Adams E R, Dratz E A, Gizachew D, Deleo F R, Yu L, Volpp B D, Vlases M, Jesaitis A J, Quinn M T
Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT59717, USA.
Biochem J. 1997 Jul 1;325 ( Pt 1)(Pt 1):249-57. doi: 10.1042/bj3250249.
During activation of the neutrophil NADPH oxidase, cytosolic p47(phox) is translocated to the membrane where it associates with flavocytochrome b via multiple binding regions, including a site in the C-terminus of gp91(phox). To investigate this binding site further, we studied the three-dimensional structure of a gp91(phox) C-terminal peptide (551SNSESGPRGVHFIFNKEN568) bound to p47(phox) using transferred nuclear Overhauser effect spectroscopy (Tr-NOESY) NMR. Using MARDIGRAS analysis and simulated annealing, five similar sets of structures of the p47(phox)-bound peptide were obtained, all containing an extended open bend from Ser5 to Phe14 (corresponding to gp91(phox) residues 555-564). The ends of the peptide were poorly defined, however, suggesting they were more flexible. Therefore further refinement was performed on the Ser5-Phe14 region of the peptide after omitting the ends of the peptide from consideration. In this case, two similar structures were obtained. Both structures again exhibited extended open-bend conformations. In addition, the amino acid side chains that showed evidence of immobilization on binding to p47(phox) correlated directly with those that were found previously to be essential for biological activity. Thus during NADPH oxidase assembly, the C-terminus of gp91(phox) binds to 47(phox) in an extended conformation between gp91(phox) residues 555 and 564, with immobilization of all of the amino acid side chains in the 558RGVHFIF564 region except for His561.
在中性粒细胞NADPH氧化酶激活过程中，胞质p47(phox)转位至膜上，通过多个结合区域与黄素细胞色素b结合，包括gp91(phox) C末端的一个位点。为了进一步研究该结合位点，我们使用转移核Overhauser效应光谱（Tr-NOESY）核磁共振技术研究了与p47(phox)结合的gp91(phox) C末端肽（551SNSESGPRGVHFIFNKEN568）的三维结构。使用MARDIGRAS分析和模拟退火，获得了五组相似的与p47(phox)结合的肽结构，所有结构均包含从Ser5到Phe14的延伸开放弯曲（对应于gp91(phox)残基555 - 564）。然而，肽的末端定义不明确，表明它们更具柔性。因此，在不考虑肽末端的情况下，对肽的Ser5 - Phe14区域进行了进一步优化。在这种情况下，获得了两个相似的结构。两个结构再次呈现延伸开放弯曲构象。此外，在与p47(phox)结合时显示出固定化迹象的氨基酸侧链与先前发现对生物活性至关重要的侧链直接相关。因此，在NADPH氧化酶组装过程中，gp91(phox)的C末端以延伸构象在gp91(phox)残基555和564之间与47(phox)结合，除His561外，558RGVHFIF564区域的所有氨基酸侧链均固定化。
