人酰基蛋白硫酯酶(APT)催化附着在质膜上的S-酰化蛋白的脱棕榈酰化,促进膜锚固和分离的可逆循环。我们之前证明了细菌APT同源物,FTT258来自革兰氏阴性病原体土拉弗朗西斯菌,基于柔性回路重叠其活动部位的结构动力学,在闭合状态和开放状态之间处于平衡状态。尽管该回路的结构动力学在人类APT中并不保守,这个环的氨基酸序列是高度保守的,表明人类APT中这一循环的基本但不同的功能。在这里,我们研究了该回路在调节催化活性中的作用,配体结合,和人类APT1的蛋白质折叠,一种与癌症有关的脱棕榈酶,免疫,和神经信号。使用替代分析与动力学的结合,结构,和生物物理表征,我们表明,即使在人类APT1中具有不同的结构位置,该环仍然通过对配体结合和底物定位的贡献来调节APT1的催化活性。我们证实了先前已知的多个残基(Phe72和Ile74)在底物结合和催化中的作用,同时在底物选择性(Pro69)中增加了新的作用,在催化稳定(Asp73和Ile75),以及在膜结合β舌和底物结合环(Trp71)之间的过渡。即使这种色氨酸(Trp71)支点的保守取代也会导致催化活性的完全丧失,总蛋白质稳定性降低13°C,配体亲和力急剧下降,表示大小的组合,形状,Trp71的芳香性对APT1的适当结构至关重要。将掩埋的疏水表面区域与对暴露的次级表面口袋的贡献混合在一起,Trp71代表α/β水解酶结构中以前未鉴定的一类必需色氨酸和人APT中潜在的变构结合位点。
Human acyl protein thioesterases (APTs) catalyze the depalmitoylation of S-acylated proteins attached to the plasma membrane, facilitating reversible cycles of membrane anchoring and detachment. We previously showed that a bacterial APT homologue, FTT258 from the gram-negative pathogen Francisella tularensis, exists in equilibrium between a closed and open state based on the structural dynamics of a flexible loop overlapping its active site. Although the structural dynamics of this loop are not conserved in human APTs, the amino acid sequence of this loop is highly conserved, indicating essential but divergent functions for this loop in human APTs. Herein, we investigated the role of this loop in regulating the catalytic activity, ligand binding, and protein folding of human APT1, a depalmitoylase connected with cancer, immune, and neurological signaling. Using a combination of substitutional analysis with kinetic, structural, and biophysical characterization, we show that even in its divergent structural location in human APT1 that this loop still regulates the catalytic activity of APT1 through contributions to ligand binding and substrate positioning. We confirmed previously known roles for multiple residues (Phe72 and Ile74) in substrate binding and catalysis while adding new roles in substrate selectivity (Pro69), in catalytic stabilization (Asp73 and Ile75), and in transitioning between the membrane binding β-tongue and substrate-binding loops (Trp71). Even conservative substitution of this tryptophan (Trp71) fulcrum led to complete loss of catalytic activity, a 13°C decrease in total protein stability, and drastic drops in ligand affinity, indicating that the combination of the size, shape, and aromaticity of Trp71 are essential to the proper structure of APT1. Mixing buried hydrophobic surface area with contributions to an exposed secondary surface pocket, Trp71 represents a previously unidentified class of essential tryptophans within α/β hydrolase structure and a potential allosteric binding site within human APTs.