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首页> 外文期刊>Biochemistry >The Antibacterial Peptide Pyrrohocoricin Inhibits the ATPase Actions of DnaK and Prevents Chaperone-Assisted Protein Folding
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The Antibacterial Peptide Pyrrohocoricin Inhibits the ATPase Actions of DnaK and Prevents Chaperone-Assisted Protein Folding

机译：抗菌肽焦红霉素抑制DnaK的ATPase活性并防止伴侣蛋白折叠

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Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin, drosocin, and apidaecin interact with the bacterial heat shock protein DnaK, and peptide binding to DnaK can be correlated with antimicrobial activity. In the current report we studied the mechanism of action of these peptides and their binding sites to Escherichia coli DnaK. Biologically active pyrrhocoricin made of L-amino acids diminished the ATPase activity of recombinant DnaK. The inactive D-pyrrhocoricin analogue and the membrane-active antibacterial peptide cecropin A or magainin 2 failed to inhibit the DnaK-mediated phosphate release from adenosine 5'-triphosphate (ATP). The effect of pyrrhocoricin on DnaK's other significant biological function, the refolding of misfolded proteins, was studied by assaying the alkaline phosphatase and f3-galactosidase activity of live bacteria. Remarkably, both enzyme activities were reduced upon incubation with L-pyrrhocoricin or drosocin. D-Pyrrhocoricin, magainin 2, or buforin II, an antimicrobial peptide involved in binding to bacterial nucleic acids, had only negligible effect. According to fluorescence polarization and dot blot analysis of synthetic DnaK fragments and labeled pyrrhocoricin analogues, pyrrhocoricin bound with a Kd of 50.8 .uM to the hinge region around the C-terminal helices D and E, at the vicinity of amino acids 583 and 615. Pyrrhocoricin binding was not observed to the homologous DnaK fragment of Staphylococcus aureus, a pyrrhocoricin nonresponsive strain. In line with the lack of A TPase inhibition, drosocin binding appears to be slightly shifted toward the D helix. Our data suggest that drosocin and pyrrhocoricin binding prevents the irequent opening and closing of the multihelicallid over the peptide-binding pocket of DnaK, permanently closes the cavity, and inhibits chaperone-assisted protein folding. The biochemical results were strongly supported by molecular modeling of DnaK -pyrrhocoricin interactions. Due to the prominent sequence variations of procaryotic and eucaryotic DnaK molecules in the multihelicallid region, our findings pave the road for the design of strain-specific antibacterial peptides and peptidomimetics. Far-fetched applications of the species-specific inhibition of chaperone-assisted protein folding include the control of not only bacteria but also fungi, parasites, insects, and perhaps rodents.
机译：最近，我们记录了短的，富含脯氨酸的抗菌肽吡咯菌素，drosocin和Apidaecin与细菌热激蛋白DnaK相互作用，并且与DnaK结合的肽可以与抗菌活性相关。在本报告中，我们研究了这些肽的作用机理及其与大肠杆菌DnaK的结合位点。由L-氨基酸制成的具有生物活性的吡咯菌素降低了重组DnaK的ATPase活性。失活的D-吡咯菌霉素类似物和具有膜活性的抗菌肽cecropin A或magainin 2不能抑制DnaK介导的从5'-三磷酸腺苷（ATP）释放的磷酸盐。通过测定活细菌的碱性磷酸酶和f3-半乳糖苷酶活性，研究了吡咯菌素对DnaK其他重要生物学功能（错误折叠的蛋白质的重新折叠）的影响。值得注意的是，与L-吡咯菌霉素或drosocin一起孵育时，两种酶的活性均降低。 D-Pyrrhocoricin，magainin 2或buforin II，一种与细菌核酸结合的抗菌肽，作用微不足道。根据合成的DnaK片段和标记的焦红霉素类似物的荧光偏振和斑点印迹分析，焦红霉素以50.8 uM的Kd结合到C末端螺旋D和E周围的铰链区，位于氨基酸583和615附近。没有观察到吡咯菌霉素与金黄色葡萄球菌（一种吡咯菌素无反应菌株）的同源DnaK片段结合。与缺乏A TPase抑制作用相一致，drosocin的结合似乎向D螺旋略微偏移。我们的数据表明，drosocin和吡咯菌素的结合可防止DnaK肽结合袋上的多螺旋盖的不必要的打开和关闭，永久关闭腔，并抑制伴侣蛋白的折叠。 DnaK-焦红霉素相互作用的分子模型为生化结果提供了有力的支持。由于原核和真核DnaK分子在多螺旋区的突出序列变异，我们的发现为菌株特异性抗菌肽和拟肽设计铺平了道路。伴侣特异性蛋白折叠的物种特异性抑制的牵强应用，不仅包括细菌的控制，而且还包括真菌，寄生虫，昆虫以及啮齿类动物的控制。

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