Biophysical characterization of full-length human phenylalanine hydroxylase provides a deeper understanding of its quaternary structure equilibrium
Biophysical characterization of full-length human phenylalanine hydroxylase provides a deeper understanding of its quaternary structure equilibrium
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DOI：
10.1074/jbc.ra119.008294
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发表时间：
2019-06-28
影响因子：
4.8
通讯作者：
Jaffe, Eileen K.
中科院分区：
文献类型：
--
作者：
Arturo, Emilia C.;Gupta, Kushol;Jaffe, Eileen K.
人苯丙氨酸羟化酶（hPAH，EC 1.14.16.1）功能障碍是苯丙酮尿症的主要原因，苯丙酮尿症是最常见的先天性氨基酸代谢缺陷。这种多聚体蛋白的动态结构域重排阻碍了对全长形式的结构研究几十年，直到现在。在这项研究中，hPAH的易处理的C29 S变体（C29 S）产生了四聚体静息态构象的3.06埃分辨率晶体结构。我们使用尺寸排阻色谱法与小角X-射线散射（SEC-SAXS）分析全长hPAH溶液结构的存在和不存在的苯丙氨酸，作为底物和变构激活剂。别构苯丙氨酸结合有利于激活的PAH四聚体构象的积累，这在溶液中是生物药理学上不同的。用酶动力学和固有荧光进行的蛋白质表征表明，C29 S变体和hPAH在对苯丙氨酸的响应方面是等同的，这进一步得到了它们在各种色谱树脂上的行为和分析超离心的支持。对SAXS数据与可用的结构数据的C29 S的静止状态和激活形式的建模创建和评估了几个新的模型之间的过渡建筑不同的构象的PAH和突出的独特的内部和亚基间的相互作用。三个最佳拟合的替代模型都将变构的Phe结合模块放置在比所有先前模型更远离四聚体中心8-10埃的位置。本文报道的hPAH变构激活的结构见解可能有助于为用新的治疗方法治疗苯丙酮尿症的持续努力提供信息。
Dysfunction of human phenylalanine hydroxylase (hPAH, EC 1.14.16.1) is the primary cause of phenylketonuria, the most common inborn error of amino acid metabolism. The dynamic domain rearrangements of this multimeric protein have thwarted structural study of the full-length form for decades, until now. In this study, a tractable C29S variant of hPAH (C29S) yielded a 3.06 angstrom resolution crystal structure of the tetrameric resting-state conformation. We used size-exclusion chromatography in line with small-angle X-ray scattering (SEC-SAXS) to analyze the full-length hPAH solution structure both in the presence and absence of Phe, which serves as both substrate and allosteric activators. Allosteric Phe binding favors accumulation of an activated PAH tetramer conformation, which is biophysically distinct in solution. Protein characterization with enzyme kinetics and intrinsic fluorescence revealed that the C29S variant and hPAH are otherwise equivalent in their response to Phe, further supported by their behavior on various chromatography resins and by analytical ultracentrifugation. Modeling of resting-state and activated forms of C29S against SAXS data with available structural data created and evaluated several new models for the transition between the architecturally distinct conformations of PAH and highlighted unique intra- and inter-subunit interactions. Three best-fitting alternative models all placed the allosteric Phe-binding module 8-10 angstrom farther from the tetramer center than do all previous models. The structural insights into allosteric activation of hPAH reported here may help inform ongoing efforts to treat phenylketonuria with novel therapeutic approaches.