Molecular defects of the glycine 41 variants of alanine glyoxylate aminotransferase associated with primary hyperoxaluria type I
Molecular defects of the glycine 41 variants of alanine glyoxylate aminotransferase associated with primary hyperoxaluria type I
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DOI：
10.1073/pnas.0908565107
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发表时间：
2010-02-16
影响因子：
11.1
通讯作者：
Voltattorni, Carla Borri
中科院分区：
文献类型：
--
作者：
Cellini, Barbara;Montioli, Riccardo;Voltattorni, Carla Borri
G41是位于丙氨酸：乙醛酸氨基转移酶（AGT）的α-螺旋34-42内的界面残基。其在主要（AGT-Ma）或次要（AGT-Mi）等位基因上的突变产生引起1型高尿症的变体G41 R-Ma、G41 R-Mi和G41 V-Ma。这些变体的二聚化受损被认为是免疫反应性缺陷、过氧化物酶体内聚集和对蛋白酶体降解敏感的原因。然而，没有实验证据支持这一观点。在这里，我们报告G41突变，除了增加二聚体单体的平衡解离常数，影响蛋白质的构象和稳定性，并扰乱其活性位点。与AGT-Ma或AGT-Mi相比，G41变体显示出不同的近UV CD和固有发射荧光光谱、更大的疏水表面暴露、对蛋白酶K引起的Met 53-Tyr 54肽键切割的敏感性、降低的热稳定性、降低的辅酶结合亲和力和催化效率。此外，与AGT-Ma和AGT-Mi不同，G41变体在生理条件下通过分子间静电相互作用形成不溶性无活性高阶聚集体（类似于5，000 nm）。AGT-Mi和G41 R-Mi的推定结构的比较分子动力学研究预测，G41 -> R突变引起34-42 α-螺旋的部分解旋和包括活性位点环24-32的前44个N-末端残基的置换。这些模拟有助于我们设想与G41突变相关的AGT功能障碍的可能结构基础。详细了解G41突变如何作用于AGT的结构-功能可能有助于实现纠正这些突变影响的最终目标。
G41 is an interfacial residue located within the alpha-helix 34-42 of alanine: glyoxylate aminotransferase (AGT). Its mutations on the major (AGT-Ma) or the minor (AGT-Mi) allele give rise to the variants G41R-Ma, G41R-Mi, and G41V-Ma causing hyperoxaluria type 1. Impairment of dimerization in these variants has been suggested to be responsible for immunoreactivity deficiency, intraperoxisomal aggregation, and sensitivity to proteasomal degradation. However, no experimental evidence supports this view. Here we report that G41 mutations, besides increasing the dimer-monomer equilibrium dissociation constant, affect the protein conformation and stability, and perturb its active site. As compared to AGT-Ma or AGT-Mi, G41 variants display different near-UV CD and intrinsic emission fluorescence spectra, larger exposure of hydrophobic surfaces, sensitivity to Met53-Tyr54 peptide bond cleavage by proteinase K, decreased thermostability, reduced coenzyme binding affinity, and catalytic efficiency. Additionally, unlike AGT-Ma and AGT-Mi, G41 variants under physiological conditions form insoluble inactive high-order aggregates (similar to 5,000 nm) through intermolecular electrostatic interactions. A comparative molecular dynamics study of the putative structures of AGT-Mi and G41R-Mi predicts that G41 -> R mutation causes a partial unwinding of the 34-42 alpha-helix and a displacement of the first 44 N-terminal residues including the active site loop 24-32. These simulations help us to envisage the possible structural basis of AGT dysfunction associated with G41 mutations. The detailed insight into how G41 mutations act on the structure-function of AGT may contribute to achieve the ultimate goal of correcting the effects of these mutations.