Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47phox in recognition of a polyproline type II helix and an adjacent alpha-helix of p22phox.
Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47phox in recognition of a polyproline type II helix and an adjacent alpha-helix of p22phox.
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DOI：
10.1042/bj20051899
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发表时间：
2006-05
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通讯作者：
I. Nobuhisa;R. Takeya;K. Ogura;N. Ueno;D. Kohda;F. Inagaki;H. Sumimoto
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文献类型：
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作者：
I. Nobuhisa;R. Takeya;K. Ogura;N. Ueno;D. Kohda;F. Inagaki;H. Sumimoto
产生超氧化物的吞噬细胞NADPH氧化酶对宿主防御至关重要，其激活需要调节蛋白p47phox与p22phox（氧化酶催化核心黄细胞色素b558的一个亚基）的SH3 （Src同源3）结构域介导的相互作用。尽管先前的晶体结构分析表明，p47phox的串联SH3结构域夹心于p22phox的短PRR（富含脯氨酸的区域）（氨基酸151-160），含有脯氨酸II螺旋，但该模型是否确实在氧化酶的激活中起作用仍然未知。在本文中，我们证明了p47phox的两个SH3结构域之间的协同作用，正如模型所期望的那样，是氧化酶激活所必需的。p47phox SH3结构域之间的连接子的缺失不仅会导致p22phox的结合缺陷，还会导致支持超氧化物产生的活性丧失。本研究利用丙氨酸扫描诱变技术鉴定出p22phox PRR中的Pro152、Pro156和Arg158是与p47phox相互作用不可或缺的残基。Pro152和Pro156被n端SH3结构域识别，而Arg158与c端SH3结构域接触。对p22phox PRR中的三个残基中的任何一个进行氨基酸替换，都可以消除完整细胞中重组的氧化酶的超氧化物产生活性。因此，bis- sh3介导的p47phox与p22phox的相互作用可以激活吞噬细胞氧化酶。此外，我们提供的证据表明，p22phox（氨基酸161-164）PRR的c端区域采用a-螺旋构象，通过加强与p47phox SH3结构域的结合，参与了吞噬细胞氧化酶的充分激活。
Activation of the superoxide-producing phagocyte NADPH oxidase, crucial for host defence, requires an SH3 (Src homology 3)-domain-mediated interaction of the regulatory protein p47phox with p22phox, a subunit of the oxidase catalytic core flavocytochrome b558. Although previous analysis of a crystal structure has demonstrated that the tandem SH3 domains of p47phox sandwich a short PRR (proline-rich region) of p22phox (amino acids 151-160), containing a polyproline II helix, it has remained unknown whether this model is indeed functional in activation of the oxidase. In the present paper we show that the co-operativity between the two SH3 domains of p47phox, as expected from the model, is required for oxidase activation. Deletion of the linker between the p47phox SH3 domains results not only in a defective binding to p22phox but also in a loss of the activity to support superoxide production. The present analysis using alanine-scanning mutagenesis identifies Pro152, Pro156 and Arg158 in the p22phox PRR as residues indispensable for the interaction with p47phox. Pro152 and Pro156 are recognized by the N-terminal SH3 domain, whereas Arg158 contacts with the C-terminal SH3 domain. Amino acid substitution for any of the three residues in the p22phox PRR abrogates the superoxide-producing activity of the oxidase reconstituted in intact cells. The bis-SH3-mediated interaction of p47phox with p22phox thus functions to activate the phagocyte oxidase. Furthermore, we provide evidence that a region C-terminal to the PRR of p22phox (amino acids 161-164), adopting an a-helical conformation, participates in full activation of the phagocyte oxidase by fortifying the association with the p47phox SH3 domains.