Vibralactone as a tool to study the activity and structure of the ClpP1P2 complex from Listeria monocytogenes.-猫眼课题宝

DOI：

10.1002/anie.201104391

发表时间：

2011-11

期刊：

Angewandte Chemie

影响因子：

通讯作者：

中科院分区：

文献类型：

作者：

E. Zeiler;Nathalie Braun;T. Böttcher;A. Kastenmüller;S. Weinkauf;S. Sieber

自然界提供了丰富的生物活性化合物来源，包括一组不同的亲电核心结构，这些结构准备与酶活性部位的相应亲核残基如丝氨酸和半胱氨酸反应。[1-3]这些残基通常与催化有关，因此对其专用底物表现出微调的反应性。[4]我们和其他人以前研究了单环β-内酯的专用靶标，结果证明它是多种疾病相关酶类的有效和选择性抑制剂。例如，共价抑制酪蛋白溶肽酶ClpP导致细菌毒力的显著减弱。高度保守的热休克蛋白，在许多病原体中具有额外的调节功能。[9-11]一些生物，如单核细胞增多性李斯特菌，在基因上编码两种功能和结构上没有特征的ClpP亚型(ClpP1和ClpP2)。到目前为止，所有的β-内酯都被报道只针对ClpP2而不是ClpP1，[2]这提出了一个问题，即单环内酯是否缺乏合适的反应性来与ClpP1活性部位的亲核性相互作用。在此，我们将天然产物衍生的β内酯的范围扩大到可能表现出增强的反应性轮廓的应变双环系统。天然产物奥穆拉里德、盐孢酰胺和维拉内酯(VL)代表了这种理想的支架，并被报道为有效的蛋白酶体或脂肪酶抑制剂。[12-14]我们利用名为“基于活性的蛋白质谱”的化学蛋白质组学策略[15-17]证明了维拉内酯(VL)与单环β-内酯相反，在单核细胞增生性李氏杆菌中既与ClpP1结合，又与ClpP2结合。此外，通过结合透射电子显微镜(TEM)和同源建模/结构预测，我们能够确定异源低聚复合体的四元结构(图1)。VL按照周和Snider[18](支持信息中的方案1)的描述合成，并在ABPP发现靶标的最后步骤中用炔柄进行修饰(图1)。[19]靶标分析始于将这种振动内酯探针(VLP)与威氏乳杆菌及其致病对应单核增多性乳杆菌的完整活细胞孵育开始。细胞裂解后，用罗丹明叠氮盐在点击化学(CC)[20-22]条件下处理蛋白质组，并通过荧光SDS-PAGE分析显示靶标(支持信息中的图1)。在威氏乳杆菌和单核细胞增多性乳杆菌(图2A)中，大约20μ蛋白质的两条强烈荧光带的强度相当，其VLP浓度为3.4kDa m(支持信息中的图1C)。与不同浓度的未修饰VL预孵育逐渐取消了这些条带的标记，表明天然产物具有类似的目标选择性(图2B)。质谱(MS)分析表明，下带对应于ClpP2，它以前曾被单环β-内酯标记过。[2]有趣的是，上带对应于ClpP1，这是任何其他β-内酯探针都不能定位的(图2A，支持信息表1)。虽然来自各种生物的ClpP2同源物在晶体结构中与十四聚体桶状组装显示出很高的序列同源性(单核细胞增多性乳杆菌和金黄色葡萄球菌之间的同源性为77%)，但ClpP1与ClpP2的同源性仅为41%(支持信息中的图2)。这就提出了这样一个问题：ClpP1是否表现出不同的折叠和功能，并与ClpP2在混合络合物中组装，正如之前对…的建议

Nature provides a rich source of bioactive compounds comprising a diverse set of electrophilic core structures that are poised to react with corresponding nucleophilic residues such as serine and cysteine in enzyme active sites.[1–3] These residues are usually relevant for catalysis and therefore display fine-tuned reactivity towards their dedicated substrates.[4] We and others previously investigated the dedicated targets of monocyclic β-lactones which turned out to be potent and selective inhibitors of diverse disease-associated enzyme classes.[2, 3, 5–8] Covalent inhibition of the caseinolytic peptidase ClpP, for instance, resulted in a dramatic attenuation of bacterial virulence.[3] ClpP is an important, highly conserved heat shock protein with additional regulatory functions in many pathogens.[9–11] Some organisms such as Listeria monocytogenes genetically encode for two functionally and structurally uncharacterized ClpP isoforms (ClpP1 and ClpP2). So far, all β-lactones were reported to target solely ClpP2 and not ClpP1,[2] raising the question whether monocyclic lactones lack suitable reactivity to interact with the ClpP1 active-site nucleophile.We herein expand the scope of natural-product-derived βlactones to strained bicyclic ring systems which may exhibit enhanced reactivity profiles. The natural products omuralide, salinosporamide, and vibralactone (VL) represent such desired scaffolds and have been reported to be potent proteasome or lipase inhibitors.[12–14] We utilized a chemical proteomic strategy termed “activity-based protein profiling (ABPP)”[15–17] to demonstrate that vibralactone (VL), contrary to monocyclic β-lactones, binds to both ClpP1 and ClpP2 in L. monocytogenes. Moreover, by combining transmission electron microscopy (TEM) and homology modeling/structure predictions, we were able to determine the quaternary structure of the hetero-oligomeric complex (Figure 1). VL was synthesized as described by Zhou and Snider [18](Scheme 1 in the Supporting Information) and modified with an alkyne handle in the final step for target discovery by ABPP (Figure 1).[19] Target analysis started by the incubation of this vibralactone probe (VLP) with intact living cells of L. welshimeri and its pathogenic counterpart L. monocytogenes. Upon cell lysis, the proteome was treated under click chemistry (CC)[20–22] conditions with rhodamine azide and the targets were visualized by fluorescent SDS-PAGE analysis (Figure 1 in the Supporting Information). Two strong fluorescent bands for approximately 20kDa proteins were present at comparable intensities in L. welshimeri as well as in L. monocytogenes (Figure 2A) down to a VLP concentration of 3.4 μm (Figure 1C in the Supporting Information). Pre-incubation with various concentrations of unmodified VL gradually abolished the labeling of these bands, demonstrating that the natural product exhibits comparable target selectivity (Figure2B). Mass spectrometric (MS) analysis revealed that the lower band corresponds to ClpP2 which has been labeled by monocyclic β-lactones before.[2] Interestingly, the upper band corresponds to ClpP1 which could not be addressed by any other β-lactone probe (Figure 2A, Table 1 in the Supporting Information). While ClpP2 orthologues from various organisms exhibit a high sequence homology (77% identity between L. monocytogenes and S. aureus) with a tetradecameric barrel-shaped assembly in crystal structures,[23–26] ClpP1 shares only 41% identity with ClpP2 (Figure2 in the Supporting Information). This raises the question whether ClpP1 exhibits a different fold and function and assembles with ClpP2 in mixed complexes, as previously suggested for …

Vibralactone as a tool to study the activity and structure of the ClpP1P2 complex from Listeria monocytogenes.

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