title: The second microtubule-binding site of monomeric kid enhances the microtubule affinity.
abstract: Chromokinesin Kid (kinesin-like DNA-binding protein) localizes on spindles and chromosomes and has important roles in generating polar ejection force on microtubules in the metaphase. To understand these functions of Kid at the molecular level, we investigated molecular properties of Kid, its oligomeric state, interaction with microtubules, and physiological activity in vitro. Kid expressed in mammalian cells, as well as Kid expressed in Escherichia coli, was found to be monomeric. However, Kid cross-linked microtubules in an ATP-sensitive manner, suggesting that Kid has a second microtubule-binding site in addition to its motor domain. This was ascertained by binding of Kid fragments lacking the motor domain to microtubules. The interaction of the second microtubule-binding site was weak in a nucleotide-insensitive manner. KmMT of the ATPase activity of Kid was lower than that of the fragments lacking the second microtubule-binding site. Moreover, the velocity of Kid movement in vitro was not affected by the second microtubule-binding site, which is consistent with the weak binding of this site to microtubules. The second microtubule-binding site would be important to enhance the affinity to microtubules for the monomeric motor, Kid. Because the amino acid sequence of this region is highly conserved among species, it seems to have essential roles for the functions of Kid in vivo.
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo, 153-8902, Japan.
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Shiroguchi
authors.forename: Miho
authors.initials: M
authors.lastname: Ohsugi
authors.forename: Masaki
authors.initials: M
authors.lastname: Edamatsu
authors.forename: Tadashi
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Yoko Y
authors.initials: YY
authors.lastname: Toyoshima
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016547:Kinesins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D004268:DNA-Binding Proteins; C098764:KIF22 protein, human; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D016547:Kinesins