Structural and mechanistic analysis of two prolyl endopeptidases: role of interdomain dynamics in catalysis and specificity.
Structural and mechanistic analysis of two prolyl endopeptidases: role of interdomain dynamics in catalysis and specificity.
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DOI：
10.1073/pnas.0408286102
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发表时间：
2005-03
影响因子：
11.1
通讯作者：
L. Shan;I. Mathews;C. Khosla
中科院分区：
文献类型：
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作者：
L. Shan;I. Mathews;C. Khosla
脯氨酰内肽酶（PEP）是一类独特的丝氨酸蛋白酶，具有相当大的治疗潜力，用于治疗乳糜泻。两个didomain PEP的晶体结构已被解决在替代配置，从而提供这些酶的作用模式的见解。的结构的鞘氨醇单胞菌capsulata PEP，解决和细化到1.8-A的分辨率，揭示了一个开放的配置的活性位点。与此相反，来自黄色粘球菌的结合有荧光素的PEP以封闭形式结晶（1.5-A分辨率）。这两种结构的比较分析突出了域界面在调节域间动力学和底物特异性中的关键作用。对M. xanthusPEP证实了几个界面残基的重要作用。Arg-572和Asp-196/Glu-197之间的盐桥似乎充当打开或关闭双结构域酶的闩锁，并且Arg-572和Ile-575也可以帮助将进入的肽底物固定到酶的开放形式。Arg-618和Asp-145负责将不变的脯氨酸残基锚定在该脯氨酸后裂解酶的活性位点中。一个模型提出了一个代表性的底物PQPQLPYPQPQLP在活性位点，其中N-末端底物残基广泛地与催化结构域相互作用，和C-末端残基伸展到螺旋桨结构域的对接。鉴于M.本研究的结果为进一步优化PEP的临床应用特性奠定了坚实的基础。
Prolyl endopeptidases (PEPs) are a unique class of serine proteases with considerable therapeutic potential for the treatment of celiac sprue. The crystal structures of two didomain PEPs have been solved in alternative configurations, thereby providing insights into the mode of action of these enzymes. The structure of the Sphingomonas capsulata PEP, solved and refined to 1.8-A resolution, revealed an open configuration of the active site. In contrast, the inhibitor-bound PEP from Myxococcus xanthus was crystallized (1.5-A resolution) in a closed form. Comparative analysis of the two structures highlights a critical role for the domain interface in regulating interdomain dynamics and substrate specificity. Structure-based mutagenesis of the M. xanthus PEP confirms an important role for several interfacial residues. A salt bridge between Arg-572 and Asp-196/Glu-197 appears to act as a latch for opening or closing the didomain enzyme, and Arg-572 and Ile-575 may also help secure the incoming peptide substrate to the open form of the enzyme. Arg-618 and Asp-145 are responsible for anchoring the invariant proline residue in the active site of this postproline-cleaving enzyme. A model is proposed for the docking of a representative substrate PQPQLPYPQPQLP in the active site, where the N-terminal substrate residues interact extensively with the catalytic domain, and the C-terminal residues stretch into the propeller domain. Given the promise of the M. xanthus PEP as an oral therapeutic enzyme for treating celiac sprue, our results provide a strong foundation for further optimization of the PEP's clinically useful features.