Protein biotinylation visualized by a complex structure of biotin protein ligase with a substrate
Protein biotinylation visualized by a complex structure of biotin protein ligase with a substrate
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DOI：
10.1074/jbc.m709116200
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发表时间：
2008-05-23
影响因子：
4.8
通讯作者：
Kunishima, Naoki
中科院分区：
文献类型：
--
作者：
Bagautdinov, Bagautdin;Matsuura, Yoshinori;Kunishima, Naoki
生物素蛋白连接酶(BPL)只在特定的赖氨酸残基上催化生物素羧基载体蛋白(BCCP)的生物素化。在这里，我们报道了BPL.BCCP络合物晶体的第一个结构，它是用两个BPL突变体R48A和R48A/K111A制备的。从详细的结构特征来看，突变体很可能保留了酶的功能，但产生反应中间体生物素基-5‘-AMP的活性降低。突变结构中观察到的生物素和部分无序的ATP可以作为底物或生物素-5‘-AMP的非反应性类似物，从而提供复杂的晶体。得到的四个结晶学上独立的BPL.BCCP络合物在结构上可以分为三类：形成阶段1和阶段2，其中包括apo-BCCP和生物素化的全BCCP的产物阶段。负责络合物形成和生物素化反应的残基已经确定。BPL的C-末端结构域表现出特别大的构象变化以适应BCCP，这表明其功能重要性。形成阶段1的络合物显示生物素的羧基碳与BCCP的特殊赖氨酸之间的距离最近，表明它与未观察到的反应阶段有关。有趣的是，结合的ATP和生物素也出现在产物阶段，这表明底物可能在Holo-BCCP释放之前被招募到产物阶段复合体中，可能是在下一个反应周期中。反应前后的形成和产物阶段的存在有利于保证反应效率和生物素化反应的极端底物专一性。
Biotin protein ligase (BPL) catalyzes the biotinylation of the biotin carboxyl carrier protein (BCCP) only at a special lysine residue. Here we report the first structure of BPL.BCCP complex crystals, which are prepared using two BPL mutants: R48A and R48A/K111A. From a detailed structural characterization, it is likely that the mutants retain functionality as enzymes but have a reduced activity to produce the reaction intermediate biotinyl-5'-AMP. The observed biotin and partly disordered ATP in the mutant structures may act as a non-reactive analog of the substrates or biotinyl-5'-AMP, thereby providing the complex crystals. The four crystallographically independent BPL.BCCP complexes obtained can be classified structurally into three groups: the formation stages 1 and 2 with apo-BCCP and the product stage with biotinylated holo-BCCP. Residues responsible for the complex formation as well as for the biotinylation reaction have been identified. The C-terminal domain of BPL shows especially large conformational changes to accommodate BCCP, suggesting its functional importance. The formation stage 1 complex shows the closest distance between the carboxyl carbon of biotin and the special lysine of BCCP, suggesting its relevance to the unobserved reaction stage. Interestingly, bound ATP and biotin are also seen in the product stage, indicating that the substrates may be recruited into the product stage complex before the release of holo-BCCP, probably for the next reaction cycle. The existence of formation and product stages before and after the reaction stage would be favorable to ensure both the reaction efficiency and the extreme substrate specificity of the biotinylation reaction.