The Kinesin Family Member BimC Contains a Second Microtubule Binding Region Attached to the N terminus of the Motor Domain
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m309419200
ISSN1083-351X
AutoresMaryanne F. Stock, Jessica Chu, David D. Hackney,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoThe kinesin family member BimC has a highly positively charged domain of ∼70 amino acids at the N terminus of the motor domain. Motor domain constructs of BimC were prepared with and without this extra domain to determine its influence. The level of microtubules needed for half saturation of the ATPase of BimC motor domain constructs is reduced by ∼7000-fold at low ionic strength upon addition of this extra N-terminal extension. Although the change in microtubule affinity is less at higher salt, addition of the N-terminal domain still produces a 20-fold increase in affinity for microtubules in 200 mm potassium acetate. A fusion protein of the N-terminal domain and thioredoxin binds tightly to MTs at low salt, consistent with the increased affinity of motor domain constructs (which contain the N-terminal domain) being due to the additional binding of the N-terminal domain to the microtubule. Hydrodynamic analysis indicates that the N-terminal extension is in a highly extended conformation, suggesting that it may be intrinsically disordered. Fusion of the N-terminal extension of BimC onto the motor domain of conventional kinesin produces a similar large increase in microtubule affinity without significant reduction in kcat or velocity in an in vitro motility assay, suggesting that the N-terminal extension can act in a modular manner to increase the microtubule affinity of kinesin motor domains without a decrease in velocity. The kinesin family member BimC has a highly positively charged domain of ∼70 amino acids at the N terminus of the motor domain. Motor domain constructs of BimC were prepared with and without this extra domain to determine its influence. The level of microtubules needed for half saturation of the ATPase of BimC motor domain constructs is reduced by ∼7000-fold at low ionic strength upon addition of this extra N-terminal extension. Although the change in microtubule affinity is less at higher salt, addition of the N-terminal domain still produces a 20-fold increase in affinity for microtubules in 200 mm potassium acetate. A fusion protein of the N-terminal domain and thioredoxin binds tightly to MTs at low salt, consistent with the increased affinity of motor domain constructs (which contain the N-terminal domain) being due to the additional binding of the N-terminal domain to the microtubule. Hydrodynamic analysis indicates that the N-terminal extension is in a highly extended conformation, suggesting that it may be intrinsically disordered. Fusion of the N-terminal extension of BimC onto the motor domain of conventional kinesin produces a similar large increase in microtubule affinity without significant reduction in kcat or velocity in an in vitro motility assay, suggesting that the N-terminal extension can act in a modular manner to increase the microtubule affinity of kinesin motor domains without a decrease in velocity. BimC is the founding member of the BimC/Eg5 or N-2 class of motor proteins in the kinesin superfamily (see Refs. 1Kim A.J. Endow S.A. J. Cell Sci. 2000; 113 (Pt. 21): 3681-3682PubMed Google Scholar and 2Miki H. Setou M. Kaneshiro K. Hirokawa N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7004-7011Crossref PubMed Scopus (495) Google Scholar). The motor domain is at the N terminus, followed by an extended region with a number of predicted coiled-coil domains. BimC was first discovered in Aspergillus nidulans (3Enos A.P. Morris N.R. Cell. 1990; 60: 1019-1027Abstract Full Text PDF PubMed Scopus (326) Google Scholar), and members of this class are widely distributed from yeast to higher organisms. The native molecule is a homotetramer that is apparently formed by antiparallel combination of two parallel dimers (see Ref. 4Kashina A.S. Rogers G.C. Scholey J.M. Biochim. Biophys. Acta. 1997; 1357: 257-271Crossref PubMed Scopus (117) Google Scholar for review). This arrangement places a pair of motor domains at each end of an elongated tetramer and is believed to facilitate cross-linking of microtubules (MTs) 1The abbreviations used are: MTmicrotubuleNteN-terminal extension of BimCTM-Ntefull Nte of BimC fused to thioredoxinMSRMAP2 similarity regionmantATP2′(3′)-O-(N-methylanthraniloyl)adenosine 5′-triphosphateMSRMAP2 similarity region. in the overlap region of the spindle. The motor domains are at each end of the tetramer and are attached to different microtubules that are capable of producing the sliding that pushes apart the poles (5Sharp D.J. McDonald K.L. Brown H.M. Matthies H.J. Walczak C. Vale R.D. Mitchison T.J. Scholey J.M. J. Cell Biol. 1999; 144: 125-138Crossref PubMed Scopus (257) Google Scholar). Because of its role in mitosis, human Eg5 is an attractive target for drug development, and monastrol is a reversible inhibitor of Eg5 that blocks mitosis (6Mayer T.U. Kapoor T.M. Haggarty S.J. King R.W. Schreiber S.L. Mitchison T.J. Science. 1999; 286: 971-974Crossref PubMed Scopus (1648) Google Scholar, 7Maliga Z. Kapoor T. Mitchison T.J. Chem. Biol. 2002; 9: 989-996Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 8DeBonis S. Simorre J.P. Crevel I. Lebeau L. Skoufias D.A. Blangy A. Ebel C. Gans P. Cross R. Hackney D.D. Wade R.H. Kozielski F. Biochemistry. 2003; 42: 338-349Crossref PubMed Scopus (176) Google Scholar). Monastrol is highly selective for Eg5 from higher organisms and does not even inhibit the closely related A. nidulans BimC (8DeBonis S. Simorre J.P. Crevel I. Lebeau L. Skoufias D.A. Blangy A. Ebel C. Gans P. Cross R. Hackney D.D. Wade R.H. Kozielski F. Biochemistry. 2003; 42: 338-349Crossref PubMed Scopus (176) Google Scholar). microtubule N-terminal extension of BimC full Nte of BimC fused to thioredoxin MAP2 similarity region 2′(3′)-O-(N-methylanthraniloyl)adenosine 5′-triphosphate MAP2 similarity region. Detailed studies of the kinetics and structural properties of BimC motor domains from lower organisms have not been performed, but the related Eg5 from higher organisms has been studied more extensively. The crystal structure of the motor domain of human Eg5 (9Turner 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 (191) Google Scholar) indicates that the overall folding of the motor domain is similar to that of other kinesins, but the neck linker is in a novel conformation. Image reconstruction of decorated MTs indicates that the attached head of an Eg5 dimer is in a similar position to that of other kinesins with an additional detached density whose position is less sensitive to nucleotide than to conventional kinesin (10Hirose K. Henningsen U. Schliwa M. Toyoshima C. Shimizu T. Alonso M. Cross R.A. Amos L.A. EMBO J. 2000; 19: 5308-5314Crossref PubMed Scopus (25) Google Scholar). Initial kinetic characterization (11Lockhart A. Cross R.A. Biochemistry. 1996; 35: 2365-2373Crossref PubMed Scopus (60) Google Scholar) established that the ATPase mechanism of Eg5 was similar to that of conventional kinesin except that it was slower, with ADP release being at least partially rate-limiting and with non-hydrolyzable ATP analogs producing tighter binding to MTs. Unlike conventional kinesin, however, there are indications that Eg5 dimers are not processive (12Crevel I.M. Lockhart A. Cross R.A. J. Mol. Biol. 1997; 273: 160-170Crossref PubMed Scopus (79) Google Scholar). Recent work has indicated how the kinetics and state of oligomerization vary with inclusion of increasing regions of the neck coil (7Maliga Z. Kapoor T. Mitchison T.J. Chem. Biol. 2002; 9: 989-996Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 8DeBonis S. Simorre J.P. Crevel I. Lebeau L. Skoufias D.A. Blangy A. Ebel C. Gans P. Cross R. Hackney D.D. Wade R.H. Kozielski F. Biochemistry. 2003; 42: 338-349Crossref PubMed Scopus (176) Google Scholar). Although BimC is classified as an N-terminal motor because of its grouping with other N-terminal motors by similarity of sequence of the motor domains, BimC from A. nidulans actually has a significant domain of ∼70 amino acids appended to the N terminus of the motor (3Enos A.P. Morris N.R. Cell. 1990; 60: 1019-1027Abstract Full Text PDF PubMed Scopus (326) Google Scholar). This N-terminal extension (Nte) is highly positively charged and has weak sequence similarity to part of the proline-rich region of MAP2 that is involved in MT binding (Fig. 1). These features suggest that the Nte of BimC may constitute a second MT-binding site that could influence the kinetics of BimC and its affinity for MTs. We report here that the Nte binds independently to MTs and that attachment of the Nte to the BimC motor domain greatly increases the affinity for MTs. Furthermore, the Nte can act in a modular manner, as attachment of the Nte to the motor domain of conventional kinesin also results in a large increase in affinity for MTs, without significant inhibition of kcat or sliding velocity. Construction of Expression Plasmids—The BimC constructs indicated in Figs. 1, 2, 3 were derived from the partial cDNA clone and the overlapping genomic clone of Enos and Morris (3Enos A.P. Morris N.R. Cell. 1990; 60: 1019-1027Abstract Full Text PDF PubMed Scopus (326) Google Scholar). The cDNA and genomic clones were fused to generate a full-length construct using the DdeI site at position 559. PCR was then used to generate a series of motor domain constructs that were cloned into pET21 (Novagen) for expression. BC72M is a nonfusion protein that contains the core motor domain and the region homologous to the neck linker of conventional kinesin but lacks the Nte of BimC. It was obtained by converting Ile72 of BimC into an initiation methionine and introducing a stop codon after Lys428. Although BC72M expresses well, constructs beginning at residue 1 and containing the Nte did not express protein at detectable levels. The 5′ coding region is highly enriched in GC base pairs, and a new primer with the sequence GCCATGCGTGGTCCGCAGCGTGCTACTTATGG was used to reduce the number of GC base pairs and to replace codons with unfavorable usage by Escherichia coli. This new construct had detectable, but low, levels of expression as a non-fusion protein. The amount of protein that could be isolated, however, was not enough for extensive biochemical characterization, and this construct was subsequently subcloned into pET32 (Novagen) as a fusion protein with thioredoxin (TT-BC1M), which expressed at a high level.Fig. 3Sequences of thioredoxin fusion proteins and the products of cleavage with thrombin. Most of the motor domain constructs described in Fig. 2 were expressed as fusion proteins with thioredoxin (Trx) and cleaved with thrombin to release the motor domain. The sequences of the fusion junctions and the final cleaved proteins are indicated. Unless otherwise indicated, all of the experiments reported here with constructs containing the full Nte were performed with the version having the S9Y substitution (see “Experimental Procedures”). More limited results with the version having the wild-type Ser-9 (see “Results”) indicated no significant difference in properties between the two versions. The TT series are thioredoxin fusions derived from pET32 (Novagen), with the DNA sequence for the terminal Gly-Ser of the thrombin cleavage site converted into a restriction site for BamH1 to facilitate cassette cloning. The location of thrombin cleavage is indicated by an arrow. Cleavage of these constructs with thrombin yields proteins with Gly-Ser appended to the N terminus, as indicated. The TM series are fusions with thioredoxin in which a BamH1 site has been introduced at the end of the spacer after thioredoxin. Consequently, the TM series has lost the His tag and the thrombin cleavage site. TM-Nte extends to residue 71, with an additional Gly appended to the C terminus.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The DNA sequence of the derived constructs did not agree completely with the published sequence (GenBankTM accession number M32075; Ref. 3Enos A.P. Morris N.R. Cell. 1990; 60: 1019-1027Abstract Full Text PDF PubMed Scopus (326) Google Scholar). A sample of whole genomic DNA from A. nidulans was also kindly provided by N. R. Morris. PCR was used to amplify the regions where discrepancies were found. The sequence of these genomic samples matched the sequence of the original clones provided by Enos and Morris (3Enos A.P. Morris N.R. Cell. 1990; 60: 1019-1027Abstract Full Text PDF PubMed Scopus (326) Google Scholar) and the above constructs derived from them but did not match the published sequence. The revised sequence has three amino acid changes from that of the published sequence as Tyr to Ser at position 9, Gly to Pro at position 12, and Met to Ile at position 393. All of the clones used for the work reported here contain the wild-type Pro12 and Ile393. Clones derived from the modified 5′-primer above, however, contain Tyr at position 9 instead of Ser because the 5′ PCR primer was originally designed to match the published sequence. Ser9 is near the N terminus in a region that is likely to be unstructured and outside of the region of sequence similarity with MAP2. Consequently the change of Ser to Tyr at position 9 is not likely to significantly affect the MT binding properties. As a control, constructs containing the wild-type Ser9 were also generated as indicated in Fig. 3, and their ATPase and hydrodynamic properties were indistinguishable for those containing the S9Y substitution (see “Results”). TT-BKinM and TT-BKin412GS contain the same N-terminal region of BimC fused to thioredoxin as described for TT-BC1M(Tyr9), but the motor domain of BimC is replaced by the motor domain of conventional kinesin as indicted in Figs. 1, 2, 3. DKH412GS and BKin412GS in addition are fused at the C terminus to cytoplasmic gelsolin derived from pKN172 (13Way M. Gooch J. Pope B. Weeds A.G. J. Cell Biol. 1989; 109: 593-605Crossref PubMed Scopus (177) Google Scholar). Protein Expression and Purification—General procedures for induction and purification on phosphocellulose were carried out as described (14Stock M.F. Hackney D.D. Vernos I. Methods in Molecular Biology-Kinesin Protocols. Humana Press, Totowa, NJ2001: 43-48Google Scholar). MgATP was maintained at ≥0.1 mm during the purification of all constructs containing the motor domain. DKH412GS and BC72M were expressed as non-fusion proteins and purified essentially as described for conventional kinesin motor domains (14Stock M.F. Hackney D.D. Vernos I. Methods in Molecular Biology-Kinesin Protocols. Humana Press, Totowa, NJ2001: 43-48Google Scholar, 15Huang T.-G. Hackney D.D. J. Biol. Chem. 1994; 269: 16493-16501Abstract Full Text PDF PubMed Google Scholar). The TT series of thioredoxin fusions (Fig. 3) contains a His tag, and they were purified by both phosphocellulose and Ni-NTA metal affinity chromatography. The fusion proteins were cleaved with thrombin, and the released motor domains were separated from thioredoxin and thrombin by chromatography on phosphocellulose at pH 6.5. TM-Nte does not contain a His tag and was purified by chromatography on phosphocellulose and gel filtration on Sephacryl S-300. Final preparations were dialyzed against 50% glycerol in A25 buffer with 50 mm KCl, 2 mm dithiothreitol and 0.1 mm MgATP (for motor domain constructs) and stored at -80 °C in small aliquots. Taxol-stabilized MTs were prepared from phosphocellulose-purified tubulin as described (16Hackney D.D. Biochemistry. 2002; 41: 4437-4446Crossref PubMed Scopus (39) Google Scholar), except that the source of the tubulin was pig brain rather than cow brain. MT concentrations are reported as the concentration of tubulin heterodimers. Hydrodynamic Characterization—Gel filtration and velocity sedimentation in a sucrose gradient were used to estimate the D20,w and s20,w values. Standards proteins were bovine catalase, bovine serum albumin, ovalbumin, bovine carbonic anhydrase, and bovine cytochrome c with s20,w values of 11.3, 4.4, 3.7, 3.0, and 1.9 S and D20,w values of 4.4, 6.1, 7.4, 9.3, and 12.7 × 107 cm2/s, respectively. Most values are from Ref. 17Sober H. Handbook of Biochemistry. Chemical Rubber Co., Cleveland, OH1968Google Scholar, but in some cases, the literature values are not self-consistent, and minor adjustments have been made so that each set of s20,w, D20,w, and Mr values obeys the Svedberg equation and yields an f/fmin > 1.10. Gel filtration chromatography was done on Sephacryl S-300 (Amersham Biosciences). Fractions were analyzed by SDS-PAGE to determine the peak positions. Cytochrome c is retarded on the column at low ionic strength and was not included in the regression in determinations in the absence of added NaCl. Ultracentrifugation was performed on 5-ml linear gradients of 9–20% sucrose in an MLS-50 rotor (Beckman-Coulter) that were approximately isokinetic at 4 °C for proteins, with a partial specific volume of 0.73 cm3/g. The buffer used was A25 buffer (25 mm potassium ACES, pH 6.9, 2 mm magnesium acetate, 2 mm K-EGTA, 0.1 mm K-EDTA and 1 mm β-mercaptoethanol) with 25 mm KCl and was supplemented with 200 mm NaCl, as indicated. For motor domains, the centrifugation was for 15 h at 44,000 rpm with catalase, serum albumin and carbonic anhydrase as standards. For the smaller TM-Nte constructs, centrifugation was for 36 h at 50,000 rpm with ovalbumin, carbonic anhydrase and cytochrome c as standards. A partial specific volume of 0.73 cm3/g was used in all calculations. Kinetic and Binding Measurements—All reactions and binding experiments were conducted at 25 °C in A25 buffer supplemented with KCl and NaCl as indicated. The coupled enzyme system of pyruvate kinase and lactic dehydrogenase were used to monitor ATP hydrolysis in the presence of 2 mm P-enolpyruvate and 1 mm MgATP as described (15Huang T.-G. Hackney D.D. J. Biol. Chem. 1994; 269: 16493-16501Abstract Full Text PDF PubMed Google Scholar, 18Stock M.F. Hackney D.D. Vernos I. Methods in Molecular Biology-Kinesin Protocols. Humana Press, Totowa, NJ2001: 65-72Google Scholar). Determination of kcat and K0.5(MT) values were typically performed at 5 or more concentrations of MTs in duplicate, with error bars indicating the range of the values. The data were fit by nonlinear regression to the full quadratic expression for mutual depletion (19Griffiths J.R. Biochem. Soc. Trans. 1979; 7: 429-439Crossref PubMed Scopus (7) Google Scholar) using the Solver routine of Excel (Microsoft). When it was not possible to go to a high enough MT concentration to accurately determine kcat and K0.5(MT) separately, only the kbi(ATPase) ratio equal to kcat/K0.5(MT) is reported. At high ionic strength when the maximum MT concentration was much less than K0.5(MT), the kbi(ATPase) value was determined from the linear increase in rate with increasing MT concentration. Motility—Short F-actin filaments were capped with complexes of kinesin-gelsolin fusion proteins and labeled with rhodamine phalloidin essentially as described by Yajima et al. (20Yajima J. Alonso M.C. Cross R.A. Toyoshima Y.Y. Curr. Biol. 2002; 12: 301-306Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The complexes with F-actin were formed in 100 mm KCl in A25 buffer without EDTA or EGTA and containing 1 mm MgATP and 1 mm CaCl2. Immediately before loading into a flow cell for observation, the complexes were diluted 1:100 into the same buffer but without CaCl2 and with 25 mm KCl and 0.4 mg/ml casein. Movement of these complexes along salt-washed sea urchin sperm axonemes was observed by fluorescence microscopy with an Olympus XI71 microscope using excitation by a 10 milliwatt laser at 532 nm (Beta Electronics) and a 550–610 nm bandpass filter for emission. A 60 × 1.45 numerical aperture objective was used with an additional 4× projection lens. Images were acquired with a PicIII intensified camera (Instrutech) at 30 frames/s, recorded on S-VHS tape, digitized, and analyzed with NIH-Image. In some cases, a rolling average of 2 or 4 video frames was used. Motility was only measured for spots that exhibited continuous movement for > 1 s. Motility was difficult to observe with BKin412GS because most of the short actin filaments rapidly became tethered to the surface of the coverslip through one end only, with the other end moving freely in solution. This was also observed with DKH412GS, but to a much reduced extent. Pretreatment of the coverslip with the basic protein lysozyme reduced the amount of surface binding, suggesting that the binding was enhanced by the interaction of the positive Nte with anionic sites on the coverslip. For all of the measurements reported here, the coverslips were washed with detergent, blocked with lysozyme at 2 mg/ml for 5 min, thoroughly rinsed with water, and air dried before assembly of a flow cell using two strips of double-sided tape. Axonemes were flowed through the cell to allow attachment, and then the cell was flushed with buffer containing 0.4 mg/ml casein to further block the surface before introduction of the motor-actin complexes. Properties of BimC Motor Domain—Previous work (21Jiang W. Stock M. Li X. Hackney D.D. J. Biol. Chem. 1997; 272: 7626-7632Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) indicated that DKH346 (residues 1–346 of Drosophila kinesin as indicated in Fig. 1A) is a good model for a motor domain of conventional kinesin that contains the core ATPase domain of Drosophila kinesin plus a complete neck linker. DHK346 is monomeric because it lacks the neck coiled-coil region that is required for dimerization. BC72M (see Figs. 1, 2, and 3) is an analogous BimC construct that begins and ends on positions that are equivalent in a sequence alignment to positions 3 and 345 of Drosophila kinesin. Hydrodynamic analysis (Table I) indicates that BC72M is also monomeric. The basal ATPase rate in the absence of MTs is ∼0.1 s-1 for all of the BimC motor domain constructs studied here. The dependence of the ATPase rate of BC72M on the concentration of MTs over a range of KCl concentrations is given in Fig. 4A, and kinetic parameters are summarized in Table II. Even in low salt buffer, saturation is far from complete at the highest MT concentration. The kcat value for the maximum ATPase rate at saturating levels of MTs and the K0.5(MT) value for the concentration of MTs producing half the maximum rate can only be estimated at ∼31 s-1 and ∼17 μm, respectively, in 3 mm KCl. At higher ionic strength, the ATPase rate is strongly inhibited, as is also observed with other kinesin family members. Separate estimation of kcat and K0.5(MT) values is not feasible at higher salt, but the bimolecular rate kbi(ATPase) = kcat/K0.5(MT) can still be determined, and the dependence of kbi(ATPase) on ionic strength is indicated in Fig. 5. For comparison, the ATPase rate of the motor domain of conventional kinesin (DKH346) was determined at 3 and 25 mm KCl as indicated in Fig. 4B. The kcat and K0.5(MT) values of ∼86 s-1 and ∼5 μm in 3 mm KCl for DKH346 indicate that BimC is slower than an equivalent construct of conventional kinesin and has weaker affinity for MTs in the presence of ATP.Table IHydrodynamic characterization Hydrodynamic parameters were determined by velocity sedimentation in a sucrose gradient and by gel filtration as described under “Experimental Procedures.” Analysis was performed in A25 buffer with 25 mm KCl for BC72M and BC1M and with 200 mm NaCl for TM-Nte and TM-S.ConstructsS20,w (S)D20,wMraCalculated by the Svedberg equation SvedbergMrbCalculated from the amino acid sequence PeptideOligomeric state×107 cm2/s×10-3×10-3BC72M3.22 ± 0.11 (4Kashina A.S. Rogers G.C. Scholey J.M. Biochim. Biophys. Acta. 1997; 1357: 257-271Crossref PubMed Scopus (117) Google Scholar)7.89 ± 0.07 (3Enos A.P. Morris N.R. Cell. 1990; 60: 1019-1027Abstract Full Text PDF PubMed Scopus (326) Google Scholar)36.839.3MonomerBC1M3.30 ± 0.03 (4Kashina A.S. Rogers G.C. Scholey J.M. Biochim. Biophys. Acta. 1997; 1357: 257-271Crossref PubMed Scopus (117) Google Scholar)NDcND, not determined–46.9MonomerTM-Nte1.69 ± 0.06 (3Enos A.P. Morris N.R. Cell. 1990; 60: 1019-1027Abstract Full Text PDF PubMed Scopus (326) Google Scholar)7.94 ± 0.04 (2Miki H. Setou M. Kaneshiro K. Hirokawa N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7004-7011Crossref PubMed Scopus (495) Google Scholar)19.220.0MonomerTM-S1.79 (1Kim A.J. Endow S.A. J. Cell Sci. 2000; 113 (Pt. 21): 3681-3682PubMed Google Scholar)ND–12.2Monomera Calculated by the Svedberg equationb Calculated from the amino acid sequencec ND, not determined Open table in a new tab Table IISummary of ATPase kinetics Kinetic parameters were determined as described under “Experimental Procedures” in A25 buffer with added salt as indicated. Data is from Figs. 4, 5, 6, 7 and other determinations. Separate values for kcat and K0.5(MT) were only determined where indicated.[NaCl]BC72MBC65MBC1MDKH346BKinMkcatK0.5(mt)kbi(ATPase)kcatK0.5(mt)kbi(ATPase)kcatK0.5(mt)kbi(ATPase)kcatK0.5(mt)kbi(ATPase)kcatK0.5(mt)kbi(ATPase)mms-1μmμm-1 s-1s-1μmμm-1 s-1s-1μmμm-1 s-1s-1μmμm-1 s-1s-1μmμm-1 s-13∼31∼171.8925.10.793224.20.00259600∼86∼517.5NDaND, not determined250.6923.44.35.425.80.005547003.965.10.00748800500.331.5427.20.01182300ND67.40.3718275NDND24.50.107230NDND1000.091ND28.41.3421NDND150NDND0.88NDND2000.0162ND0.148NDND200 (K-acetate)bWith 200 mm potassium acetate and no KCl0.0510.0851.01NDNDa ND, not determinedb With 200 mm potassium acetate and no KCl Open table in a new tab Fig. 5Dependence of kbi(ATPase) on ionic strength. ♦, BC72M; ♦, BC65M; ▪, BC1M. Slopes for BC72M and BC1M are -2.8 and -9.8, respectively. Data at <50 mm KCl were not included in the fit for BC1M. The ionic strength of the ATPase reaction is 0.042 in the absence of added KCl or NaCl.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Influence of Nte—BC1M contains the full Nte domain of BimC in addition to the motor domain and neck linker of BC72M as indicated in Figs. 1, 2, 3. The D20,w value of BC1M could not be obtained by gel filtration because it binds to the column, even in the presence of 200 mm NaCl, but the sedimentation coefficient of 3.3 S (Table I) is similar to that of BC72M and indicates that BC1M is also a monomer under these conditions. Inclusion of the Nte in BC1M produces a striking increase in the affinity of the motor for MTs during ATP hydrolysis as indicated in Fig. 6. At 3 mm KCl in Fig. 6A, the activation by MTs is close to stoichiometric, even at a BimC concentration of only 8.3 nm. Exact determination of the K0.5(MT) value under these conditions is complicated by the tight binding with K0.5(MT) < Etotal and the possibility that the affinity for BimC may change as the MT lattice approaches saturation with bound BimC. However, the 7000-fold decrease in K0.5(MT) on addition of the Nte (∼0.0025 μm for BC1M versus ∼17 μm for BC72M) clearly indicates a dramatic increase in MT affinity. The kcat value of 24 s-1 for BC1M is similar to the value of ∼33 s-1 for BC72M, indicating that addition of the Nte does not prevent catalytic turnover at close to the normal rate. The kbi(ATPase) of BC1M is also strongly dependent on ionic strength (Fig. 6), and the dependence is even greater than for BC72M, as indicated in Fig. 5. BC1M with wild-type Ser at position 9 has kcat and K0.5(MT) values of 24.2 s-1 and 0.118 μm, respectively, in 75 mm KCl. These values are essentially identical to the corresponding values of 24.5 s-1 and 0.107 μm for BC1M with Tyr at position 9 (Table II); thus, this substitution has no influence on kcat or MT affinity. BimC contains a region with a high concentration of mixed negative and positively charged groups that immediately adjoins the motor domain (Fig. 1). BC65M contains this highly charged region but lacks the highly positively charged MSR region with similarity to MAP2. The MT-stimulated ATPase of BC65M is elevated over that of BC72M as indicated in Fig. 5 and Table II. However, the increase represents only a fraction of the total increase produced by the full Nte. In the linear phase of the ionic strength dependence of BC1M at 50 mm KCl, the kbi(ATPase) value of 1.54 μm-1 s-1 for BC65M is 5-fold larger than for BC72M but still 1300-fold smaller than for BC1M. Ionic Strength Dependence—The influence of the Nte decreases with increasing ionic strength as seen by the convergence of the lines for BC72M and BC1M in Fig. 5 at high ionic strength. This suggests that the Nte has a diminished influence at physiological ionic strength, but even at 200 mm KCl the Nte still produces almost a 10-fold increase in kbi(ATPase) (Table II). Also, MT-stimulated ADP release of conventional kinesin has a large dependence on the nature of the anion, with chloride being more inhibitory than acetate or aspartate (22Cheng J.Q. Jiang W. Hackney D.D. Biochemistry. 1998; 37: 5288-5295Crossref PubMed Scopus (43) Google Scholar), which are more typical of physiological conditions. The kbi(ATPase) of BC1M, BC65M, and BC72M are 1.01 ± 0.07 (3Enos A.P. Morris N.R. Cell. 1990; 60: 1019-1027Abstract Full Text PDF PubMed Scopus (326) Google Scholar), 0.085 ± 0.004 (3Enos A.P. Morris N.R. Cell. 1990; 60: 1019-1027Abstract Full Text PDF PubMed Scopus (326) Google Scholar), and 0.051 ± 0.004 (3Enos A.P. Morris N.R. Cell. 1990; 60: 1019-1027Abstract Full Text PDF PubMed Scopus (326) Google Scholar) μm-1 s-1, respectively, in A25 buffer with 200 mm potassium acetate at a total ionic strength of 0.24. These rates wi
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