Cheung Wai Ling, Chen Maria Y, Maksimov Mikhail O, Link A James
Department of Chemical and Biological Engineering and Department of Molecular Biology, Princeton University , Princeton, New Jersey 08544, United States.
ACS Cent Sci. 2016 Oct 26;2(10):702-709. doi: 10.1021/acscentsci.6b00184. Epub 2016 Sep 29.
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 , which produces the antimicrobial lasso peptide lariatin. The B enzyme in is split between two open reading frames, and , 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 -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.
套索肽是核糖体合成和翻译后修饰肽（RiPPs）超类的成员。与所有RiPPs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切割，由B酶完成；以及ATP依赖性异肽键形成，由C酶完成。在革兰氏阳性生物的一部分套索肽生物合成基因簇中，B酶被分成两种蛋白质。在产生抗微生物套索肽拉里亚汀的生物体中发现了这样一个基因簇。该生物体中的B酶被分成两个开放阅读框LarB1和LarB2，两者都是拉里亚汀生物合成所必需的。虽然半胱氨酸催化三联体存在于LarB2蛋白中，但基于其与其他RiPP前导肽结合域的结构同源性，LarB1是一种预期与拉里亚汀前体LarA结合的PqqD同源物。我们表明，LarB1以亚微摩尔亲和力与拉里亚汀前体蛋白LarA的前导肽结合。我们使用非天然氨基酸叠氮苯丙氨酸进行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，出乎意料的是，LarA的核心肽也在几个位置与LarB1结合。根据光交联实验的距离限制构建的Rosetta模型表明，LarA中前导肽和核心肽之间的可裂解键处于溶剂暴露环中。
