Li W, Wang J C
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
J Biol Chem. 1997 Dec 5;272(49):31190-5. doi: 10.1074/jbc.272.49.31190.
Footprinting of yeast DNA topoisomerase II and its NH2- and COOH-terminal truncation derivatives was carried out to map the locations of lysyl side chains that are involved in enzyme-DNA interaction, in the binding of ATP, or in interaction between domains of the same enzyme molecule. Several conclusions were drawn based on these measurements and the crystal structures of a 92-kDa fragment of the yeast enzyme and a 43-kDa fragment of Escherichia coli gyrase B-subunit. First, the footprinting results support the model previously inferred from the 92-kDa fragment crystal structure that the main site of DNA binding is comprised of a pair of semicircular grooves. Second, the binding of a nonhydrolyzable ATP analog to the yeast enzyme appears to affect citraconylation at a minimum of six lysines in the ATPase domain of each polypeptide. Two of these lysines are probably involved in contacting the nucleotide directly, and one probably becomes buried when the two ATPase domains of a dimeric enzyme come into contact upon ATP binding; for the others, changes in lysine reactivity appear to reflect allosteric changes following ATP binding. Third, from a comparison of the footprint of the intact enzyme and those of the truncated polypeptides comprised of either the NH2- or the COOH-terminal half of the intact polypeptide, it appears that there are few contacts between the NH2- and COOH-terminal half of yeast DNA topoisomerase II.
对酵母DNA拓扑异构酶II及其NH2端和COOH端截短衍生物进行了足迹分析，以确定参与酶与DNA相互作用、ATP结合或同一酶分子结构域间相互作用的赖氨酰侧链的位置。基于这些测量结果以及酵母酶92 kDa片段和大肠杆菌gyrase B亚基43 kDa片段的晶体结构，得出了几个结论。首先，足迹分析结果支持先前从92 kDa片段晶体结构推断出的模型，即DNA结合的主要位点由一对半圆形凹槽组成。其次，不可水解的ATP类似物与酵母酶的结合似乎至少影响每个多肽ATP酶结构域中六个赖氨酸的柠康酰化。其中两个赖氨酸可能直接参与与核苷酸的接触，一个赖氨酸可能在二聚体酶的两个ATP酶结构域在ATP结合时相互接触时被掩埋；对于其他赖氨酸，赖氨酸反应性的变化似乎反映了ATP结合后的变构变化。第三，通过比较完整酶的足迹与由完整多肽的NH2端或COOH端一半组成的截短多肽的足迹，似乎酵母DNA拓扑异构酶II的NH2端和COOH端一半之间几乎没有接触。
