A Single Aspartate Coordinates Two Catalytic Steps in Hedgehog Autoprocessing.-猫眼课题宝

单个天冬氨酸协调刺猬自动加工中的两个催化步骤。

DOI：

10.1021/jacs.6b06928

发表时间：

2016

期刊：

Journal of the American Chemical Society

影响因子：

通讯作者：

中科院分区：

化学1区

文献类型：

作者：

Xie,Jian;Owen,Timothy;Xia,Ke;Callahan,Brian;Wang,Chunyu

Hedgehog（Hh）信号传导由胆固醇修饰的Hh配体驱动，该配体通过Hh前体蛋白的自动加工产生。Hh自动加工中的两个步骤，N-S酰基转移和酯交换，必须耦合以有效地Hh胆固醇化和下游信号转导。在本研究中，我们表明，一个保守的天冬氨酸残基，D46的Hh自动加工域，协调这两个催化步骤。突变表明，D46抑制非天然Hh前体的自动加工，是必不可少的酯交换与胆固醇。NMR测量表明，由于质子化的D46和催化半胱氨酸残基之间的氢键，D46的pKa为5.6，比天冬氨酸的预期pKa高出2.2个单位。然而，D46侧链的去质子化形式也是必不可少的，因为D46 N突变不能介导胆固醇化。在这些数据的基础上，我们提出，质子穿梭的D46侧链的机械耦合的两个步骤的Hh胆固醇化。

Hedgehog (Hh) signaling is driven by the cholesterol-modified Hh ligand, generated by autoprocessing of Hh precursor protein. Two steps in Hh autoprocessing, N–S acyl shift and transesterification, must be coupled for efficient Hh cholesteroylation and downstream signal transduction. In the present study, we show that a conserved aspartate residue, D46 of the Hh autoprocessing domain, coordinates these two catalytic steps. Mutagenesis demonstrated that D46 suppresses non-native Hh precursor autoprocessing and is indispensable for transesterification with cholesterol. NMR measurements indicated that D46 has a pKaof 5.6, ∼2 units above the expected pKaof aspartate, due to a hydrogen-bond between protonated D46 and a catalytic cysteine residue. However, the deprotonated form of D46 side chain is also essential, because a D46N mutation cannot mediate cholesteroylation. On the basis of these data, we propose that the proton shuttling of D46 side chain mechanistically couples the two steps of Hh cholesteroylation.