Identification of residues in the human guanylate-binding protein 1 critical for nucleotide binding and cooperative GTP hydrolysis
Identification of residues in the human guanylate-binding protein 1 critical for nucleotide binding and cooperative GTP hydrolysis
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DOI：
10.1016/j.jmb.2004.09.026
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发表时间：
2004-11-12
影响因子：
5.6
通讯作者：
Herrmann, C
中科院分区：
文献类型：
--
作者：
Praefcke, GJK;Kloep, S;Herrmann, C
鸟苷酸结合蛋白（GBP）是一组干扰素-γ诱导的GTP结合蛋白，属于动力蛋白相关蛋白家族。与该家族的其他成员一样，人鸟苷酸结合蛋白1（hGBP 1）显示出刺激蛋白质的GT3活性的核苷酸依赖性寡聚化。GBP的一个独特特征是它们能够将GTP水解为GDP和GMP。为了阐明这些发现之间的关系，我们基于hGBP 1的晶体结构设计了磷酸结合环（P环）以及蛋白质的开关I和开关II区域中的点突变体。分析这些突变蛋白与荧光染料标记的鸟嘌呤核苷酸的相互作用以及它们以协同方式水解GTP的能力。我们确定了氨基酸残基的突变，降低GTP酶活性的数量级，其中一部分是保守的GTP结合蛋白。此外，P环中的突变体的特征在于强烈损害核苷酸的结合。因此，与改变的GTP酶活性和给定的细胞核苷酸浓度一起，这导致hGBP 1突变体分别以无核苷酸（K51 A和S52 N）或GTP结合形式（R48 A）稳定地静止。使用尺寸排阻色谱和分析超离心。我们讨论了对蛋白质寡聚化的影响。总之，鉴定了hGBP 1的突变体，并对其进行了生化表征，从而将hGBP 1锁定在限定的状态，以研究其在未来细胞生物学研究中的功能作用。(C)2004爱思唯尔有限公司保留所有权利。
The guanylate-binding proteins (GBPs) form a group of interferon-gamma inducible GTP-binding proteins which belong to the family of dynamin-related proteins. Like other members of this family, human guanylate-binding protein 1 (hGBP1) shows nucleotide-dependent oligomerisation that stimulates the GTPase activity of the protein. A unique feature of the GBPs is their ability to hydrolyse GTP to GDP and GMP In order to elucidate the relationship between these findings, we designed point mutants in the phosphate-binding loop (P-loop) as well as in the switch I and switch II regions of the protein based on the crystal structure of hGBP1. These mutant proteins were analysed for their interaction with guanine nucleotides labeled with a fluorescence dye and for their ability to hydrolyse GTP in a cooperative manner. We identified mutations of amino acid residues that decrease GTPase activity by orders of magnitude a part of which are conserved in GTP-binding proteins. In addition, mutants in the P-loop were characterized that strongly impair binding of nucleotide. In consequence, together with altered GTPase activity and given cellular nucleotide concentrations this results in hGBP1 mutants prevailingly resting in the nucleotide-free (K51A and S52N) or the GTP bound form (R48A), respectively. Using size-exclusion chromatography and analytical ultracentrifugation. we addressed the impact on protein oligomerisation. In summary, mutants of hGBP1 were identified and biochemically characterized providing hGBP1 locked in defined states in order to investigate their functional role in future cell biology studies. (C) 2004 Elsevier Ltd. All rights reserved.