Rac binding to p67(phox) - Structural basis for interactions of the Rac1 effector region and insert region with components of the respiratory burst oxidase
Rac binding to p67(phox) - Structural basis for interactions of the Rac1 effector region and insert region with components of the respiratory burst oxidase
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DOI：
10.1074/jbc.272.30.18834
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发表时间：
1997-07-25
影响因子：
4.8
通讯作者：
Lambeth, JD
中科院分区：
文献类型：
--
作者：
Nisimoto, Y;Freeman, JLR;Lambeth, JD
呼吸爆发氧化酶的激活涉及膜相关的黄色素b(558)与胞浆组分P47(Phox)、P67(Phox)和小GTP酶Rac的组装。在这里，我们使用功能和物理方法来探索RAC和p67(Phox)之间的相互作用。用测量依赖于NADPH的超氧化物生成的稳态动力学方法证明了rac1和p67(Phox)在NADPH氧化酶复合体内的相互促进结合(EC50)。以RAC结合的荧光类似物GTP(甲基邻氨基甲酰鸟苷-5‘-[β，γ-亚胺]三磷酸)为报告基团，研究了rac1和rac2与p67(Phox)的直接结合。加入P67(Phox)后，甲基邻氨基苯甲酰基荧光增强，而P47(Phox)未见增强，最大发射波长由445 nm移至440 nm。Rac1和rac2以1：1的化学计量比与p67(Phox)结合，k(D)值分别为120和60 nm。突变研究(Freeman，J.，Kreck，M.，Uhlinger，D.J.和Lambeth，J.D.(1994年)生物化学33,13431-13435；Freeman，J.L.，Abo，A，and Lambeth，J.D.(1996)J.Biol.化学。19794-19801)以前发现了RAC1中对活性重要的两个区域：‘’效应区‘’(残基26-45)和‘’插入区‘’(残基124-135)。在效应区突变的蛋白质(rac1(N26H)、rac1(133N)和rac1(D38N))显示K-d和EC50显著增加，表明该区域的突变通过抑制RAC与p67(Phox)的结合而影响活性。插入区突变(rac1(K132E)和L134R)，但EC50值显著升高，与p67(Phox)结合正常。由X射线结晶学测定的rac1的结构表明，效应区和插入区位于rac1表面的特定扇区内。讨论了一种模型，其中rac1效应区与p67(Phox)结合，C末端与膜结合，插入区与不同的蛋白质组分相互作用，可能是细胞色素b(558)。
Activation of the respiratory burst oxidase involves the assembly of the membrane associated flavocyto chrome b(558) with the cytosolic components p47(phox), p67(phox), and the small GTPase Rac. Herein, the interaction between Rac and p67(phox) is explored using functional and physical methods. Mutually facilitated binding (EC50) of Rac1 and p67(phox) within the NADPH oxidase complex was demonstrated using steady state kinetic methods measuring NADPH-dependent superoxide generation. Direct binding of Rac1 and Rac2 to p67(phox) was shown using a fluorescent analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]triphosphate) bound to Rac as a reporter group. An increase in the methylanthraniloyl fluorescence was seen with added p67(phox) but not p47(phox), and the emission maximum shifted from 445 to 440 nm. Rac1 and Rac2 bound to p67(phox) with a 1:1 stoichiometry and with k(d) values of 120 and 60 nM, respectively. Mutational studies (Freeman, J., Kreck, M., Uhlinger, D. J., and Lambeth, J. D. (1994) Biochemistry 33, 13431-13435; Freeman, J. L., Abo, A, and Lambeth, J. D. (1996) J. Biol. Chem. 271, 19794-19801) previously identified two regions in Rac1 that are important for activity: the ''effector region'' (residues 26-45) and the ''insert region'' (residues 124-135). Proteins mutated in the effector region (Rac1(N26H), Rac1(133N), and Rac1(D38N)) showed a marked increase in both the K-d and the EC50, indicating that mutations in this region affect activity by inhibiting Rac binding to p67(phox). Insert region mutations (Rac1(K132E) and L134R), while showing markedly elevated EC50 values, bound with normal affinity to p67(phox). The structure of Rac1 determined by x-ray crystallography reveals that the effector region and the insert region are located in defined sectors on the surface of Rac1. A model is discussed in which the Rac1 effector region binds to p67(phox), the C terminus binds to the membrane, and the insert region interacts with a different protein component, possibly cytochrome b(558).