NMR solution structure of the tandem Src homology 3 domains of p47phox complexed with a p22phox-derived proline-rich peptide
NMR solution structure of the tandem Src homology 3 domains of p47phox complexed with a p22phox-derived proline-rich peptide
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DOI：
10.1074/jbc.m505193200
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发表时间：
2006-02-10
影响因子：
4.8
通讯作者：
Inagaki, F
中科院分区：
文献类型：
--
作者：
Ogura, K;Nobuhisa, I;Inagaki, F
吞噬细胞 NADPH 氧化酶通过产生活性氧，在宿主防御微生物感染方面发挥着至关重要的作用。它是一种多亚基酶，由膜结合的黄细胞色素 b(558) 以及胞质成分（包括 p47(phox)）组成，p47(phox) 对于复合物的组装至关重要。当吞噬细胞被激活时，由于 p47(phox) 的串联 Src 同源 3 (SH3) 结构域与黄素细胞色素 b558 亚基 p22(phox) 中富含脯氨酸的区域结合，NADPH 氧化酶的胞质成分易位至黄素细胞色素 b558。通过 NMR 滴定，我们首先鉴定了 p22(phox) 富含脯氨酸的区域，该区域对于与 p47(phox) 串联 SH3 结构域的结合至关重要。随后，我们使用核磁共振波谱确定了与富含脯氨酸的 p22(phox) 肽复合的 p47(phox) 串联 SH3 结构域的溶液结构。与报道的晶态交织二聚体相反，溶液结构是单体。 p22phox 肽的中心区域形成聚脯氨酸 II 型螺旋，夹在 N 端和 C 端 SH3 结构域之间，正如在晶体结构中观察到的那样，而肽的 C 端区域呈现短 α 螺旋构象，为 N 端 SH3 结构域提供了额外的结合位点。因此，p22(phox) 肽的 C 端 α 螺旋区域将 p47(phox) 串联 SH3 结构域的结合亲和力提高了 10 倍以上。
The phagocyte NADPH oxidase plays a crucial role in host defense against microbial infections by generating reactive oxygen species. It is a multisubunit enzyme composed of membrane-bound flavocytochrome b(558) as well as cytosolic components, including p47(phox), which is essential for assembly of the complex. When phagocytes are activated, the cytosolic components of the NADPH oxidase translocate to flavocytochrome b558 due to binding of the tandem Src homology 3 (SH3) domains of p47(phox) to a proline-rich region in p22(phox), a subunit of flavocytochrome b558. Using NMR titration, we first identified the proline-rich region of p22(phox) that is essential for binding to the tandem SH3 domains of p47(phox). We subsequently determined the solution structure of the p47(phox) tandem SH3 domains complexed with the proline-rich peptide of p22(phox) using NMR spectroscopy. In contrast to the intertwined dimer reported for the crystal state, the solution structure is a monomer. The central region of the p22phox peptide forms a polyproline type II helix that is sandwiched by the N- and C-terminal SH3 domains, as was observed in the crystal structure, whereas the C-terminal region of the peptide takes on a short alpha-helical conformation that provides an additional binding site with the N- terminal SH3 domain. Thus, the C-terminal alpha-helical region of the p22(phox) peptide increases the binding affinity for the tandem SH3 domains of p47(phox) more than 10-fold.