Conformation of a Cdc42/Rac interactive binding peptide in complex with Cdc42 and analysis of the binding interface

Biochemistry. 1999 May 11;38(19):5968-75. doi: 10.1021/bi990426u.

Abstract

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-gammaS-loaded Cdc42 in a saturable manner, with an apparent Kd of 0.6 microM, as determined by fluorescence titration using sNBD-labeled Cdc42. Interaction of the 22-residue peptide PAK2(71-92) with GTP-gammaS-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-gammaS-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • 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
  • cdc42 GTP-Binding Protein

Substances

  • Cell Cycle Proteins
  • Peptides
  • Guanosine Diphosphate
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • GTP-Binding Proteins
  • cdc42 GTP-Binding Protein