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• W2024078970 abstract "Background: CENP-E is a processive kinesin that is required for chromosome congression and stable chromosome biorientation.Results: CENP-E exhibits unusually slow microtubule collision coupled with slow ADP release.Conclusion: This novel ATPase cycle may favor CENP-E binding of stable kinetochore microtubules over dynamic microtubules.Significance: This study may enhance our understanding of the role of CENP-E in mitosis and meiosis. Background: CENP-E is a processive kinesin that is required for chromosome congression and stable chromosome biorientation. Results: CENP-E exhibits unusually slow microtubule collision coupled with slow ADP release. Conclusion: This novel ATPase cycle may favor CENP-E binding of stable kinetochore microtubules over dynamic microtubules. Significance: This study may enhance our understanding of the role of CENP-E in mitosis and meiosis. Accurate chromosome segregation requires that sister kinetochores on each chromosome establish stable attachments to opposite spindle poles by microtubules (MTs) 2The abbreviations used are: MTmicrotubulekMTkinetochore MTCENP-Ecentromere protein EAMPPNPadenosine 5′-(β, γ-imido)triphosphateATPγSadenosine-5′-(γ-thio)-triphosphateMant2′(3′)-O-(N-methylanthraniloyl). (reviewed in Ref. 1.Walczak C.E. Cai S. Khodjakov A. Mechanisms of chromosome behavior during mitosis.Nat. Rev. Mol. Cell Biol. 2010; 11: 91-102Crossref PubMed Google Scholar). This process of biorientation is mediated by MT bundles or k-fibers attached to the sister kinetochores of each chromosome (2.Rieder C.L. Salmon E.D. The vertebrate cell kinetochore and its roles during mitosis.Trends Cell Biol. 1998; 8: 310-318Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). CENP-E is a mitotic kinesin whose expression is up-regulated during G2 and M phases of the cell cycle, and it localizes to kinetochores from early prometaphase through metaphase moving to the antiparallel midzone MTs at anaphase (3.Yen T.J. Li G. Schaar B.T. Szilak I. Cleveland D.W. CENP-E is a putative kinetochore motor that accumulates just before mitosis.Nature. 1992; 359: 536-539Crossref PubMed Scopus (335) Google Scholar, 4.Brown K.D. Coulson R.M. Yen T.J. Cleveland D.W. Cyclin-like accumulation and loss of the putative kinetochore motor CENP-E results from coupling continuous synthesis with specific degradation at the end of mitosis.J. Cell Biol. 1994; 125: 1303-1312Crossref PubMed Scopus (118) Google Scholar, 5.Brown K.D. Wood K.W. Cleveland D.W. The kinesin-like protein CENP-E is kinetochore-associated throughout poleward chromosome segregation during anaphase-A.J. Cell Sci. 1996; 109: 961-969Crossref PubMed Google Scholar, 6.Cooke C.A. Schaar B. Yen T.J. Earnshaw W.C. Localization of CENP-E in the fibrous corona and outer plate of mammalian kinetochores from prometaphase through anaphase.Chromosoma. 1997; 106: 446-455Crossref PubMed Scopus (129) Google Scholar, 7.Hoffman D.B. Pearson C.G. Yen T.J. Howell B.J. Salmon E.D. Microtubule-dependent changes in assembly of microtubule motor proteins and mitotic spindle checkpoint proteins at PtK1 kinetochores.Mol. Biol. Cell. 2001; 12: 1995-2009Crossref PubMed Scopus (280) Google Scholar). CENP-E is well conserved throughout the animal kingdom, and reduced levels of CENP-E in cells and mice have been linked to aneuploidy and chromosome instability (8.Putkey F.R. Cramer T. Morphew M.K. Silk A.D. Johnson R.S. McIntosh J.R. Cleveland D.W. Unstable kinetochore-microtubule capture and chromosomal instability following deletion of CENP-E.Dev. Cell. 2002; 3: 351-365Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 9.Weaver B.A. Bonday Z.Q. Putkey F.R. Kops G.J. Silk A.D. Cleveland D.W. Centromere-associated protein-E is essential for the mammalian mitotic checkpoint to prevent aneuploidy due to single chromosome loss.J. Cell Biol. 2003; 162: 551-563Crossref PubMed Scopus (214) Google Scholar, 10.Weaver B.A. Silk A.D. Montagna C. Verdier-Pinard P. Cleveland D.W. Aneuploidy acts both oncogenically and as a tumor suppressor.Cancer Cell. 2007; 11: 25-36Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). In 2006, Kapoor et al. (11.Kapoor T.M. Lampson M.A. Hergert P. Cameron L. Cimini D. Salmon E.D. McEwen B.F. Khodjakov A. Chromosomes can congress to the metaphase plate before biorientation.Science. 2006; 311: 388-391Crossref PubMed Scopus (320) Google Scholar) made the remarkable discovery that chromosomes can congress to the spindle equator prior to biorientation through a novel mechanism in which monooriented chromosomes glide to the metaphase plate along kinetochore MTs (kMTs) of chromosomes that were already bioriented (11.Kapoor T.M. Lampson M.A. Hergert P. Cameron L. Cimini D. Salmon E.D. McEwen B.F. Khodjakov A. Chromosomes can congress to the metaphase plate before biorientation.Science. 2006; 311: 388-391Crossref PubMed Scopus (320) Google Scholar, 12.Magidson V. O'Connell C.B. Lončarek J. Paul R. Mogilner A. Khodjakov A. The spatial arrangement of chromosomes during prometaphase facilitates spindle assembly.Cell. 2011; 146: 555-567Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Their results revealed that this movement of monooriented chromosomes was dependent upon CENP-E. microtubule kinetochore MT centromere protein E adenosine 5′-(β, γ-imido)triphosphate adenosine-5′-(γ-thio)-triphosphate 2′(3′)-O-(N-methylanthraniloyl). CENP-E is an N-terminal homodimeric kinesin that promotes slow processive MT plus-end-directed movement (13.Kim Y. Heuser J.E. Waterman C.M. Cleveland D.W. CENP-E combines a slow, processive motor and a flexible coiled-coil to produce an essential motile kinetochore tether.J. Cell Biol. 2008; 181: 411-419Crossref PubMed Scopus (111) Google Scholar, 14.Yardimci H. van Duffelen M. Mao Y. Rosenfeld S.S. Selvin P.R. The mitotic kinesin CENP-E is a processive transport motor.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 6016-6021Crossref PubMed Scopus (55) Google Scholar) like conventional kinesin-1 (15.Block S.M. Goldstein L.S. Schnapp B.J. Bead movement by single kinesin molecules studied with optical tweezers.Nature. 1990; 348: 348-352Crossref PubMed Scopus (851) Google Scholar, 16.Howard J. Hudspeth A.J. Vale R.D. Movement of microtubules by single kinesin molecules.Nature. 1989; 342: 154-158Crossref PubMed Scopus (740) Google Scholar, 17.Clancy B.E. Behnke-Parks W.M. Andreasson J.O. Rosenfeld S.S. Block S.M. A universal pathway for kinesin stepping.Nat. Struct. Mol. Biol. 2011; 18: 1020-1027Crossref PubMed Scopus (159) Google Scholar) and kinesin-5 Eg5/kinesin-5 Eg5/KSP (18.Valentine M.T. Fordyce P.M. Krzysiak T.C. Gilbert S.P. Block S.M. Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro.Nat. Cell Biol. 2006; 8: 470-476Crossref PubMed Scopus (210) Google Scholar). CENP-E processive run lengths have been reported at 1.5–2.6 μm with the rate of stepping varying from 8 to 342 nm/s, depending on the specific construct expressed and the surface conditions of the assay (13.Kim Y. Heuser J.E. Waterman C.M. Cleveland D.W. CENP-E combines a slow, processive motor and a flexible coiled-coil to produce an essential motile kinetochore tether.J. Cell Biol. 2008; 181: 411-419Crossref PubMed Scopus (111) Google Scholar, 14.Yardimci H. van Duffelen M. Mao Y. Rosenfeld S.S. Selvin P.R. The mitotic kinesin CENP-E is a processive transport motor.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 6016-6021Crossref PubMed Scopus (55) Google Scholar, 19.Espeut J. Gaussen A. Bieling P. Morin V. Prieto S. Fesquet D. Surrey T. Abrieu A. Phosphorylation relieves autoinhibition of the kinetochore motor Cenp-E.Mol. Cell. 2008; 29: 637-643Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 20.Sardar H.S. Luczak V.G. Lopez M.M. Lister B.C. Gilbert S.P. Mitotic kinesin CENP-E promotes microtubule plus-end elongation.Curr. Biol. 2010; 20: 1648-1653Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 21.Shastry S. Hancock W.O. Interhead tension determines processivity across diverse N-terminal kinesins.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 16253-16258Crossref PubMed Scopus (68) Google Scholar). The stall force for CENP-E at 6 piconewtons is comparable with the stall force of kinesin-1 (14.Yardimci H. van Duffelen M. Mao Y. Rosenfeld S.S. Selvin P.R. The mitotic kinesin CENP-E is a processive transport motor.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 6016-6021Crossref PubMed Scopus (55) Google Scholar), and Rosenfeld et al. (22.Rosenfeld S.S. van Duffelen M. Behnke-Parks W.M. Beadle C. Corrreia J. Xing J. The ATPase cycle of the mitotic motor CENP-E.J. Biol. Chem. 2009; 284: 32858-32868Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) reported that CENP-E exhibits a kinetics profile similar to kinesin-1 where ATP binding to the lead head was gated by intramolecular strain such that ATP binding on the leading head can only occur once strain is relieved by detachment of the trailing head. Comparative studies of processive kinesins revealed that neck linker length, sequence, and orientation modulate interhead tension and therefore regulate head-head communication for processive stepping (17.Clancy B.E. Behnke-Parks W.M. Andreasson J.O. Rosenfeld S.S. Block S.M. A universal pathway for kinesin stepping.Nat. Struct. Mol. Biol. 2011; 18: 1020-1027Crossref PubMed Scopus (159) Google Scholar, 21.Shastry S. Hancock W.O. Interhead tension determines processivity across diverse N-terminal kinesins.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 16253-16258Crossref PubMed Scopus (68) Google Scholar, 23.Yildiz A. Tomishige M. Gennerich A. Vale R.D. Intramolecular strain coordinates kinesin stepping behavior along microtubules.Cell. 2008; 134: 1030-1041Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). However, unlike kinesin-1 and Eg5, CENP-E can also promote MT plus-end elongation in vitro (20.Sardar H.S. Luczak V.G. Lopez M.M. Lister B.C. Gilbert S.P. Mitotic kinesin CENP-E promotes microtubule plus-end elongation.Curr. Biol. 2010; 20: 1648-1653Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), suggesting that the mechanochemistry of CENP-E is specific for its functional roles in mitosis. More recently, a key threonine residue near the CENP-E motor domain and conserved across species (Thr422 in human and Thr424 in Xenopus laevis) has been identified as the site of a phospho-regulatory switch that is critical for chromosome congression and correct biorientation (24.Kim Y. Holland A.J. Lan W. Cleveland D.W. Aurora kinases and protein phosphatase 1 mediate chromosome congression through regulation of CENP-E.Cell. 2010; 142: 444-455Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Phosphorylation of this threonine by Aurora kinases A and B occurs during prometaphase when the chromosomes are near the spindle poles and where the gradient of Aurora kinases is high. Because CENP-E phosphorylation weakens its affinity for MTs, this mechanism is believed to contribute to correction of inappropriate kinetochore MT attachments. In contrast, as chromosomes move away from the spindle poles and congress to the equator, CENP-E becomes dephosphorylated by protein phosphatase 1, leading to higher MT affinity and stabilization of kinetochore end-on kMT amphitelic attachments (24.Kim Y. Holland A.J. Lan W. Cleveland D.W. Aurora kinases and protein phosphatase 1 mediate chromosome congression through regulation of CENP-E.Cell. 2010; 142: 444-455Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 25.Hendrickx A. Beullens M. Ceulemans H. Den Abt T. Van Eynde A. Nicolaescu E. Lesage B. Bollen M. Docking motif-guided mapping of the interactome of protein phosphatase-1.Chem. Biol. 2009; 16: 365-371Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). To identify mechanistic differences for CENP-E that contribute to its role in chromosome congression, we pursued a pre-steady-state kinetics study using a recombinant, truncated human CENP-E dimer (20.Sardar H.S. Luczak V.G. Lopez M.M. Lister B.C. Gilbert S.P. Mitotic kinesin CENP-E promotes microtubule plus-end elongation.Curr. Biol. 2010; 20: 1648-1653Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The results show that the ATPase mechanism accounts for the high processivity of CENP-E and its ability to elongate MTs in vitro. More importantly, this study has revealed that productive CENP-E association with the MT is unusually slow and may bias CENP-E selection for stable kMTs, a characteristic that could enhance the role of CENP-E in chromosome congression. The experiments reported in this study were performed at room temperature (22–25 °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, and 5% sucrose). On the morning of each experiment, bovine brain tubulin was cold-depolymerized, clarified, and polymerized with 1 mm GTP at 34 °C. These MTs were stabilized with 40 μm paclitaxel, and MT concentrations reported are the paclitaxel-stabilized tubulin polymer. All reported concentrations of nucleotides (ATP, ADP, GTP) and nucleotide analogues (mantATP, mantADP, AMPPNP, ATPγS) also include the equivalent concentration of magnesium acetate. Errors are reported as S.E. A human CENP-E construct was expressed in Escherichia coli and purified as described previously (20.Sardar H.S. Luczak V.G. Lopez M.M. Lister B.C. Gilbert S.P. Mitotic kinesin CENP-E promotes microtubule plus-end elongation.Curr. Biol. 2010; 20: 1648-1653Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The CENP-E-His6 protein includes the N-terminal residues, Met1–Leu407, followed by 22 additional residues at the C terminus from the pET30c(+) plasmid (RDPNSSSVDKLAAALEHHHHHH). The predicted Mr is 48,467 per polypeptide chain and 96,934 for the CENP-E dimer. On the day of each experiment, CENP-E was clarified (90,000 rpm for 5 min at 4 °C, Beckman Coulter Optima TLX ultracentrifuge, TLA 100 rotor) followed by protein concentration determination using the Bio-Rad protein assay with IgG as the standard. The CENP-E concentration is reported based on the concentration of a single CENP-E polypeptide. For the experiments in Figs. 1 and 4, the MT·CENP-E complex was preformed and treated with apyrase (0.02 unit/ml; grade VII, Sigma-Aldrich) for 20 min to generate a nucleotide-free complex (26.Krzysiak T.C. Grabe M. Gilbert S.P. Getting in sync with dimeric Eg5: initiation and regulation of the processive run.J. Biol. Chem. 2008; 283: 2078-2087Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 27.Chen C.J. Rayment I. Gilbert S.P. Kinesin Kar3Cik1 ATPase pathway for microtubule cross-linking.J. Biol. Chem. 2011; 286: 29261-29272Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The grade VII apyrase isoform preferentially selects ADP to convert to AMP, and the binding affinity of AMP for CENP-E is so weak that CENP-E becomes essentially nucleotide-free. Because the concentration of apyrase used is so low, it does not compete with CENP-E for nucleotide during the experiments.FIGURE 4ATP-triggered mantADP release kinetics. A preformed MT·CENP-E·mantADP (mADP) complex was rapidly mixed in the stopped-flow instrument with ATP. Final concentrations: 5 μm CENP-E, 10 μm MTs, 2 μm mantATP (mATP), 5–500 μm ATP. A, experimental design to establish the processive stepping intermediate. B, representative transients at 0, 10, 25, 50, 100 μm ATP (top to bottom). C, the observed rates of the initial exponential phase were plotted as a function of ATP concentration, and the hyperbolic fit of the data provided kmax = 19.4 ± 0.6 s−1, K½,ATP = 26.6 ± 3.2 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The nucleotide-free MT·CENP-E complex was rapidly mixed with mantATP (Invitrogen Molecular Probes) in a KinTek SF2003 stopped-flow instrument, and the fluorescence change was monitored (λex = 360 nm, λem = 450 nm, 409-nm long pass filter). The fluorescence increase as a function of time is correlated with mantATP binding because the fluorescence of mantATP is enhanced by the hydrophobic environment of the active site. Each transient was fit to a double exponential function, the observed rate of the initial exponential phase was plotted as a function of mantATP concentration, and the data were fit to the following equationkobs= k+1[MantATP]+ k-1(Eq. 1) where kobs represents the observed rate of the initial exponential phase, k+1 is the second-order rate constant for mantATP binding, and k−1 is the dissociation rate constant obtained from the y-intercept (see Fig. 1). The MT·CENP-E complex was preformed and rapidly mixed in the KinTek chemical quench-flow instrument with ATP plus trace [α-32P]ATP. The reaction was subsequently chased with 30 mm nonradiolabeled ATP (syringe concentration) and allowed to proceed for 15 s (>10 turnovers). The reaction was expelled from the instrument into a 1.5-ml tube containing 100 μl of 22 n formic acid. The acid terminates the reaction and releases any nucleotide at the active site. This experimental design quantifies [α-32P]ATP stably bound at the active site because during the time of the chase, the stably bound [α-32P]ATP proceeds through ATP hydrolysis. Any weakly bound [α-32P]ATP or [α-32P]ATP in solution is diluted by the high concentration of nonradiolabeled ATP. Therefore, this experiment measures the kinetics of ATP binding. The products [α-32P]ADP + Pi were separated from [α-32P]ATP by thin layer chromatography and quantified (27.Chen C.J. Rayment I. Gilbert S.P. Kinesin Kar3Cik1 ATPase pathway for microtubule cross-linking.J. Biol. Chem. 2011; 286: 29261-29272Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 28.Gilbert S.P. Mackey A.T. Kinetics: a tool to study molecular motors.Methods. 2000; 22: 337-354Crossref PubMed Scopus (67) Google Scholar). The individual transients were fit to the following burst equationProduct = A0[1-exp(-kbt)]+kslowt(Eq. 2) where A0 is the amplitude of the initial exponential burst phase representing the concentration of ADP·Pi formed at the active site during the first ATP turnover, kb is the observed rate of the exponential burst phase, kslow represents the rate of subsequent ATP binding events corresponding to steady-state turnover, and t is the time in seconds. For these experiments, 200 mm KCl was added to the [α-32P]ATP syringe, resulting in a final concentration of 100 mm KCl after mixing and prior to the unlabeled ATP chase. This strategy is commonly used for processive kinesins to slow the rate of subsequent ATP turnovers without affecting the first turnover (27.Chen C.J. Rayment I. Gilbert S.P. Kinesin Kar3Cik1 ATPase pathway for microtubule cross-linking.J. Biol. Chem. 2011; 286: 29261-29272Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 29.Gilbert S.P. Webb M.R. Brune M. Johnson K.A. Pathway of processive ATP hydrolysis by kinesin.Nature. 1995; 373: 671-676Crossref PubMed Scopus (246) Google Scholar, 30.Moyer M.L. Gilbert S.P. Johnson K.A. Pathway of ATP hydrolysis by monomeric and dimeric kinesin.Biochemistry. 1998; 37: 800-813Crossref PubMed Scopus (127) Google Scholar, 31.Klumpp L.M. Brendza K.M. Rosenberg J.M. Hoenger A. Gilbert S.P. Motor domain mutation traps kinesin as a microtubule rigor complex.Biochemistry. 2003; 42: 2595-2606Crossref PubMed Scopus (27) Google Scholar, 32.Klumpp L.M. Hoenger A. Gilbert S.P. Kinesin's second step.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 3444-3449Crossref PubMed Scopus (96) Google Scholar, 33.Cochran J.C. Sontag C.A. Maliga Z. Kapoor T.M. Correia J.J. Gilbert S.P. Mechanistic analysis of the mitotic kinesin Eg5.J. Biol. Chem. 2004; 279: 38861-38870Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 34.Krzysiak T.C. Gilbert S.P. Dimeric Eg5 maintains processivity through alternating-site catalysis with rate-limiting ATP hydrolysis.J. Biol. Chem. 2006; 281: 39444-39454Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The exponential burst phase therefore becomes better defined to quantify the kinetics of the first ATP turnover. The observed rates of the initial exponential burst phase were plotted as a function of ATP concentration, and the data were fit to the following equationkobs=[K1k+1'[ATP]/(K1[ATP]+1)](Eq. 3) where K1 represents the equilibrium association constant for formation of the collision complex, and k+1′ is the first-order rate constant for the ATP-promoted isomerization with Kd = 1/K1 (see Fig. 2). The pre-steady-state kinetic experiments to determine the time course of ATP hydrolysis were performed in the KinTek chemical quench-flow instrument as described (27.Chen C.J. Rayment I. Gilbert S.P. Kinesin Kar3Cik1 ATPase pathway for microtubule cross-linking.J. Biol. Chem. 2011; 286: 29261-29272Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 28.Gilbert S.P. Mackey A.T. Kinetics: a tool to study molecular motors.Methods. 2000; 22: 337-354Crossref PubMed Scopus (67) Google Scholar). The MT·CENP-E complex was rapidly mixed with ATP plus trace [α-32P]ATP for times ranging from 5 to 600 ms. The ATP syringe also contained 200 mm KCl (100 mm after mixing). At varying times of incubation, the reaction was quenched with 10 n formic acid (syringe concentration). The formic acid quench terminated the reaction and unfolded the protein, releasing nucleotide from the active site. The products [α-32P]ADP and Pi were separated from [α-32P]ATP and quantified as described above for the pulse-chase experiments. The transients from these experiments were fit to Equation 2, and the observed rates of the initial exponential burst phase were plotted as a function of ATP concentration. The hyperbolic fit of the data provided the rate constant of ATP hydrolysis, k+2 (see Fig. 3). To determine the mantADP release kinetics from the trailing head (see Fig. 4) and correlated with processive stepping required that we generate an intermediate in which the leading head was nucleotide-free and tightly bound to the MT with the trailing head detached from the MT and mantADP tightly bound at its active site (see Fig. 4A). The strategy used was previously developed for Eg5 (26.Krzysiak T.C. Grabe M. Gilbert S.P. Getting in sync with dimeric Eg5: initiation and regulation of the processive run.J. Biol. Chem. 2008; 283: 2078-2087Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The preformed MT·CENP-E complex (10 μm CENP-E, 20 μm MTs, syringe concentrations) was treated with apyrase to generate a nucleotide-free complex. This complex was incubated with 4 μm mantATP, a concentration less than half the CENP-E active sites, and therefore, each mantATP was assumed to bind to the nucleotide binding site of the trailing head because of strain-induced gating of the leading head (17.Clancy B.E. Behnke-Parks W.M. Andreasson J.O. Rosenfeld S.S. Block S.M. A universal pathway for kinesin stepping.Nat. Struct. Mol. Biol. 2011; 18: 1020-1027Crossref PubMed Scopus (159) Google Scholar, 22.Rosenfeld S.S. van Duffelen M. Behnke-Parks W.M. Beadle C. Corrreia J. Xing J. The ATPase cycle of the mitotic motor CENP-E.J. Biol. Chem. 2009; 284: 32858-32868Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 23.Yildiz A. Tomishige M. Gennerich A. Vale R.D. Intramolecular strain coordinates kinesin stepping behavior along microtubules.Cell. 2008; 134: 1030-1041Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 32.Klumpp L.M. Hoenger A. Gilbert S.P. Kinesin's second step.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 3444-3449Crossref PubMed Scopus (96) Google Scholar, 35.Guydosh N.R. Block S.M. Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8054-8059Crossref PubMed Scopus (109) Google Scholar). The mantATP was subsequently hydrolyzed to mantADP (see Fig. 4A). This MT·CENP-E·mantADP complex was then rapidly mixed with increasing concentrations of ATP in the stopped-flow instrument, and the fluorescence change over time was monitored and correlated with mantADP release from the active site. The observed exponential rates were plotted as a function of ATP concentration and fit to a hyperbolic function (see Fig. 4C). The preformed MT·CENP-E complex was rapidly mixed in the stopped-flow instrument with increasing concentrations of ATP plus 300 mm KCl (syringe concentration), and turbidity (λϵx = 340 nm) was monitored. Because the MT·CENP-E complex has a greater mass than MTs without motors or CENP-E in solution, the detachment of CENP-E from MTs results in a decrease in turbidity. The observed initial exponential rates from the transients were plotted as a function of increasing ATP concentration, and a hyperbolic fit of these data provided k+3, the rate constant of ATP-promoted CENP-E detachment from the MT (see Fig. 5). The stopped-flow instrument was used to measure the MT·CENP-E association kinetics and mantADP release kinetics (see Fig. 6). A CENP-E·mantADP complex was preformed and rapidly mixed in the stopped-flow instrument with varying concentrations of MTs plus 2 mm ATP (syringe concentration), and the fluorescence change was monitored over time. The transients were fit to a double exponential function, and the observed rates of the initial exponential phase were plotted as a function of increasing MT concentrations. The data from 0.5 to 4 μm were fit to the following linear equationkobs=k+4[MTs]+k-4(Eq. 4) where k+4 is the second-order rate constant for MT association, and k−4 is the CENP-E off rate as defined by the y axis. The apparent Kd,MT is defined by k−4/k+4. This experiment was designed to determine whether CENP-E bound ADP tightly at both nucleotide sites of the dimer, or whether the dimer exhibited a high affinity site and a low affinity site. Nucleotide-free CENP-E at 5 μm was incubated with either 5 μm or 2.5 μm mantADP to form the CENP-E·mantADP intermediate. It was assumed that if one nucleotide site bound mantADP tightly and the other weakly, the addition of 2.5 μm mantADP to 5 μm CENP-E would result in mantADP partitioning to the high affinity site. The CENP-E·mantADP intermediate was rapidly mixed in the stopped-flow instrument, and data were collected in two time domains: 1–10 and 10–250 s with fluorescence expressed in arbitrary units. Each transient was fit to a double exponential function (see Fig. 7). This study analyzed the ATPase cycle of a CENP-E dimeric motor (20.Sardar H.S. Luczak V.G. Lopez M.M. Lister B.C. Gilbert S.P. Mitotic kinesin CENP-E promotes microtubule plus-end elongation.Curr. Biol. 2010; 20: 1648-1653Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We showed previously that this CENP-E dimer promotes MT plus-end-directed MT gliding at 11 nm/s, and the steady-state ATPase kcat was 0.9 s−1. Furthermore, MT plus-end elongation promoted by CENP-E required ATP turnover, and the kinetics revealed that the elongation rate occurred at 1.03 αβ-tubulin subunits/s. The overall goal for the pre-steady-state kinetics study presented here was to determine whether the ATPase mechanism of CENP-E was consistent with its processivity, the CENP-E-promoted MT elongation, and whether the catalytic cycle was in some way tuned for its role in chromosome congression. Substrate binding by dimeric CENP-E was measured by two approaches: fluorescent mantATP binding using the stopped-flow instrument (Fig. 1) and pulse-chase kinetics with [α-32P]ATP using the rapid quench-flow instrument (Fig. 2). Fig. 1A shows representative transients of mantATP binding to the nucleotide-free MT·CENP-E complex at increasing concentrations of mantATP. The transients are biphasic, and the observed rates of the initial exponential phase were plotted as a function of mantATP concentration, providing the second-order rate constant of mantATP binding at 1.4 μm−1 s−1 (Fig. 1B, Table 1). The observation that the mantATP binding kinetics were biphasic (Fig. 1A) suggests that the second phase represents mantATP binding to the second head of the CENP-E dimer. The relative amplitude associated with each phase at ∼50% (47–56% initial phase and 43–53% for the second phase) is consistent with this interpretation. Fig. 1C reveals that observed rates of mantATP binding during the second phase of fluorescence enhancement were also dependent upon the concentration of mantATP but were not linear as observed for the first phase (Fig. 1B). The hyperbolic fit of the data provided the maximum rate of mantATP binding to the second head at 1.6 s−1, therefore implying that mantATP binding to the second head was limited by an event that occurred after mantATP binding to the first head. These results are consistent with an alternating head catalysis model for processive kinesins.TABLE 1Experimentally determined constants for the MT·CENP-E ATPaseMantATP bindingk+1 = 1.4 ± 0.06 μm−1 s−1k−1 = 1.6 ± 0.7 s−1ATP binding (pulse-chase)K1 = 0.13 ± 0.01 μm−1k+1′ = 47.5 ± 0.8 s−1K1k+1′ = 6.2 μm−1 s−1Kd,ATP = 7.7 μmA0 = 1.96 ± 0.1 ADP·Pi per CENP-E siteKd,ATP = 16.7 ± 1.7 μmATP hydrolysisk+2 = 24.7 ± 0.5 s−1Kd,ATP = 12.3 ± 1.1 μmA0 = 1.93 ± 0.1 ADP·Pi per CENP-E siteKd,ATP = 44.8 ± 4.4 μmMT·CENP-E dissociationk+3 = 1.4 ± 0.01 s−1K½,ATP = 6.4 ± 0.5 μmMT·CENP-E associationk+4 = 0.08 ± 0.004 μm−1 s−1k−4 = 0.07 ± 0.01 s−1Kd,MTs = 0.9 μmMantADP releaseHead 1k+5 = 0.9 ± 0.01 s−1K½,MTs = 5.1 ± 0.1 μmHead 2kmax = 19.4 ± 0.6 s−1K½,ATP = 26.6 ± 3.2 μmMantADP affinityKd,Site 1 = 3.6 ± 0.3 nmKd,Site 2 = 31.8 ± 1.4 nmSteady-state parametersaData from Sardar et al. (20). Errors reported ar" @default.
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• W2024078970 title "Microtubule Capture by Mitotic Kinesin Centromere Protein E (CENP-E)" @default.
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• W2024078970 doi "https://doi.org/10.1074/jbc.m112.376830" @default.
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