Structure of the N-Terminal Gyrase B Fragment in Complex with ADP⋅Pi Reveals Rigid-Body Motion Induced by ATP Hydrolysis

Type II DNA topoisomerases are essential enzymes that catalyze topological rearrangement of double-stranded DNA using the free energy generated by ATP hydrolysis. Bacterial DNA gyrase is a prototype of this family and is composed of two subunits (GyrA, GyrB) that form a GyrA2GyrB2 heterotetramer. The N-terminal 43-kDa fragment of GyrB (GyrB43) from E. coli comprising the ATPase and the transducer domains has been studied extensively. The dimeric fragment is competent for ATP hydrolysis and its structure in complex with the substrate analog AMPPNP is known. Here, we have determined the remaining conformational states of the enzyme along the ATP hydrolysis reaction path by solving crystal structures of GyrB43 in complex with ADP⋅BeF3, ADP⋅Pi, and ADP. Upon hydrolysis, the enzyme undergoes an obligatory 12° domain rearrangement to accommodate the 1.5 Å increase in distance between the γ- and β-phosphate of the nucleotide within the sealed binding site at the domain interface. Conserved residues from the QTK loop of the transducer domain (also part of the domain interface) couple the small structural change within the binding site with the rigid body motion. The domain reorientation is reflected in a significant 7 Å increase in the separation of the two transducer domains of the dimer that would embrace one of the DNA segments in full-length gyrase. The observed conformational change is likely to be relevant for the allosteric coordination of ATP hydrolysis with DNA binding, cleavage/re-ligation and/or strand passage.

II型DNA拓扑异构酶（Type II DNA topoisomerases）是一类不可或缺的酶，可借助ATP水解所释放的自由能催化双链DNA发生拓扑重排。细菌DNA旋转酶（Bacterial DNA gyrase）是该酶家族的原型成员，由两个亚基（GyrA、GyrB）组装为GyrA₂GyrB₂异四聚体。大肠杆菌（E. coli）来源的GyrB的N端43 kDa片段（GyrB43）包含ATP酶结构域与转导结构域，该片段已被广泛研究。该二聚体片段具备ATP水解活性，其与底物类似物AMP-PNP结合的复合物结构已有报道。本研究通过解析GyrB43分别与ADP⋅BeF₃、ADP⋅Pi及ADP结合的晶体结构，确定了该酶沿ATP水解反应通路的剩余构象状态。在水解过程中，酶会发生强制性的12°结构域重排，以适配封闭的结构域界面结合位点内核苷酸γ-与β-磷酸之间1.5 Å的间距增量。来自转导结构域QTK环（同时也是结构域界面的组成部分）的保守残基，可将结合位点内的微小结构变化与刚体运动相耦联。该结构域重定向体现在二聚体的两个转导结构域之间的间距显著增大7 Å，这一变化在全长旋转酶中会包裹其中一段DNA链。本次观测到的构象变化，可能与ATP水解、DNA结合、切割/再连接以及链传递的变构调控密切相关。