Structures of Pathogenic Fungal FKBP12s Reveal Possible Self-Catalysis Function.
Structures of Pathogenic Fungal FKBP12s Reveal Possible Self-Catalysis Function.
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DOI：
10.1128/mbio.00492-16
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发表时间：
2016-04-26
期刊：
影响因子：
6.4
通讯作者：
Schumacher MA
中科院分区：
文献类型：
--
作者：
Tonthat NK;Juvvadi PR;Zhang H;Lee SC;Venters R;Spicer L;Steinbach WJ;Heitman J;Schumacher MA
侵袭性真菌感染仍然难以治疗，需要新的靶向策略。 12-kDa FK506 结合蛋白 (FKBP12) 是一种普遍表达的肽基-脯氨酰异构酶，在真菌病原体之间具有相当大的同源性，因此是未来生成泛真菌策略的靶向工作的主要候选者。尽管对 FKBP 进行了数十年的研究，但其底物和作用机制仍不清楚。在这里，我们描述了来自致病真菌白色念珠菌、光滑念珠菌和烟曲霉的 FKBP12 的结构、生化和体内分析。引人注目的是，多个 apo A. fumigatus 和 C. albicans FKBP12 晶体结构揭示了对称的分子间相互作用，涉及活性位点环脯氨酸深度插入相邻亚基的活性位点袋中。在以前的 FKBP 结构中尚未观察到这种相互作用。这一发现表明，这可能是含有该中心脯氨酸的烟曲霉和白色念珠菌真菌蛋白所特有的自底物相互作用。用顺式和反式状态的脯氨酸获得的结构提供了更多支持自催化的数据。此外，半胱氨酸交联实验捕获了相互作用的二聚体，支持了它在溶液中形成的想法。最后，探索改变中央脯氨酸和相邻残基的突变影响的遗传学研究提供了证据，表明体内形成的任何二聚体状态（其中 FKBP12 浓度较低）都是短暂的。总而言之，这些发现表明真菌 FKBP12 具有独特的自身底物调节机制，为未来的药物靶向工作提供了对该蛋白质的进一步新颖的理解。 FKBP12 是一种顺反肽基脯氨酰异构酶，在细胞蛋白质稳态中发挥关键作用。 FKBP12 还结合免疫抑制药物 FK506 来抑制磷酸酶钙调神经磷酸酶 (CaN)。 CaN 是烟曲霉、白色念珠菌、光滑念珠菌和其他致命真菌病原体的毒力所必需的，这使得 FKBP12 和 CaN 成为潜在的广谱药物靶点。在这里，我们描述了真菌 FKBP12 的结构。多个 apo A. fumigatus 和 C. albicans FKBP12 结构揭示了这些蛋白质中明显保守的脯氨酸插入到相邻分子的活性位点中。这表明这些蛋白质可能作为它们自己的底物。半胱氨酸二硫化物捕获实验为这种自相互作用以及这些酶可能的分子间催化提供了支持。
Invasive fungal infections remain difficult to treat and require novel targeting strategies. The 12-kDa FK506-binding protein (FKBP12) is a ubiquitously expressed peptidyl-prolyl isomerase with considerable homology between fungal pathogens and is thus a prime candidate for future targeting efforts to generate a panfungal strategy. Despite decades of research on FKBPs, their substrates and mechanisms of action remain unclear. Here we describe structural, biochemical, and in vivo analyses of FKBP12s from the pathogenic fungi Candida albicans, Candida glabrata, and Aspergillus fumigatus. Strikingly, multiple apo A. fumigatus and C. albicans FKBP12 crystal structures revealed a symmetric, intermolecular interaction involving the deep insertion of an active-site loop proline into the active-site pocket of an adjacent subunit. Such interactions have not been observed in previous FKBP structures. This finding indicates the possibility that this is a self-substrate interaction unique to the A. fumigatus and C. albicans fungal proteins that contain this central proline. Structures obtained with the proline in the cis and trans states provide more data in support of self-catalysis. Moreover, cysteine cross-linking experiments captured the interacting dimer, supporting the idea that it forms in solution. Finally, genetic studies exploring the impact of mutations altering the central proline and an adjacent residue provide evidence that any dimeric state formed in vivo, where FKBP12 concentrations are low, is transient. Taken together, these findings suggest a unique mechanism of self-substrate regulation by fungal FKBP12s, lending further novel understanding of this protein for future drug-targeting efforts. FKBP12 is a cis-trans peptidyl-prolyl isomerase that plays key roles in cellular protein homeostasis. FKBP12s also bind the immunosuppressive drug FK506 to inhibit the phosphatase calcineurin (CaN). CaN is required for virulence of A. fumigatus, C. albicans, C. glabrata, and other deadly fungal pathogens, marking FKBP12 and CaN as potential broad-spectrum drug targets. Here we describe structures of fungal FKBP12s. Multiple apo A. fumigatus and C. albicans FKBP12 structures reveal the insertion of a proline, conspicuously conserved in these proteins, into the active sites of adjacent molecules. This suggests that these proteins might serve as their own substrates. Cysteine disulfide trapping experiments provide support for this self-interaction and hence possible intermolecular catalysis by these enzymes.