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Mapping the binding site for the GTP-binding protein Rac-1 on its inhibitor RhoGDI-1.

BACKGROUND: Members of the Rho family of small GTP-binding proteins, such as Rho, Rac and Cdc42, have a role in a wide range of cell responses. These proteins function as molecular switches by virtue of a conformational change between the GTP-bound (active) and GDP-bound (inactive) forms. In addition, most members of the Rho and Rac subfamilies cycle between the cytosol and membrane. The cytosolic guanine nucleotide dissociation inhibitors, RhoGDIs, regulate both the GDP/GTP exchange cycle and the membrane association/dissociation cycle. RESULTS: We have used NMR spectroscopy and site-directed mutagenesis to identify the regions of human RhoGDI-1 that are involved in binding Rac-1. The results emphasise the importance of the flexible regions of both proteins in the interaction. At least one specific region (residues 46-57) of the flexible N-terminal domain of RhoGDI, which has a tendency to form an amphipathic helix in the free protein, makes a major contribution to the binding energy of the complex. In addition, the primary site of Rac-1 binding on the folded domain of RhoGDI involves the beta4-beta5 and beta6-beta7 loops, with a slight movement of the 3(10) helix accompanying the interaction. This binding site is on the same face of the protein as the binding site for the isoprenyl group of post-translationally modified Rac-1, but is distinct from this site. CONCLUSIONS: Isoprenylated Rac-1 appears to interact with three distinct sites on RhoGDI. The isoprenyl group attached to the C terminus of Rac-1 binds in a pocket in the folded domain of RhoGDI. This is distinct from the major site on this domain occupied by Rac-1 itself, which involves two loops at the opposite end to the isoprenyl-binding site. It is probable that the flexible C-terminal region of Rac-1 extends from the site at which Rac-1 contacts the folded domain of RhoGDI to allow the isoprenyl group to bind in the pocket at the other end of the RhoGDI molecule. Finally, the flexible N terminus of RhoGDI-1, and particularly residues 48-58, makes a specific interaction with Rac-1 which contributes substantially to the binding affinity.

Pubmed ID: 10673424


  • Lian LY
  • Barsukov I
  • Golovanov AP
  • Hawkins DI
  • Badii R
  • Sze KH
  • Keep NH
  • Bokoch GM
  • Roberts GC


Structure (London, England : 1993)

Publication Data

January 15, 2000

Associated Grants


Mesh Terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Guanine Nucleotide Dissociation Inhibitors
  • Humans
  • In Vitro Techniques
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Protein Conformation
  • Protein Folding
  • Protein Structure, Tertiary
  • Recombinant Proteins
  • Sequence Homology, Amino Acid
  • Thermodynamics
  • rac1 GTP-Binding Protein
  • rho Guanine Nucleotide Dissociation Inhibitor alpha
  • rho-Specific Guanine Nucleotide Dissociation Inhibitors