Role of Individual Domains and Identification of Internal Gap in Human Guanylate Binding Protein-1
Role of Individual Domains and Identification of Internal Gap in Human Guanylate Binding Protein-1
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DOI：
10.1016/j.jmb.2008.12.060
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发表时间：
2009-02-27
影响因子：
5.6
通讯作者：
Sau, Apurba Kumar
中科院分区：
文献类型：
--
作者：
Abdullah, Nazish;Srinivasan, Bharani;Sau, Apurba Kumar
与其他GTP酶不同，干扰素γ诱导的人鸟苷酸结合蛋白-1具有将GTP水解为GDP和GMP的能力，其中GMP是该反应的主要产物。该蛋白有两个结构域，一个n端球形结构域和一个c端螺旋结构域。这两个结构域由一个短的中间区域连接，该区域由一个双链β -片和一个螺旋组成。由于人类鸟苷酸结合蛋白-1已被证明在没有外部GTPase激活蛋白的情况下经历刺激的GTPase活性，我们试图了解两个单独结构域，中间区域，保守基序(（103)DXEKGD(108)）的作用，以及刺激GTPase活性的机制。用放射性标记的[α - p -32]GTP对野生型蛋白进行的稳态分析表明，活性的刺激主要发生在GTP的第二磷酸的裂解过程中，而不是通过变弹性相互作用。使用几个截断和突变的蛋白，我们首次证明了中间区域的a-螺旋和（103）DXEKGD（108）基序在水解成GMP中发挥关键作用，但它们似乎以不同的方式起作用：a-螺旋通过变构相互作用的结构稳定起作用，因此，作为内部gtpase激活蛋白，而基序可能通过提供必要的催化残基起作用。我们的数据还表明，n端球形结构域只能进行第一次催化（GTP转化为GDP，活性与基础水平相关），但全长蛋白中的螺旋结构域通过阻止GDP结合酶二聚体的解离，刺激GTP水解为GMP，从而提高GMP的形成。(c) 2009 Elsevier Ltd.版权所有。
Unlike other GTPases, interferon-gamma-induced human guanylate binding protein-1 has the ability to hydrolyze GTP to both GDP and GMP, with GMP being the major product of the reaction. This protein has two domains, an N-terminal globular domain and a C-terminal helical domain. These two domains are connected by a short intermediate region consisting of a two-stranded beta-sheet and a helix. As human guanylate binding protein-1 has been shown to undergo stimulated GTPase activity without external GTPase-activating protein, we sought to understand the roles of each of the two individual domains, the intermediate region, a conserved motif ((103)DXEKGD(108)), and the mechanism of the stimulation of GTPase activity. The steady-state assays using radiolabeled [alpha-P-32]GTP on the wild-type protein suggest that the stimulation of activity primarily occurs during the cleavage of the second phosphate of GTP rather than the first, through allosteric interaction. Using several truncated and mutant proteins, we demonstrate for the first time that both the a-helix of the intermediate region and the (103)DXEKGD(108) motif play critical roles for the hydrolysis to GMP, but they appear to act in different ways: a-helix acts through structural stabilization by allosteric interaction and, thus, acts as an internal GTPase-activating protein, whereas the motif might act by providing necessary catalytic residues. Our data also show that the N-terminal globular domain is able to perform only the first catalysis (GTP to GDP, an activity associated with basal level), but the helical domain in the full-length protein stimulates the hydrolysis of GTP to GMP with higher GMP formation by preventing the dissociation of GDP-bound enzyme dimer. (c) 2009 Elsevier Ltd. All rights reserved.