ジャーナル: Cell / 年: 2009 タイトル: ATPase cycle of the nonmotile kinesin NOD allows microtubule end tracking and drives chromosome movement. 著者: Jared C Cochran / Charles V Sindelar / Natasha K Mulko / Kimberly A Collins / Stephanie E Kong / R Scott Hawley / F Jon Kull / 要旨: Segregation of nonexchange chromosomes during Drosophila melanogaster meiosis requires the proper function of NOD, a nonmotile kinesin-10. We have determined the X-ray crystal structure of the NOD ...Segregation of nonexchange chromosomes during Drosophila melanogaster meiosis requires the proper function of NOD, a nonmotile kinesin-10. We have determined the X-ray crystal structure of the NOD catalytic domain in the ADP- and AMPPNP-bound states. These structures reveal an alternate conformation of the microtubule binding region as well as a nucleotide-sensitive relay of hydrogen bonds at the active site. Additionally, a cryo-electron microscopy reconstruction of the nucleotide-free microtubule-NOD complex shows an atypical binding orientation. Thermodynamic studies show that NOD binds tightly to microtubules in the nucleotide-free state, yet other nucleotide states, including AMPPNP, are weakened. Our pre-steady-state kinetic analysis demonstrates that NOD interaction with microtubules occurs slowly with weak activation of ADP product release. Upon rapid substrate binding, NOD detaches from the microtubule prior to the rate-limiting step of ATP hydrolysis, which is also atypical for a kinesin. We propose a model for NOD's microtubule plus-end tracking that drives chromosome movement.
SEQUENCES OF TUBULIN A AND B (CHAINS A AND B) IN THE SAMPLE WERE DERIVED FROM COW, WHILE THE MODELS ...SEQUENCES OF TUBULIN A AND B (CHAINS A AND B) IN THE SAMPLE WERE DERIVED FROM COW, WHILE THE MODELS USED FOR FITTING THE MAP WERE FROM PIG.
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実験情報
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実験
実験
手法: 電子顕微鏡法 / 使用した結晶の数: 1
EM実験
試料の集合状態: HELICAL ARRAY / 3次元再構成法: 単粒子再構成法
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試料調製
構成要素
名称: Nucleotide-Free NOD (3DC4)Complexed to Microtubule / タイプ: COMPLEX
緩衝液
pH: 6.9 詳細: 10 mM PIPES [pH 6.9], 1 mM EGTA, 2 mM MgCl2, 1 mM DTT, 5% sucrose