National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM124847
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM100008
米国
引用
ジャーナル: Nat Commun / 年: 2019 タイトル: Convergent allostery in ribonucleotide reductase. 著者: William C Thomas / F Phil Brooks / Audrey A Burnim / John-Paul Bacik / JoAnne Stubbe / Jason T Kaelber / James Z Chen / Nozomi Ando / 要旨: Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for ...Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a distinct and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis. Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible active tetramer and two inhibited helical filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery.
温度: 293 K / 手法: 蒸気拡散法, シッティングドロップ法 / pH: 7.6 詳細: The crystal was grown by vapor diffusion from 4.5 mg/ml holo-NrdE in 50 mM HEPES (pH 7.6), 50 mM NaCl, 5 mM MgCl2, 2 mM TCEP, and 1% glycerol, supplemented with 5 mM ATP, 0.5 mM TTP, and 1 mM ...詳細: The crystal was grown by vapor diffusion from 4.5 mg/ml holo-NrdE in 50 mM HEPES (pH 7.6), 50 mM NaCl, 5 mM MgCl2, 2 mM TCEP, and 1% glycerol, supplemented with 5 mM ATP, 0.5 mM TTP, and 1 mM GDP. The protein solution was incubated for 10 minutes with freshly added nucleotides prior to being mixed in a 1:1 hanging drop with a precipitating solution of 6% PEG 3350, 1% w/v tryptone, and 50 mM HEPES at pH 7. Crystals were cryoprotected by soaking for 5-10 seconds in well solution mixed with 8% w/v sucrose, 2% w/v glucose, 8% v/v glycerol, 8% v/v ethylene glycol and supplemented with nucleotides, TCEP, and MgCl2 adjusted to the same concentrations used in the original protein solutions.
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データ収集
回折
平均測定温度: 100 K / Serial crystal experiment: N
放射光源
由来: シンクロトロン / サイト: CHESS / ビームライン: F1 / 波長: 0.9775 Å