Ligand-linked structural changes in the Escherichia coli biotin repressor: the significance of surface loops for binding and allostery.
Ligand-linked structural changes in the Escherichia coli biotin repressor: the significance of surface loops for binding and allostery.
复制标题
大肠杆菌生物素阻遏物中配体连接的结构变化：表面环对于结合和变构的重要性。
DOI：
10.1006/jmbi.1999.3086
复制
发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Beckett,D
中科院分区：
文献类型：
--
作者：
Streaker,ED;Beckett,D
大肠杆菌生物素生物合成阻遏物（BirA）是一种变构位点特异性DNA结合蛋白。BirA催化底物生物素和ATP合成生物素基-5 ′-AMP，腺苷酸在阻遏物与生物素操纵基因序列的结合中充当正变构效应物。虽然已经通过X射线晶体学技术确定了脱辅基阻遏物的三维结构，但还没有任何配体结合形式的阻遏物的结构。先前发表的溶液研究结果与伴随配体结合的蛋白质构象变化的发生一致。在这项工作中，羟基自由基足迹技术已被用来探测伴随配体结合的BirA的肽骨架的反应性的变化。这些研究的结果表明，生物素与蛋白质的结合导致保护活性位点附近的中心结构域区域和C-末端结构域免受化学切割。生物素相关的反应性变化构成与腺苷酸结合相关的变化的子集。bio-5′-AMP和生物素操纵基因DNA的结合抑制了蛋白质氨基和羧基末端结构域中其他位点的切割。在所有复合物中观察到BirA上的五个表面环从羟基自由基介导的裂解的不同程度的保护。这些结果牵连的C-末端结构域的BirA，其中没有功能以前是已知的，在小的配体和位点特异性的DNA结合和突出的表面环的意义，其中一些是无序的apoBirA结构，配体结合和蛋白质中的变构信息的传输。
The Escherichia coli repressor of biotin biosynthesis (BirA) is an allosteric site-specific DNA-binding protein. BirA catalyzes synthesis of biotinyl-5′-AMP from substrates biotin and ATP and the adenylate serves as the positive allosteric effector in binding of the repressor to the biotin operator sequence. Although a three-dimensional structure of the apo-repressor has been determined by X-ray crystallographic techniques, no structures of any ligand-bound forms of the repressor are yet available. Results of previously published solution studies are consistent with the occurrence of conformational changes in the protein concomitant with ligand binding. In this work the hydroxyl radical footprinting technique has been used to probe changes in reactivity of the peptide backbone of BirA that accompany ligand binding. Results of these studies indicate that binding of biotin to the protein results in protection of regions of the central domain in the vicinity of the active site and the C-terminal domain from chemical cleavage. Biotin-linked changes in reactivity constitute a subset of those linked to adenylate binding. Binding of both bio-5′-AMP and biotin operator DNA suppresses cleavage at additional sites in the amino and carboxy-terminal domains of the protein. Varying degrees of protection of the five surface loops on BirA from hydroxyl radical-mediated cleavage are observed in all complexes. These results implicate the C-terminal domain of BirA, for which no function has previously been known, in small ligand and site-specific DNA binding and highlight the significance of surface loops, some of which are disordered in the apoBirA structure, for ligand binding and transmission of allosteric information in the protein.