Characterization of caulonodin lasso peptides revealed unprecedented N-terminal residues and a precursor motif essential for peptide maturation
Characterization of caulonodin lasso peptides revealed unprecedented N-terminal residues and a precursor motif essential for peptide maturation
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DOI：
10.1039/c4sc01428f
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发表时间：
2014-01-01
期刊：
影响因子：
8.4
通讯作者：
Marahiel, Mohamed A.
中科院分区：
文献类型：
--
作者：
Zimmermann, Marcel;Hegemann, Julian D.;Marahiel, Mohamed A.
Lasso 肽是核糖体组装和翻译后修饰肽 (RiPP) 的特殊家族，具有令人着迷的 3D 结构，可以赋予抗化学和热变性的刚性和稳定性。它们独特的“套索结”结构决定了它们的抗菌、酶抑制和受体拮抗活性。虽然生物合成机制最近得到了表征，但有关基于肽序列形成这种独特套索结构的规则仍然难以捉摸。最近，通过鉴定该 RiPP 家族的新成员，对肽的长度、环的大小或与套索折叠稳定相关的氨基酸的性质等限制进行了彻底修改。在这项工作中，我们展示了四种基因组挖掘预测的套索肽的分离，其特征在于核心肽的位置 1 具有前所未有的氨基酸丝氨酸或丙氨酸。通过突变方法，我们能够预测四种肽（caulonodins IV 至 VII）的套索折叠。通过 NMR 全面阐明 caulonodin V 的 3D 结构，证实了这一预测；通过长程 NOE 接触的测定，证实了 caulonodin VI 的预测。此外，还探讨了生物合成机制对非典型位置 1 的底物特异性。此外，利用最近生长的功能性套索肽前体序列，我们能够通过生物信息学分析识别前导肽 C 末端部分的保守基序。对这个保守区域中生物合成机制的取代耐受性进行了广泛的体内分析，证实了几个残基的重要性，表明预测的基序很可能是套索肽成熟特异的通用前导肽识别序列。
Lasso peptides, a peculiar family of ribosomally assembled and post-translationally modified peptides (RiPPs), possess a fascinating 3D structure, which can confer rigidity and stability against chemical and thermal denaturation. Their distinctive "lariat knot" structure is accountable for their antibacterial, enzyme inhibitory and receptor antagonist activities. While the biosynthetic machinery was recently characterized, the rules concerning the formation of this unique lasso structure on the basis of their peptide sequences remain elusive. Restrictions such as the length of the peptide, the size of the ring, or the nature of the amino acids associated with the lasso fold stabilization were recently overhauled by the identification of new members of this RiPP family. In this work we demonstrate the isolation of four genome-mining-predicted lasso peptides featuring the unprecedented amino acids serine or alanine at position 1 of the core peptide. By a mutational approach we were able to predict the lasso fold for four peptides (caulonodins IV to VII). This prediction was confirmed for caulonodin V by the full elucidation of its 3D-structure via NMR and for caulonodin VI by the determination of long range NOE-contacts. Furthermore, the substrate specificity of the biosynthetic machinery for the atypical position 1 was probed. Additionally, utilizing the recent growth of functional lasso peptide precursor sequences we were able to identify a conserved motif in the C-terminal part of the leader peptide through bioinformatics analysis. Employing an extensive in vivo analysis for substitution tolerance of the biosynthetic machinery in this conserved region confirmed the significance of several residues, indicating that the predicted motif is very likely a general leader peptide recognition sequence specific for lasso peptide maturation.