title: The dynein stalk head, the microtubule binding-domain of dynein: NMR assignment and ligand binding.
abstract: Dynein is a motor ATPase, and the C-terminal two-thirds of its heavy chain form a ring structure. One of protrudings from this ring structure is a stalk whose tip, the dynein stalk head (DSH), is thought to be the microtubule-binding domain. As a first step toward elucidating the functional mechanisms of DSH, we aimed at the NMR structural analysis of an isolated DSH from mouse cytoplasmic dynein. The DSH expressed in bacteria and purified was coprecipitated with microtubules, suggesting its proper folding. Chemical shifts of the DSH were obtained from NMR measurements, and backbone assignment identified 94% of the main-chain N-H signals. Secondary structural prediction programs showed that about 60% of the residues formed alpha-helices. A region with cationic residues K58 and R61 (and possibly R66 as well), and another with R86, K88, K90, and K91, were found to form alpha-helices. Both of these regions may be important in the formation of the DSH-binding site to a microtubule that has a low pI with a number of acidic residues. Two synthetic peptides containing the sequence of the alpha-helix 12 of beta-tubulin, considered to be important in binding to DSH, were investigated. Of these two peptides, the one with higher helix-formation propensity appeared to bind to DSH, since it precipitated with DSH in a nearly stoichiometric manner. This suggested that the alpha-helicity of this region would be important in its binding to DSH.
authors.affiliation: Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Japan.
authors.forename: Youské
authors.initials: Y
authors.lastname: Shimizu
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Kato
authors.forename: Hisayuki
authors.initials: H
authors.lastname: Morii
authors.forename: Masaki
authors.initials: M
authors.lastname: Edamatsu
authors.forename: Yoko Yano
authors.initials: YY
authors.lastname: Toyoshima
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004398:Dyneins / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D047468:Immunoprecipitation; D008024:Ligands; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013552:Swine; D014404:Tubulin / Q000737:chemistry
chemical_list: D008024:Ligands; D010455:Peptides; D014404:Tubulin; D004398:Dyneins