RAC BINDING TO P67(PHOX) - STRUCTURAL BASIS FOR INTERACTIONS OF THE RAC1 EFFECTOR REGION AND INSERT REGION WITH COMPONENTS OF THE RESPIRATORY BURST OXIDASE

Activation of the respiratory burst oxidase involves the assembly of t he membrane associated flavocyto chrome b(558) with the cytosolic comp onents p47(phox), p67(phox), and the small GTPase Rac. Herein, the int eraction between Rac and p67(phox) is explored using functional and ph ysical methods. Mutually facilitated binding (EC50) of Rac1 and p67(ph ox) within the NADPH oxidase complex was demonstrated using steady sta te kinetic methods measuring NADPH-dependent superoxide generation. Di rect binding of Rac1 and Rac2 to p67(phox) was shown using a fluoresce nt analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]tr iphosphate) bound to Rac as a reporter group. An increase in the methy lanthraniloyl fluorescence was seen with added p67(phox) but not p47(p hox), and the emission maximum shifted from 445 to 440 nm. Rac1 and Ra c2 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 effe ctor region (Rac1(N26H), Rac1(133N), and Rac1(D38N)) showed a marked i ncrease in both the K-d and the EC50, indicating that mutations in thi s region affect activity by inhibiting Rac binding to p67(phox). Inser t region mutations (Rac1(K132E) and L134R), while showing markedly ele vated EC50 values, bound with normal affinity to p67(phox). The struct ure of Rac1 determined by x-ray crystallography reveals that the effec tor region and the insert region are located in defined sectors on the surface of Rac1. A model is discussed in which the Rac1 effector regi on binds to p67(phox), the C terminus binds to the membrane, and the i nsert region interacts with a different protein component, possibly cy tochrome b(558).