Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2).
Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2).
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DOI：
10.1021/acschembio.6b00720
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发表时间：
2016-12-16
影响因子：
4
通讯作者：
Martin BR
中科院分区：
文献类型：
--
作者：
Won SJ;Davda D;Labby KJ;Hwang SY;Pricer R;Majmudar JD;Armacost KA;Rodriguez LA;Rodriguez CL;Chong FS;Torossian KA;Palakurthi J;Hur ES;Meagher JL;Brooks CL 3rd;Stuckey JA;Martin BR
翻译后 S-棕榈酰化指导数百种细胞蛋白的运输和膜定位，通常涉及协调的棕榈酰化循环，需要蛋白酰基转移酶 (PAT) 和酰基蛋白硫酯酶 (APT) 主动将 S-棕榈酰化蛋白重新分配到不同的细胞膜区室。这一过程对于 S-棕榈酰化 Ras 亚型以及许多潜在的外周膜蛋白的运输和致癌信号传导是必需的。去棕榈酰化酶 APT1 和 APT2 在所有脊椎动物中分别保守，表明每种酶具有独特的功能作用。最近发现的 APT 同工型选择性抑制剂 ML348 和 ML349 为探索每种酶的功能开辟了新的可能性，但仍不清楚每种抑制剂如何实现正交抑制。在此，我们报告了人 APT2 与 ML349 (1.64 Å) 复合物的高分辨率结构，以及人 APT1 与 ML348 (1.55 Å) 结合的互补结构。尽管整体肽主链结构几乎相同，但每种抑制剂在每个活性位点内采用不同的构象。在APT1中，ML348位于催化三联体上方，但在APT2中，ML349的磺酰基与活性位点驻留水形成氢键，间接接合催化三联体和氧阴离子空穴。相互诱变和活性分析揭示了活性位点周围的几个不同残基，这些残基作为亚型可及性和动力学的关键守门人。结构和生化分析表明，抑制剂占据了假定的酰基结合区域，建立了异构体特异性抑制、酰基底物水解以及对未来探针开发重要的结构正交性的机制。
Post-translational S-palmitoylation directs the trafficking and membrane localization of hundreds of cellular proteins, often involving a coordinated palmitoylation cycle that requires both protein acyl transferases (PATs) and acyl protein thioesterases (APTs) to actively re-distribute S-palmitoylated proteins towards different cellular membrane compartments. This process is necessary for the trafficking and oncogenic signaling of S-palmitoylated Ras isoforms, and potentially many peripheral membrane proteins. The de-palmitoylating enzymes APT1 and APT2 are separately conserved in all vertebrates, suggesting unique functional roles for each enzyme. The recent discovery of the APT isoform-selective inhibitors ML348 and ML349 has opened new possibilities to probe the function of each enzyme, yet it remains unclear how each inhibitor achieves orthogonal inhibition. Herein we report the high-resolution structure of human APT2 in complex with ML349 (1.64 Å), as well as the complementary structure of human APT1 bound to ML348 (1.55 Å). Although the overall peptide backbone structures are nearly identical, each inhibitor adopts a distinct conformation within each active site. In APT1, ML348 is positioned above the catalytic triad, but in APT2, the sulfonyl group of ML349 forms hydrogen bonds with active site resident waters to indirectly engage the catalytic triad and oxyanion hole. Reciprocal mutagenesis and activity profiling revealed several differing residues surrounding the active site that serve as critical gatekeepers for isoform accessibility and dynamics. Structural and biochemical analysis suggests the inhibitors occupy a putative acyl-binding region, establishing the mechanism for isoform-specific inhibition, hydrolysis of acyl substrates, and structural orthogonality important for future probe development.