Backbone dynamics of Escherichia coli adenylate kinase at the extreme stages of the catalytic cycle studied by 15N NMR relaxation
Backbone dynamics of Escherichia coli adenylate kinase at the extreme stages of the catalytic cycle studied by 15N NMR relaxation
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DOI：
10.1021/bi992076h
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发表时间：
2000-06-06
期刊：
影响因子：
2.9
通讯作者：
Meirovitch, E
中科院分区：
文献类型：
--
作者：
Shapiro, YE;Sinev, MA;Meirovitch, E
来自大肠杆菌的腺苷酸激酶（AKeco）由折叠成结构域CORE、AMPbd和LID的单个23.6kDa多肽链组成，催化AMP + ATP → 2ADP的反应。结构域LID和AMPbd在催化过程中执行大规模运动。采用N-15 NMR弛豫方法比较研究了无配体和AP（5）A抑制剂结合的AKeco的骨架动力学。测定了AKeco（AKeco*AP（5）A）的总体扩散，相关时间为15.05（11.42）ns，各向异性D-平行/D-perp = 1.25（1.10），快速内部运动的相关时间高达100 ps（50 ps）。快速的内部运动影响93%的AKeco位点，明显偏好于结构域AMPbd和LLD，以及47%的AKeco*AP（5）A位点，沿着链沿着具有有限的可变性。二级结构元素和环的均方广义序参数[S-2]受配体结合的影响差异，并在域特异性的方式。AMPbd内的纳秒运动占主导地位。突出的交换贡献，特别是与残基G10的核苷酸结合的P-环基序，被解释为反映氢键动力学在寡核苷酸结合位点。基于晶体学数据的底物结合的能量平衡的假设得到了溶液NMR结果的有力支持。主链动力学和域位移之间的相关性建立。
Adenylate kinase from Escherichia coli (AKeco), consisting of a single 23.6 kDa polypeptide chain folded into domains CORE, AMPbd, and LID, catalyzes the reaction AMP + ATP --> 2ADP. Domains LID and AMPbd execute large-scale movements during catalysis. Backbone dynamics of ligand-free and AP(5)A-inhibitor-bound AKeco were studied comparatively with N-15 NMR relaxation methods. Overall diffusion with correlation times of 15.05 (11.42) ns and anisotropy D-parallel/D-perp = 1.25 (1.10), and fast internal motions with correlation times up to 100 ps (50 ps), were determined for AKeco (AKeco*AP(5)A). Fast internal motions affect 93% of the AKeco sites, with pronounced preference for domains AMPbd and LLD, and 47% of the AKeco*AP(5)A sites, with limited variability along the chain. The mean squared generalized order parameters, [S-2], Of secondary structure elements and loops are affected by ligand binding differentially and in a domain-specific manner. Nanosecond motions predominate within AMPbd. Prominent exchange contributions, associated in particular with residue G10 of the nucleotide-binding P-loop motif, are interpreted to reflect hydrogen bond dynamics at the inhibitor-binding site. The hypothesis of energetic counter balancing of substrate binding based on crystallographic data is strongly supported by the solution NMR results. Correlations between backbone dynamics and domain displacement are established.