Lasso Peptide Biosynthetic Protein LarB1 Binds Both Leader and Core Peptide Regions of the Precursor Protein LarA.
Lasso Peptide Biosynthetic Protein LarB1 Binds Both Leader and Core Peptide Regions of the Precursor Protein LarA.
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DOI：
10.1021/acscentsci.6b00184
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发表时间：
2016-10-26
影响因子：
18.2
通讯作者：
Link, A. James
中科院分区：
文献类型：
--
作者：
Cheung, Wai Ling;Chen, Maria Y.;Maksimov, Mikhail O.;Link, A. James
Lasso 肽是核糖体合成和翻译后修饰肽 (RiPP) 超类的成员。与所有 RiPP 一样，套索肽源自基因编码的前体蛋白。套索肽的生物合成需要两种酶活性：前体蛋白中前导肽和核心肽之间的蛋白水解裂解（由 B 酶完成）和 ATP 依赖性异肽键形成（由 C 酶完成）。在来自革兰氏阳性生物的套索肽生物合成基因簇的子集中，B 酶被分成两种蛋白质。在红球菌 jostii 生物体中发现了这样一个基因簇，它产生抗菌套索肽 lariatin。 R. jostii 中的 B 酶分为两个开放阅读框 larB1 和 larB2，这两个框都是 lariatin 生物合成所必需的。虽然半胱氨酸催化三联体存在于 LarB2 蛋白中，但 LarB1 是 PqqD 同源物，基于其与其他 RiPP 前导肽结合域的结构同源性，预计会与 lariatin 前体 LarA 结合。我们发现 LarB1 以亚微摩尔亲和力与 lariatin 前体蛋白 LarA 的前导肽结合。我们使用非经典氨基酸对叠氮基苯丙氨酸的光交联和质谱来绘制 LarA 和 LarB1 的相互作用。该分析表明 LarA 前导肽与 LarB1 内的保守基序相互作用，并且出乎意料的是，LarA 的核心肽也在多个位置与 LarB1 结合。根据光交联实验的距离限制建立的 Rosetta 模型表明，LarA 中前导肽和核心肽之间的易裂键位于暴露于溶剂的环中。光交联用于绘制套索肽前体蛋白 LarA 和生物合成蛋白 LarB1 之间的界面。 LarA 前导肽区和核心肽区均与 LarB1 结合。
Lasso peptides are a member of the superclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Like all RiPPs, lasso peptides are derived from a gene-encoded precursor protein. The biosynthesis of lasso peptides requires two enzymatic activities: proteolytic cleavage between the leader peptide and the core peptide in the precursor protein, accomplished by the B enzymes, and ATP-dependent isopeptide bond formation, accomplished by the C enzymes. In a subset of lasso peptide biosynthetic gene clusters from Gram-positive organisms, the B enzyme is split between two proteins. One such gene cluster is found in the organism Rhodococcus jostii, which produces the antimicrobial lasso peptide lariatin. The B enzyme in R. jostii is split between two open reading frames, larB1 and larB2, both of which are required for lariatin biosynthesis. While the cysteine catalytic triad is found within the LarB2 protein, LarB1 is a PqqD homologue expected to bind to the lariatin precursor LarA based on its structural homology to other RiPP leader peptide binding domains. We show that LarB1 binds to the leader peptide of the lariatin precursor protein LarA with a sub-micromolar affinity. We used photocrosslinking with the noncanonical amino acid p-azidophenylalanine and mass spectrometry to map the interaction of LarA and LarB1. This analysis shows that the LarA leader peptide interacts with a conserved motif within LarB1 and, unexpectedly, the core peptide of LarA also binds to LarB1 in several positions. A Rosetta model built from distance restraints from the photocrosslinking experiments shows that the scissile bond between the leader peptide and core peptide in LarA is in a solvent-exposed loop. Photocrosslinking was used to map the interface between the lasso peptide precursor protein LarA and biosynthetic protein LarB1. Both the LarA leader and core peptide regions are bound by LarB1.
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影响因子：
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Naismith JH
影响因子：
3.2
作者：
Pan, Si Jia;Rajniak, Jakub;Link, A. James
通讯作者：
Link, A. James