Conformation of a Cdc42/Rac Interactive Binding Peptide in Complex with Cdc42 and Analysis of the Binding Interface

• Published in: Biochemistry (Easton) 1999-05, Vol.38 (19), p.5968-5975
• Main Authors: Stevens, Willem K, Vranken, Wim, Goudreau, Nathalie, Xiang, Hui, Xu, Ping, Ni, Feng
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites; cdc42 GTP-Binding Protein; Cell Cycle Proteins - chemistry; Cell Cycle Proteins - metabolism; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Guanosine Diphosphate - metabolism; Humans; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Peptides - chemistry; Peptides - metabolism; Protein Conformation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi990426u

Most of the putative effectors for the Rho-family small GTPases Cdc42 and Rac share a common sequence motif referred to as the Cdc42/Rac interactive binding (CRIB) motif. This sequence, with a consensus of I-S-x-P-(x)2 - 4-F-x-H-x-x-H-V-G [Burbelo, P. D., et al. (1995) J. Biol. Chem. 270, 29071−29074], has been shown to be essential for the functional interactions between these effector proteins and Cdc42. We have characterized the interactions of a 22-residue CRIB peptide derived from human PAK2 [PAK2(71−92)] with Cdc42 using proton and heteronuclear NMR spectroscopy. This CRIB peptide binds to GTP-γS-loaded Cdc42 in a saturable manner, with an apparent K d of 0.6 μM, as determined by fluorescence titration using sNBD-labeled Cdc42. Interaction of the 22-residue peptide PAK2(71−92) with GTP-γS-loaded Cdc42 causes resonance perturbations in the 1H−15N HSQC spectrum of Cdc42 that are similar to those observed for a longer (46-amino acid) CRIB-containing protein fragment [Guo, W., et al. (1998) Biochemistry 37, 14030−14037]. Proton NMR studies of PAK2(71−92) demonstrate structuring of PAK2(71−92) in the presence of GTP-γS-loaded Cdc42, through the observation of many nonsequential transferred NOEs. Structure calculations based on the observed transferred NOEs show that the central portion of the Cdc42-bound CRIB peptide assumes a loop conformation in which the side chains of consensus residues Phe80, His82, Ile84, His85, and Val86 are brought into proximity. The CRIB motif may therefore represent a minimal interfacial region in the complexes between Cdc42 and its effector proteins.