Goldberg Allison B, Cho Eunice, Miller Chad J, Lou Hua Jane, Turk Benjamin E
From the Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520.
From the Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520
J Biol Chem. 2017 Jan 20;292(3):814-825. doi: 10.1074/jbc.M116.761734. Epub 2016 Dec 1.
The metalloproteinase anthrax lethal factor (LF) is secreted by Bacillus anthracis to promote disease virulence through disruption of host signaling pathways. LF is a highly specific protease, exclusively cleaving mitogen-activated protein kinase kinases (MKKs) and rodent NLRP1B (NACHT leucine-rich repeat and pyrin domain-containing protein 1B). How LF achieves such restricted substrate specificity is not understood. Previous studies have suggested the existence of an exosite interaction between LF and MKKs that promotes cleavage efficiency and specificity. Through a combination of in silico prediction and site-directed mutagenesis, we have mapped an exosite to a non-catalytic region of LF. Mutations within this site selectively impair proteolysis of full-length MKKs yet have no impact on cleavage of short peptide substrates. Although this region appears important for cleaving all LF protein substrates, we found that mutation of specific residues within the exosite differentially affects MKK and NLRP1B cleavage in vitro and in cultured cells. One residue in particular, Trp-271, is essential for cleavage of MKK3, MKK4, and MKK6 but dispensable for targeting of MEK1, MEK2, and NLRP1B. Analysis of chimeric substrates suggests that this residue interacts with the MKK catalytic domain. We found that LF-W271A blocked ERK phosphorylation and growth in a melanoma cell line, suggesting that it may provide a highly selective inhibitor of MEK1/2 for use as a cancer therapeutic. These findings provide insight into how a bacterial toxin functions to specifically impair host signaling pathways and suggest a general strategy for mapping protease exosite interactions.
金属蛋白酶炭疽致死因子（LF）由炭疽芽孢杆菌分泌，通过破坏宿主信号通路来增强疾病毒力。LF是一种高度特异性的蛋白酶，专门切割丝裂原活化蛋白激酶激酶（MKKs）和啮齿动物NLRP1B（含NACHT富含亮氨酸重复序列和吡啉结构域的蛋白1B）。LF如何实现如此有限的底物特异性尚不清楚。先前的研究表明，LF与MKKs之间存在一种别构位点相互作用，可提高切割效率和特异性。通过计算机模拟预测和定点诱变相结合的方法，我们已将一个别构位点定位到LF的一个非催化区域。该位点内的突变选择性地损害全长MKKs的蛋白水解作用，但对短肽底物的切割没有影响。尽管该区域对于切割所有LF蛋白底物似乎都很重要，但我们发现别构位点内特定残基的突变在体外和培养细胞中对MKK和NLRP1B的切割有不同影响。特别是一个残基Trp-271，对于MKK3、MKK4和MKK6的切割至关重要，但对于MEK1、MEK2和NLRP1B的靶向作用是可有可无的。嵌合底物分析表明，该残基与MKK催化结构域相互作用。我们发现LF-W271A阻断了黑色素瘤细胞系中的ERK磷酸化和生长，这表明它可能是一种高度选择性的MEK1/2抑制剂，可用作癌症治疗药物。这些发现为细菌毒素如何特异性损害宿主信号通路提供了见解，并提出了一种绘制蛋白酶别构位点相互作用的通用策略。
