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• W2116895384 abstract "Switch I and II are key active site structural elements of kinesins, myosins, and G-proteins. Our analysis of a switch I mutant (R210A) in Drosophila melanogaster kinesin showed a reduction in microtubule affinity, a loss in cooperativity between the motor domains, and an ATP hydrolysis defect leading to aberrant detachment from the microtubule. To investigate the conserved arginine in switch I further, a lysine substitution mutant was generated. The R210K dimeric motor has lost the ability to hydrolyze ATP; however, it has rescued microtubule function. Our results show that R210K has restored microtubule association kinetics, microtubule affinity, ADP release kinetics, and motor domain cooperativity. Moreover, the active site at head 1 is able to distinguish ATP, ADP, and AMP-PNP to signal head 2 to bind the microtubule and release mantADP with kinetics comparable with wild-type. Therefore, the structural pathway of communication from head 1 to head 2 is restored, and head 2 can respond to this signal by binding the microtubule and releasing mantADP. Structural modeling revealed that lysine could retain some of the hydrogen bonds made by arginine but not all, suggesting a structural hypothesis for the ability of lysine to rescue microtubule function in the Arg210 mutant. Switch I and II are key active site structural elements of kinesins, myosins, and G-proteins. Our analysis of a switch I mutant (R210A) in Drosophila melanogaster kinesin showed a reduction in microtubule affinity, a loss in cooperativity between the motor domains, and an ATP hydrolysis defect leading to aberrant detachment from the microtubule. To investigate the conserved arginine in switch I further, a lysine substitution mutant was generated. The R210K dimeric motor has lost the ability to hydrolyze ATP; however, it has rescued microtubule function. Our results show that R210K has restored microtubule association kinetics, microtubule affinity, ADP release kinetics, and motor domain cooperativity. Moreover, the active site at head 1 is able to distinguish ATP, ADP, and AMP-PNP to signal head 2 to bind the microtubule and release mantADP with kinetics comparable with wild-type. Therefore, the structural pathway of communication from head 1 to head 2 is restored, and head 2 can respond to this signal by binding the microtubule and releasing mantADP. Structural modeling revealed that lysine could retain some of the hydrogen bonds made by arginine but not all, suggesting a structural hypothesis for the ability of lysine to rescue microtubule function in the Arg210 mutant. The ATPase mechanism of kinesin requires that the active site hydrolyze ATP to ADP·Pi and communicate the nucleotide state at the active site to the microtubule to mediate specific conformational changes that generate movement. ATPase activity is stimulated by the microtubule filament (1Kuznetsov S.A. Gelfand V.I. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8530-8534Crossref PubMed Scopus (165) Google Scholar); and therefore, there must also be communication from the microtubule to the active site. The three-dimensional structure of the active site can be organized into a variety of structural motifs that are common to kinesins, myosins, and G-proteins including the P loop (GXXXXGKS/T), switch I (NXXSSRSH), and switch II (DLAGXE) (2Vale R.D. J. Cell Biol. 1996; 135: 291-302Crossref PubMed Scopus (242) Google Scholar, 3Smith C.A. Rayment I. Biophys. J. 1996; 70: 1590-1602Abstract Full Text PDF PubMed Scopus (215) Google Scholar, 4Kull F.J. Vale R.D. Fletterick R.J. J. Muscle Res. Cell Motil. 1998; 19: 877-886Crossref PubMed Scopus (139) Google Scholar, 5Sack S. Kull F.J. Mandelkow E. Eur. J. Biochem. 1999; 262: 1-11Crossref PubMed Scopus (85) Google Scholar, 6Vale R.D. Milligan R.A. Science. 2000; 288: 88-95Crossref PubMed Scopus (1218) Google Scholar, 7Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (632) Google Scholar). Several recent reports have examined the roles of switch I and switch II in kinesins and myosins (8Shimada T. Sasaki N. Ohkura R. Sutoh K. Biochemistry. 1997; 36: 14037-14043Crossref PubMed Scopus (78) Google Scholar, 9Pate E. Naber N. Matuska M. Franks-Skiba K. Cooke R. Biochemistry. 1997; 36: 12155-12166Crossref PubMed Scopus (28) Google Scholar, 10Minehardt T.J. Cooke R. Pate E. Kollman P.A. Biophys. J. 2001; 80: 1151-1168Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 11Onishi H. Kojima S. Katoh K. Fujiwara K. Martinez H.M. Morales M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6653-6658Crossref PubMed Scopus (45) Google Scholar, 12Suzuki Y. Yasunaga T. Ohkura R. Wakabayashi T. Sutoh K. Nature. 1998; 396: 380-383Crossref PubMed Scopus (156) Google Scholar, 13Furch M. Fujita-Becker S. Geeves M.A. Holmes K.C. Manstein D.J. J. Mol. Biol. 1999; 290: 797-809Crossref PubMed Scopus (70) Google Scholar, 14Muller J. Marx A. Sack S. Song Y.H. Mandelkow E. Biol. Chem. 1999; 380: 981-992Crossref PubMed Scopus (21) Google Scholar, 15Wriggers W. Schulten K. Biophys. J. 1998; 75: 646-661Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 16Yun M. Zhang X. Park C.G. Park H.W. Endow S.A. EMBO J. 2001; 20: 2611-2618Crossref PubMed Scopus (85) Google Scholar, 17Song Y.H. Marx A. Muller J. Woehlke G. Schliwa M. Krebs A. Hoenger A. Mandelkow E. EMBO J. 2001; 20: 6213-6225Crossref PubMed Scopus (76) Google Scholar, 18Kull F.J. Sablin E.P. Lau R. Fletterick R.J. Vale R.D. Nature. 1996; 380: 550-555Crossref PubMed Scopus (578) Google Scholar, 19Kozielski F. Sack S. Marx A. Thormahlen M. Schonbrunn E. Biou V. Thompson A. Mandelkow E.M. Mandelkow E. Cell. 1997; 91: 985-994Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 20Sack S. Muller J. Marx A. Thormahlen M. Mandelkow E.M. Brady S.T. Mandelkow E. Biochemistry. 1997; 36: 16155-16165Crossref PubMed Scopus (181) Google Scholar, 21Sasaki N. Shimada T. Sutoh K. J. Biol. Chem. 1998; 273: 20334-20340Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 22Kull F.J. Endow S.A. J. Cell Sci. 2002; 115: 15-23Crossref PubMed Google Scholar, 23Onishi H. Ohki T. Mochizuki N. Morales M.F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15339-15344Crossref PubMed Scopus (37) Google Scholar, 24Kliche W. Fujita-Becker S. Kollmar M. Manstein D.J. Kull F.J. EMBO J. 2001; 20: 40-46Crossref PubMed Scopus (54) Google Scholar). These studies focused on the proposed role of switch I and switch II in positioning the water molecule that is critical for the hydrolysis of the phosphodiester bond of the γ-phosphate of ATP. The positioning of the water molecule is thought to be through a salt bridge between the conserved arginine in switch I and the conserved glutamic acid in switch II. Mutants in kinesins at either position exhibit dramatic reductions in the steady-state k cat which are attributed to the ATP hydrolysis defect (16Yun M. Zhang X. Park C.G. Park H.W. Endow S.A. EMBO J. 2001; 20: 2611-2618Crossref PubMed Scopus (85) Google Scholar, 25Rice 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 (646) Google Scholar, 26Farrell C.M. Mackey A.T. Klumpp L.M. Gilbert S.P. J. Biol. Chem. 2002; 277: 17079-17087Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The neck linker has been shown to specify plus-end directionality of kinesin (25Rice 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 (646) Google Scholar, 27Sablin 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 (183) Google Scholar, 28Case R.B. Pierce D.W. Hom-Booher N. Hart C.L. Vale R.D. Cell. 1997; 90: 959-966Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 29Endow S.A. Waligora K.W. Science. 1998; 281: 1200-1202Crossref PubMed Scopus (135) Google Scholar, 30Henningsen U. Schliwa M. Nature. 1997; 389: 93-96Crossref PubMed Scopus (180) Google Scholar) as well as docking to the catalytic core near the microtubule binding face, loop 12, and switch II relay helix α4 (25Rice 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 (646) Google Scholar, 31Tomishige M. Vale R.D. J. Cell Biol. 2000; 151: 1081-1092Crossref PubMed Scopus (112) Google Scholar). These results suggest a role for communication between the nucleotide and microtubule binding sites. Bound to microtubules, the kinesin neck linker exhibits an ATP-promoted docking transition (25Rice 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 (646) Google Scholar, 32Case R.B. Rice S. Hart C.L. Ly B. Vale R.D. Cur. Biol. 2000; 10: 157-160Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 33Rosenfeld S.S. Jefferson G.M. King P.H. J. Biol. Chem. 2001; 276: 40167-40174Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 34Skiniotis G. Surrey T. Altmann S. Gross H. Song Y.H. Mandelkow E. Hoenger A. EMBO J. 2003; 22: 1518-1528Crossref PubMed Scopus (62) Google Scholar); however, in the absence of microtubules the neck linker can exist in two states without nucleotide discrimination (35Sindelar C.V. Budny M.J. Rice S. Naber N. Fletterick R. Cooke R. Nat. Struct. Biol. 2002; 9: 844-848PubMed Google Scholar). Crystallization of kinesin with a docked neck linker suggests that the switch II cluster (downstream element of switch II not involved in nucleotide sensing) moves loop 11, and the flexibility in loop 11 permits switch II cluster movement that in turn allows neck linker docking without shifting the switch II nucleotide sensor. Microtubule binding may order loop 11, allowing for the coupled movement of the switch II cluster with the active site switch II element (35Sindelar C.V. Budny M.J. Rice S. Naber N. Fletterick R. Cooke R. Nat. Struct. Biol. 2002; 9: 844-848PubMed Google Scholar, 36Naber N. Rice S. Matuska M. Vale R.D. Cooke R. Pate E. Biophys. J. 2003; 84: 3190-3196Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). We have recently explored the role of ATP hydrolysis for kinesin cooperativity (26Farrell C.M. Mackey A.T. Klumpp L.M. Gilbert S.P. J. Biol. Chem. 2002; 277: 17079-17087Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Using the Drosophila conventional kinesin construct K401-wt, 1The abbreviations used are: K401-wt, wild-type kinesin heavy chain fragment containing the N-terminal 401 amino acids; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; mant, 2′(3′)-O-(N-methylanthraniloyl); Mt, microtubule.1The abbreviations used are: K401-wt, wild-type kinesin heavy chain fragment containing the N-terminal 401 amino acids; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; mant, 2′(3′)-O-(N-methylanthraniloyl); Mt, microtubule. a mutant was constructed where the conserved arginine of switch I (Arg210 in the Drosophila melanogaster sequence) was changed to an alanine. This mutant did not show a pre-steady-state burst of ADP·Pi product formation in acid quench experiments, indicative of a defect in ATP hydrolysis. The analysis also indicated that ATP hydrolysis was necessary for motor detachment. The experiments outlined a mechanism for kinesin in which ATP hydrolysis occurred after ADP release from the second head, consistent with other published reports (25Rice 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 (646) Google Scholar, 37Gilbert S.P. Moyer M.L. Johnson K.A. Biochemistry. 1998; 37: 792-799Crossref PubMed Scopus (154) Google Scholar, 38Moyer M.L. Gilbert S.P. Johnson K.A. Biochemistry. 1998; 37: 800-813Crossref PubMed Scopus (126) Google Scholar, 39Crevel I. Carter N. Schliwa M. Cross R. EMBO J. 1999; 18: 5863-5872Crossref PubMed Scopus (58) Google Scholar). The rate of ADP release from the second head of the Mt·R210A complex was similar using ATP, ADP, or AMP-PNP, whereas conventional wild-type kinesin shows discrimination among these different nucleotides in activating ADP release from the second head (37Gilbert S.P. Moyer M.L. Johnson K.A. Biochemistry. 1998; 37: 792-799Crossref PubMed Scopus (154) Google Scholar, 38Moyer M.L. Gilbert S.P. Johnson K.A. Biochemistry. 1998; 37: 800-813Crossref PubMed Scopus (126) Google Scholar, 40Gilbert S.P. Webb M.R. Brune M. Johnson K.A. Nature. 1995; 373: 671-676Crossref PubMed Scopus (246) Google Scholar, 41Brendza K.M. Sontag C.A. Saxton W.M. Gilbert S.P. J. Biol. Chem. 2000; 275: 22187-22195Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 42Hackney D.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6865-6869Crossref PubMed Scopus (306) Google Scholar, 43Ma Y.Z. Taylor E.W. J. Biol. Chem. 1997; 272: 724-730Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The R210A mutant also exhibited a microtubule binding defect, in both pre-steady-state microtubule association experiments and equilibrium microtubule binding experiments. To understand the role of the switch I arginine in greater detail, we examined the kinetics of an arginine to lysine mutation at position 210, referred to as R210K. The results presented here show that the R210K dimeric motor is defective in steady-state ATP turnover with a reduction in the k cat. There was also no pre-steady-state burst of product formation, indicative that replacement of the side chain geometry by the lysine is not sufficient to restore proper ATP hydrolysis. However, replacement with the lysine was able to restore microtubule affinity and the ability of mutant motor to discriminate among ATP, AMP-PNP, and ADP in mantADP release experiments from the second head. In addition, the rates at which mantADP release occurred were near wild-type levels. These data indicate that the mutant kinesin R210K can communicate the nucleotide state to the partner motor domain, and the partner motor domain can respond to the signal by binding the microtubule and releasing mantADP. Furthermore, the structural pathway for communication between the active site and the microtubule has been restored even though the ATP hydrolysis defect is not corrected. Through structural modeling we identified crystal structures of monomeric kinesin which represent an ATP-like structure and a true ADP structure. These structural differences are supported by our kinetic data. We hypothesize that the ability of lysine to rescue microtubule function is the result of its ability to maintain a key hydrogen bond in the ADP state and its inability to form critical hydrogen bonds in the ATP-like state. Materials—Paclitaxel (Taxol, Taxus brevifolia) was purchased from Sigma. Polyethylenimine-cellulose TLC plates (EM Science of Merck, 20 × 20 cm, plastic-backed) were from VWR Scientific (West Chester, PA), and mantATP and mantADP were from Molecular Probes (Eugene, OR). Buffer Conditions—The kinetic and equilibrium binding experiments were performed in ATPase buffer (20 mm Hepes pH 7.2 with KOH, 5 mm magnesium acetate, 0.1 mm EGTA, 0.1 mm EDTA, 50 mm potassium acetate, 1 mm dithiothreitol) at 25 °C. Concentrations (proteins, nucleotides, etc.) reported are final concentrations after mixing. Expression and Purification of R210K Mutant Kinesin Motor—The construction of the R210K plasmid and the expression and the purification of the R210K mutant kinesin were performed as described previously (26Farrell C.M. Mackey A.T. Klumpp L.M. Gilbert S.P. J. Biol. Chem. 2002; 277: 17079-17087Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The K401-wt plasmid (44Gilbert S.P. Johnson K.A. Biochemistry. 1993; 32: 4677-4684Crossref PubMed Scopus (76) Google Scholar) was used to construct the R210K mutant kinesin by introducing a single amino acid change at position 210 using the Chameleon mutagenesis protocol (Stratagene, Inc., La Jolla, CA). DNA sequencing confirmed the arginine to lysine substitution. The conventional kinesin construct, K401, contains the first 401 amino acids of the D. melanogaster conventional kinesin heavy chain and produces a dimeric kinesin motor when expressed in Escherichia coli (45Correia J.J. Gilbert S.P. Moyer M.L. Johnson K.A. Biochemistry. 1995; 34: 4898-4907Crossref PubMed Scopus (51) Google Scholar). The R210K plasmid was transformed into BL21(DE3)pLysS, expressed in E. coli, and purified as described previously (44Gilbert S.P. Johnson K.A. Biochemistry. 1993; 32: 4677-4684Crossref PubMed Scopus (76) Google Scholar, 46Brendza 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 (62) Google Scholar). Determination of protein concentration of the purified R210K kinesin mutant motor was performed by the Bradford method using the Bio-Rad protein assay with IgG as a protein standard. Active site experiments were performed as described (26Farrell C.M. Mackey A.T. Klumpp L.M. Gilbert S.P. J. Biol. Chem. 2002; 277: 17079-17087Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 47Gilbert S.P. Mackey A.T. Methods. 2000; 22: 337-354Crossref PubMed Scopus (67) Google Scholar, 48Klumpp L.M. Brendza K.M. Rosenberg J.M. Hoenger A. Gilbert S.P. Biochemistry. 2003; 42: 2595-2606Crossref PubMed Scopus (27) Google Scholar). Microtubule Preparation—Microtubules were polymerized from bovine brain tubulin and stabilized with 20 μm Taxol as described previously (44Gilbert S.P. Johnson K.A. Biochemistry. 1993; 32: 4677-4684Crossref PubMed Scopus (76) Google Scholar). The Taxol-treated microtubules were stable as polymers as determined by sedimentation assays and SDS-PAGE analysis. Steady-state ATPase Assays—ATPase measurements were performed by following the turnover of [α-32P]ATP to [α-32P]ADP·Pi as described previously (44Gilbert S.P. Johnson K.A. Biochemistry. 1993; 32: 4677-4684Crossref PubMed Scopus (76) Google Scholar, 47Gilbert S.P. Mackey A.T. Methods. 2000; 22: 337-354Crossref PubMed Scopus (67) Google Scholar). Equilibrium Binding to the Microtubule—These experiments were performed as described previously (26Farrell C.M. Mackey A.T. Klumpp L.M. Gilbert S.P. J. Biol. Chem. 2002; 277: 17079-17087Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 48Klumpp L.M. Brendza K.M. Rosenberg J.M. Hoenger A. Gilbert S.P. Biochemistry. 2003; 42: 2595-2606Crossref PubMed Scopus (27) Google Scholar, 49Foster 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, 50Mackey A.T. Gilbert S.P. Biochemistry. 2000; 39: 1346-1355Crossref PubMed Scopus (25) Google Scholar). R210K at 2 μm was incubated with 0-7 μm microtubules in the absence of added nucleotides for 30 min and subjected to high speed centrifugation. The supernatant was removed, and the microtubule pellet was resuspended in ATPase buffer to the same volume as the supernatant. Laemmli sample buffer (5×) was added to samples of the supernatant and resuspended pellet, and the proteins were resolved by SDS-PAGE (8% acrylamide and 2 m urea). The gel was stained with Coomassie Blue and scanned by a Microtek Scan Maker X6EL scanner (Microtek, Redondo Beach, CA). The scanned image was quantified (NIH Image version 1.62) to determine the concentration of R210K in the supernatant and pellet at each microtubule concentration. Fig. 2 presents the data as fractional binding, which is defined as the ratio of R210K in the pellet to total R210K (2 μm), plotted as a function of microtubule concentration. The data were fit to quadratic Equation 1, Mt·E/E0=0.5((E0+Mt0+Kd)-((E0+Mt0+Kd)2-(4E0Mt0))1/2)(Eq. 1) where Mt·E/E 0 is the fraction of R210K that sediments with the microtubules, E 0 is total R210K concentration, Mt0 is the total tubulin concentration as microtubule polymer, and K d is the dissociation constant. Rapid Quench Experiments—R210A was noted to have an ATP hydrolysis defect (26Farrell C.M. Mackey A.T. Klumpp L.M. Gilbert S.P. J. Biol. Chem. 2002; 277: 17079-17087Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). We performed similar experiments with R210K in comparison to wild-type K401 (51Gilbert S.P. Johnson K.A. Biochemistry. 1994; 33: 1951-1960Crossref PubMed Scopus (86) Google Scholar) to determine whether the two mutants shared the same ATP hydrolysis defect. The preformed Mt·R210K complex (syringe concentrations: 16 μm R210K, 30 μm microtubules, 40 μm Taxol) was mixed rapidly in a chemical quenched-flow instrument (RQF-3, Kintek Corp., Austin, TX) with 100 μm [α-32P]ATP (see Fig. 3). The reaction was terminated with 5 m formic acid (syringe concentration) and expelled from the instrument. The radiolabeled product (ADP·Pi) was separated from the radiolabeled reactant (ATP) by thin layer chromatography, and the data were quantified. The concentration of [α-32P]ADP was determined for each time point and plotted as a function of time (KaleidaGraph, Synergy Software, Reading, PA). The data were then fit to the burst equation, product=A*(1-exp(-kbt))+ksst(Eq. 2) where A is the amplitude of the pre-steady-state burst phase which represents the formation of [α-32P]ADP·Pi at the active site during the first ATP turnover; k b is the rate constant of the exponential burst phase; t is time in seconds; and k ss is the rate constant of the linear phase (μm ADP·s-1). The rate constant k ss, when divided by enzyme concentration, corresponds to the rate of steady-state turnover at the same ATP and microtubule concentrations. Stopped-flow Kinetics—The pre-steady-state kinetics of mantATP binding, R210K binding to microtubules, ATP-promoted dissociation of R210K, and mantADP release were all conducted using the SF-2001 KinTek stopped-flow instrument in ATPase buffer at 25 °C. For the mantATP and mantADP experiments, the excitation wavelength was 360 nm (mercury arc lamp) with emitted light measured through a 400 nm cutoff filter (mant λemm = 450 nm). The mantATP binding data in the inset of Fig. 4A were fit Equation 3, kobs=k1[mantATP]+koff(Eq. 3) where k obs is the rate of the initial exponential increase in fluorescence, k 1 is the second-order rate constant for mantATP binding (see Scheme 1), and k off obtained from the y intercept is the rate of mantATP dissociation from the Mt·R210K·mantATP complex.Scheme 1View Large Image Figure ViewerDownload Hi-res image Download (PPT) The microtubule association kinetics (see Fig. 5) and the ATP-promoted dissociation kinetics (see Fig. 6) were performed by observation of the change in turbidity at 340 nm. The exponential rate constants (k obs) for microtubule association were plotted as a function of microtubule concentration and fit to Equation 4, kobs=k5[tubulin]+k-5(Eq. 4) where k obs is the rate of the observed exponential process, k 5 is the second-order rate constant for microtubule association (see Scheme 1), and k -5 obtained from the y intercept is the rate constant for motor dissociation from the Mt·R210K complex.Fig. 6ATP-promoted dissociation kinetics of Mt·R210K and Mt·K401. In the stopped-flow, the Mt·R210K or the Mt·K401 complex (both at 6 μm motor, 6 μm tubulin, 10 μm Taxol) was mixed rapidly with 1 mm MgATP + 100 mm KCl, and a change in turbidity was monitored. Both transients were fit to two exponential functions. For R210K, the amplitude of the fast initial phase was 0.0107 ± 0.0002 with k obs = 17.7 ± 0.7 s-1. For K401, the fit of the data yielded an amplitude of the fast initial phase = 0.049 ± 0.001 and the k obs = 14.9 ± 0.3 s-1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Structural Modeling—The structural modeling of Fig. 10 was performed on a Silicon Graphics work station using the program O (52Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar) and rendered using PyMOL (53DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar). The hydrogen bonding capability of Arg210 was compared in the monomeric kinesin rat structure, 2KIN (20Sack S. Muller J. Marx A. Thormahlen M. Mandelkow E.M. Brady S.T. Mandelkow E. Biochemistry. 1997; 36: 16155-16165Crossref PubMed Scopus (181) Google Scholar) and the monomeric kinesin human structure, 1BG2 (18Kull F.J. Sablin E.P. Lau R. Fletterick R.J. Vale R.D. Nature. 1996; 380: 550-555Crossref PubMed Scopus (578) Google Scholar). Asn256 in 2KIN was rotated by 180° about χ2 such that Nδ2 and Oδ1 are switched (this produces a model more consistent with the hydrogen bonding potential of this residue and its immediate environment). Mutant models were generated by redecorating the polypeptide backbone, followed by rotomer selection based on visual inspection using O (52Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar). Rotomers were also examined for adjacent residues. A stereochemically sensible model could be obtained by rotomer selection at Lys203 and Glu199, followed by minimal manual readjustment of the side chains (see Fig. 10C). Steady-state and Equilibrium Properties of R210K—We began our analysis by examining the steady-state kinetics of R210K (Fig. 1). For R210K, the rate of ATP turnover increased as a function of ATP concentration with the k cat = 0.03 s-1. Compared with K401-wt, there is a 700-fold decrease in the steady-state ATPase from 19.5 to 0.03 s-1 (Fig. 1B). The depressed k cat was similar to the constant observed for the R210A switch I mutant at 0.12 s-1, but the K m,ATP for R210K at 38 μm indicated higher affinity for ATP compared with R210A at 118 μm and K401-wt at 107 μm (Fig. 1 and Table I).Table IMicrotubule-kinesin constantsExperimentally observeda20 mm Hepes pH 7.2, with KOH, 5 mm magnesium acetate, 0.1 mm EGTA, 0.1 mm EDTA, 50 mm potassium acetate, 1 mm dithiothreitol at 25 °C.Computer simulation,bExperimentally determined rate constants refined by computer simulation (37, 38, 62). K401-wtRate constantsR210AcR210A rate constants are from Ref. 26.R210KK401-wtk 1ATP bindingdMantATP binding (38).eAcid and pulse-chase rapid quench (51).0.71 ± 0.08 μm−1s−11.29 ± 0.05 μm−1s−11.1 μm−1s−1dMantATP binding (38).eAcid and pulse-chase rapid quench (51).2 μm−1s−1k 1k max80.7 ± 4.2 s−182.0 ± 4.0 s−1240 s−1K 0.5,ATP8.8 ± 2.0 μm ATP27.4 ± 4.6 μm ATP85 μm ATPk −1ATP dissociationeAcid and pulse-chase rapid quench (51).NDfND, not determined.ND200 s−1eAcid and pulse-chase rapid quench (51).120 s−1k 2Acid quencheAcid and pulse-chase rapid quench (51).0.2 ± 0.09 s−10.12 ± 0.08 s−1100 s−1eAcid and pulse-chase rapid quench (51).100 s−1k 3ATP-promoted microtubule dissociationgTurbidity (38, 40).No dissociationNo dissociation12-16 s−1gTurbidity (38, 40).50 s−1k 4Pi; releasehMDCC-PBP (38, 40).NDND13 s−1hMDCC-PBP (38, 40).>150 s−1k 5Microtubule associationgTurbidity (38, 40).0.83 ± 0.04 μm−1s−17.51 ± 0.17 μm−1s−110-20 μm−1s−111 μm−1s−1k 6ADP release both headsiMantADP competed with excess unlabeled MgATP, MgAMP-PNP, or MgADP (37, 38, 40, 41).57.2 ± 2.9 s−179.4 ± 3.0 s−1>200 s−1iMantADP competed with excess unlabeled MgATP, MgAMP-PNP, or MgADP (37, 38, 40, 41).300 s−1K 0.5,mt16.2 ± 1.9 μm Mt6.2 ± 0.7 μm Mt15 μm MtADP release head 2iMantADP competed with excess unlabeled MgATP, MgAMP-PNP, or MgADP (37, 38, 40, 41).ATP:ATP:ATP:200 s−130-42 s−169.3 ± 1.3 s−1>100 s−10.45 ± 0.12 μm76.3 ± 6.3 μm ATPk maxAMP-PNP:AMP-PNP:AMP-PNP:K 0.530-40 s−126.2 ± 1.9 s−130-40 s−10.35 ± 0.14 μm857 ± 165 μm AMP-PNPADP:ADP:ADP:25 s−18.7 ± 0.2 s−16 s−175.4 ± 5.0 μm ADPk cat0.12 ± 0.05 s−10.11 ± 0.05 s−120.6 ± 0.9 s−1K m,ATP118 ± 63 μm38 ± 9.9 μm94.4 ± 5.9 μmK d,mt950 ± 28 nm75 ± 14 nm37 ± 6 nmjK d,mt from Ref. 63.a 20 mm Hepes pH 7.2, with KOH, 5 mm magnesium acetate, 0.1 mm EGTA, 0.1 mm EDTA, 50 mm potassium acetate, 1 mm dithiothreitol at 25 °C.b Experimentally determined rate constants refined by computer simulation (37Gilbert S.P. Moyer M.L. Johnson K.A. Biochemistry. 1998; 37: 792-799Crossref PubMed Scopus (154) Google Scholar, 38Moyer M.L. Gilbert S.P. Johnson K.A. Biochemistry. 1998; 37: 800-813Crossref PubMed Scopus (126) Google Scholar, 62Mandelkow E. Johnson K.A. Trends Biochem. Sci. 1998; 23: 429-433Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar).c R210A rate constants are from Ref. 26Farrell C.M. Mackey A.T. Klumpp L.M. Gilbert S.P. J. Biol. Chem. 2002; 277: 17079-17087Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar.d MantATP binding (38Moyer M.L. Gilbert S.P. Johnson K.A. Biochemistry. 1998; 37: 800-813Crossref PubMed Scopus (126) Google Scholar).e Acid and pulse-chase rapid quench (51Gilbert S.P. Johnson K.A. Biochemistry. 1994; 33: 1951-1960Crossref PubMed Scopus (86) Google Scholar).f ND, not determined.g Turbidity (38Moyer M.L. Gilbert S.P. Johnson K.A. Biochemistry. 1998; 37: 800-813Crossref PubMed Scopus (126) Google Scholar, 40Gilbert S.P. Webb M.R. Brune M. Johnson K.A. Nature. 1995; 373: 671-676Crossref PubMed Scopus (246) Google Scholar).h MDCC-P" @default.
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