title: Mapping the binding site for the GTP-binding protein Rac-1 on its inhibitor RhoGDI-1.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biological NMR Centre, University of Leicester, Leicester, LE1 7RH, UK. yun@le.ac.uk
authors.forename: L Y
authors.initials: LY
authors.lastname: Lian
authors.forename: I
authors.initials: I
authors.lastname: Barsukov
authors.forename: A P
authors.initials: AP
authors.lastname: Golovanov
authors.forename: D I
authors.initials: DI
authors.lastname: Hawkins
authors.forename: R
authors.initials: R
authors.lastname: Badii
authors.forename: K H
authors.initials: KH
authors.lastname: Sze
authors.forename: N H
authors.initials: NH
authors.lastname: Keep
authors.forename: G M
authors.initials: GM
authors.lastname: Bokoch
authors.forename: G C
authors.initials: GC
authors.lastname: Roberts
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
chemical_list: C569638:ARHGDIA protein, human; D020730:Guanine Nucleotide Dissociation Inhibitors; D011994:Recombinant Proteins; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020830:rac1 GTP-Binding Protein