The active N-terminal region of p67phox -: Structure at 1.8 Å resolution and biochemical characterizations of the A128V mutant implicated in chronic granulomatous disease
The active N-terminal region of p67phox -: Structure at 1.8 Å resolution and biochemical characterizations of the A128V mutant implicated in chronic granulomatous disease
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DOI：
10.1074/jbc.m100893200
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发表时间：
2001-06-15
影响因子：
4.8
通讯作者：
Pebay-Peyroula, E
中科院分区：
文献类型：
--
作者：
Grizot, S;Fieschi, F;Pebay-Peyroula, E
在激活时，来自嗜中性粒细胞的NADPH氧化酶响应于微生物感染而产生超氧阴离子。这种酶复合物通过其胞质因子p67（phox）、p47（phox）和小G蛋白Rac与膜相关黄细胞色素B的结合而被激活（558）。在这里，我们报告的晶体结构的活性N-末端片段的p67（phox）在1.8埃分辨率，以及功能的p67（phox）突变体的研究。该N-末端区域（残基1-213）主要由四个TPR（tetratricopeptide repeat）基序组成，其中C末端折叠回到由TPR结构域形成的疏水沟中。结构是非常相似的非活性截短形式的p67（phox）绑定到小C：蛋白Rac以前报道，但不同的是存在一个短的C-末端螺旋（残基187-193），可能是激活域的一部分。所有p67（phox）突变体负责慢性肉芽肿病（CGD），NADPH氧化酶功能的严重缺陷，定位在N-末端区域。我们研究了两个CGD突变，G78 E和A128 V。令人惊讶的是，A128 V CGD突变体能够在25 ℃体外完全激活NADPH氧化酶。然而，该点突变代表了p67（phox）中的温度敏感性缺陷，这解释了其在生理温度下的表型。
Upon activation, the NADPH oxidase from neutrophils produces superoxide anions in response to microbial infection. This enzymatic complex is activated by association of its cytosolic factors p67(phox), p47(phox), and the small G protein Rac with a membrane-associated flavocytochrome b(558). Here we report the crystal structure of the active N-terminal fragment of p67(phox) at 1.8 Angstrom resolution, as well as functional studies of p67(phox) mutants. This N-terminal region (residues 1-213) consists mainly of four TPR (tetratricopeptide repeat) motifs in which the C terminus folds back into a hydrophobic groove formed by the TPR domain. The structure is very similar to that of the inactive truncated form of p67(phox) bound to the small C: protein Rac previously reported, but differs by the presence of a short C-terminal helix (residues 187-193) that might be part of the activation domain. All p67(phox) mutants responsible for Chronic Granulomatous Disease (CGD), a severe defect of NADPH oxidase function, are localized in the N-terminal region. We investigated two CGD mutations, G78E and A128V, Surprisingly, the A128V CGD mutant is able to fully activate the NADPH oxidase in vitro at 25 degreesC, However, this point mutation represents a temperature-sensitive defect in p67(phox) that explains its phenotype at physiological temperature.