quench-flow instrument. The reaction mixture was terminated with formic acid, and the radiolabeled product was quantified. The data in Fig. 6 (A and C) were fit to Equation 3, and each data set in Fig. 6B was fit to a hyperbola. Stopped-flow Experiments—The pre-steady-state kinetics of mantAT" @default.
• W2099687412 created "2016-06-24" @default.
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• W2099687412 date "2004-09-01" @default.
• W2099687412 modified "2023-10-14" @default.
• W2099687412 title "Mechanistic Analysis of the Mitotic Kinesin Eg5" @default.
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• W2099687412 doi "https://doi.org/10.1074/jbc.m404203200" @default.
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