Ireland David C, Colgrave Michelle L, Nguyencong Philip, Daly Norelle L, Craik David J
Institute for Molecular Bioscience and Australian Research Council Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Qld 4072, Australia.
J Mol Biol. 2006 Apr 14;357(5):1522-35. doi: 10.1016/j.jmb.2006.01.051. Epub 2006 Feb 2.
Cyclotides are mini-proteins of 28-37 amino acid residues that have the unusual feature of a head-to-tail cyclic backbone surrounding a cystine knot. This molecular architecture gives the cyclotides heightened resistance to thermal, chemical and enzymatic degradation and has prompted investigations into their use as scaffolds in peptide therapeutics. There are now more than 80 reported cyclotide sequences from plants in the families Rubiaceae, Violaceae and Cucurbitaceae, with a wide variety of biological activities observed. However, potentially limiting the development of cyclotide-based therapeutics is a lack of understanding of the mechanism by which these peptides are cyclized in vivo. Until now, no linear versions of cyclotides have been reported, limiting our understanding of the cyclization mechanism. This study reports the discovery of a naturally occurring linear cyclotide, violacin A, from the plant Viola odorata and discusses the implications for in vivo cyclization of peptides. The elucidation of the cDNA clone of violacin A revealed a point mutation that introduces a stop codon, which inhibits the translation of a key Asn residue that is thought to be required for cyclization. The three-dimensional solution structure of violacin A was determined and found to adopt the cystine knot fold of native cyclotides. Enzymatic stability assays on violacin A indicate that despite an increase in the flexibility of the structure relative to cyclic counterparts, the cystine knot preserves the overall stability of the molecule.
环肽是由28至37个氨基酸残基组成的微型蛋白质，其具有独特的头尾相连的环状骨架，围绕着一个胱氨酸结。这种分子结构使环肽对热、化学和酶促降解具有更高的抗性，并促使人们研究其作为肽类治疗药物支架的用途。目前已报道了来自茜草科、堇菜科和葫芦科植物的80多种环肽序列，并观察到它们具有广泛的生物活性。然而，对这些肽在体内环化机制的缺乏了解可能会限制基于环肽的治疗药物的开发。到目前为止，尚未报道环肽的线性版本，这限制了我们对环化机制的理解。本研究报道了从植物香堇中发现的一种天然存在的线性环肽——紫堇素A，并讨论了其对肽体内环化的影响。紫堇素A的cDNA克隆的阐明揭示了一个引入终止密码子的点突变，该突变抑制了一个关键天冬酰胺残基的翻译，而该残基被认为是环化所必需的。测定了紫堇素A的三维溶液结构，发现其采用了天然环肽的胱氨酸结折叠。对紫堇素A的酶稳定性分析表明，尽管相对于环状对应物，该结构的灵活性有所增加，但胱氨酸结保留了分子的整体稳定性。
