title: The kinesin family member BimC contains a second microtubule binding region attached to the N terminus of the motor domain.
abstract: The kinesin family member BimC has a highly positively charged domain of approximately 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 approximately 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.
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Maryanne F
authors.initials: MF
authors.lastname: Stock
authors.forename: Jessica
authors.initials: J
authors.lastname: Chu
authors.forename: David D
authors.initials: DD
authors.lastname: Hackney
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001233:Aspergillus nidulans / Q000378:metabolism; D002465:Cell Movement; D002850:Chromatography, Gel; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry*; D007477:Ions; D016547:Kinesins / Q000737:chemistry*; D007700:Kinetics; D007701:Kinetin; D008870:Microtubules / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D019347:Potassium Acetate / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013879:Thioredoxins / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: C094560:BimC protein, Aspergillus nidulans; D018076:DNA, Complementary; D005656:Fungal Proteins; D007477:Ions; D011993:Recombinant Fusion Proteins; D014867:Water; D013879:Thioredoxins; D000251:Adenosine Triphosphatases; D016547:Kinesins; D000225:Adenine; D019347:Potassium Acetate; D007701:Kinetin