Structural, Dynamic, and Functional Characterization of a DnaX Mini-intein Derived from Spirulina platensis Provides Important Insights into Intein-Mediated Catalysis of Protein Splicing
Structural, Dynamic, and Functional Characterization of a DnaX Mini-intein Derived from Spirulina platensis Provides Important Insights into Intein-Mediated Catalysis of Protein Splicing
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DOI：
10.1021/acs.biochem.0c00828
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发表时间：
2020-12-22
期刊：
影响因子：
2.9
通讯作者：
De, Soumya
中科院分区：
文献类型：
--
作者：
Boral, Soumendu;Maiti, Snigdha;De, Soumya
蛋白质剪接是一种自催化的翻译后修饰，其中内含肽酶从前体蛋白质中切除自身并连接侧翼序列以产生成熟蛋白质。我们报告的解决方案结构的136个残基DnaX迷你内含肽酶来自蓝藻螺旋藻。该序列采用明确定义的球状结构，并形成HINT（刺猬内含肽）拓扑结构中常见的马蹄形折叠。骨架动力学和氢交换实验揭示了各种时间尺度上的保守运动，这被认为是内含肽折叠的一个特征。有趣的是，在蛋白质结构中的对称等价位置发现了几个动态运动，这可能是对称内含肽折叠的结果。在细胞内剪接活性表明Spl DnaX mini-intein是一种高活性的酶。在试验的任何时间点均未检出前体蛋白。除了剪接反应外，还观察到前体蛋白的N-和C-末端的催化裂解。为了确定催化残基在剪接和切割反应中的作用，产生这些残基的丙氨酸突变的所有组合并进行功能表征。这种深入的分析揭示了这些催化残基之间的协同性，这抑制了N-和C-末端裂解反应，并提高了剪接产物的产率。总的来说，这项研究提供了一个彻底的结构，动态和功能表征的一个新的内含肽序列，并增加了这些独特的酶，发现在生物化学和生物技术的巨大应用的集合。
Protein splicing is a self-catalyzed post-translational modification in which the intein enzyme excises itself from a precursor protein and ligates the flanking sequences to produce a mature protein. We report the solution structure of a 136-residue DnaX mini-intein enzyme derived from the cyanobacterium Spirulina platensis. This sequence adopts a well-defined globular structure and forms a horseshoe-shaped fold commonly found in the HINT (hedgehog intein) topology. Backbone dynamics and hydrogen exchange experiments revealed conserved motions on various time scales, which is proposed to be a characteristic of the intein fold. Interestingly, several dynamic motions were found in symmetrically equivalent positions within the protein structure, which might be a consequence of the symmetrical intein fold. In cell splicing activity showed that Spl DnaX mini-intein is a highly active enzyme. The precursor protein was not detected at any timepoint of the assay. Apart from the splicing reaction, catalytic cleavage at the N- and C-termini of the precursor protein was also observed. To determine the roles of the catalytic residues in splicing and cleavage reactions, all combinations of alanine mutations of these residues were generated and functionally characterized. This in-depth analysis revealed cooperativity between these catalytic residues, which suppresses the N- and C-terminal cleavage reactions and enhances the yield of the spliced product. Overall, this study provides a thorough structural, dynamic, and functional characterization of a new intein sequence and adds to the collection of these unique enzymes that have found tremendous applications in biochemistry and biotechnology.