The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding
The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding
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DOI：
10.1021/bi002656a
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发表时间：
2001-03-13
期刊：
影响因子：
2.9
通讯作者：
Otvos, L
中科院分区：
文献类型：
--
作者：
Kragol, G;Lovas, S;Otvos, L
最近，我们发现短的富含脯氨酸的抗菌肽pyrrhocoricin、drosocin和apidaecin与细菌热休克蛋白DnaK相互作用，并且与DnaK结合的肽与抗菌活性相关。在本报告中，我们研究了这些肽的作用机制及其与大肠杆菌DnaK的结合位点。由l -氨基酸组成的具有生物活性的红火杆菌素降低了重组dna的atp酶活性。无活性的D-pyrrhocoricin类似物和膜活性抗菌肽cecropin A或magainin 2未能抑制dnak介导的5'-三磷酸腺苷（ATP）的磷酸盐释放。通过测定活菌的碱性磷酸酶和β -半乳糖苷酶活性，研究了pyrhocoricin对DnaK的其他重要生物学功能（错误折叠蛋白的再折叠）的影响。值得注意的是，这两种酶的活性在与l -火红霉素或红霉素孵育后都降低了。D-Pyrrhocoricin， magainin2或buforin II，一种与细菌核酸结合的抗菌肽，只有微不足道的作用。通过对合成的DnaK片段和标记的红火杆菌素类似物的荧光偏振和斑点斑点分析，红火杆菌素以50.8 muM的K-d结合在c端螺旋D和E周围的铰链区域，位于氨基酸583和615附近。在金黄色葡萄球菌的同源DnaK片段中未观察到与红火霉素结合，这是一种对红火霉素无反应的菌株。由于缺乏atp酶的抑制，促生蛋白的结合似乎稍微向D螺旋方向移动。我们的数据表明，促肽素和红心杆菌素的结合可以阻止DnaK肽结合口袋上的多螺旋盖的频繁打开和关闭，永久关闭空腔，并抑制伴侣辅助的蛋白质折叠。生物化学结果得到了dna -火红霉素相互作用的分子模型的有力支持。由于原核生物和真核生物dna分子在多螺旋盖区具有显著的序列差异，我们的研究结果为设计菌株特异性抗菌肽和拟肽物铺平了道路。对伴侣辅助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，还包括真菌、寄生虫、昆虫，也许还有啮齿动物的控制。
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 beta -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 K-d of 50.8 muM 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 ATPase inhibition, drosocin binding appears to be slightly shifted toward the D helix. Our data suggest that drosocin and pyrrhocoricin binding prevents the frequent opening and closing of the multihelical lid 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 multihelical lid 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.