Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase
Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase
复制标题
DOI：
10.1016/j.chembiol.2019.03.008
复制
发表时间：
2019-07-18
影响因子：
8.6
通讯作者：
Yokoyama, Shigeyuki
中科院分区：
文献类型：
--
作者：
Yanagisawa, Tatsuo;Kuratani, Mitsuo;Yokoyama, Shigeyuki
吡咯赖氨酰-tRNA合成酶（PylRS）和tRNA（Pyl）已被广泛用于遗传密码扩增。携带Y306 A和Y384 F突变的马氏甲烷八叠球菌PylRS突变体（PylRS（Y306 A/Y384 F））通过UAG编码各种庞大的非天然赖氨酸衍生物。在这项研究中，我们研究了PylRS（Y306 A/Y384 F）如何识别许多氨基酸。在17个非天然赖氨酸衍生物中，N-α-（苄氧羰基）赖氨酸（ZLys）和10个邻位/Meta/对位取代的ZLys衍生物被有效地连接到tRNA（Pyl）上，并通过PylRS（Y306 A/Y384 F）整合到蛋白质中。我们确定了14个非天然赖氨酸衍生物结合的PylRS（Y306 A/Y384 F）催化片段的晶体结构。间位和对位取代的ZLys衍生物被紧密地容纳在生产模式中。相比之下，ZLys和未取代或邻位取代的ZLys衍生物显示出除了生产模式之外的另一种结合模式。PylRS（Y306 A/Y384 F）显示出ZLys的高氨酰化速率，表明双结合模式对氨酰化的影响最小。PylRS（Y306 A/Y384 F）的这些精确的底物识别机制可以促进新的非天然氨基酸的基于结构的设计。
Pyrrolysyl-tRNA synthetase (PylRS) and tRNA(Pyl) have been extensively used for genetic-code expansion. A Methanosarcina mazei PylRS mutant bearing the Y306A and Y384F mutations (PylRS(Y306A/Y384F)) encodes various bulky non-natural lysine derivatives by UAG. In this study, we examined how PylRS(Y306A/Y384F) recognizes many amino acids. Among 17 non-natural lysine derivatives, N-epsilon-(benzyl-oxycarbonyl)lysine (ZLys) and 10 ortho/meta/parasubstituted ZLys derivatives were efficiently ligated to tRNA(Pyl) and were incorporated into proteins by PylRS(Y306A/Y384F). We determined crystal structures of 14 non-natural lysine derivatives bound to the PylRS(Y306A/Y384F) catalytic fragment. The meta-and para-substituted ZLys derivatives are snugly accommodated in the productive mode. In contrast, ZLys and the unsubstituted or ortho-substituted ZLys derivatives exhibited an alternative binding mode in addition to the productive mode. PylRS(Y306A/Y384F) displayed a high aminoacylation rate for ZLys, indicating that the double-binding mode minimally affects aminoacylation. These precise substrate recognition mechanisms by PylRS (Y306A/Y384F) may facilitate the structure-based design of novel non-natural amino acids.