title: How RhoGDI binds Rho.
abstract: Like all Rho (Ras homology) GTPases, RhoA functions as a molecular switch in cell signaling, alternating between GTP- and GDP-bound states, with its biologically inactive GDP-bound form maintained as a cytosolic complex with RhoGDI (guanine nucleotide-exchange inhibitor). The crystal structures of RhoA-GDP and of the C-terminal immunoglobulin-like domain of RhoGDI (residues 67-203) are known, but the mechanism by which the two proteins interact is not known. The functional human RhoA-RhoGDI complex has been expressed in yeast and crystallized (P6(5)22, unit-cell parameters a = b = 139, c = 253 A, two complexes in the asymmetric unit). Although diffraction from these crystals extends to 3.5 A and is highly anisotropic, the experimentally phased (MAD plus MIR) electron-density map was adequate to reveal the mutual disposition of the two molecules. The result was validated by molecular-replacement calculations when data were corrected for anisotropy. Furthermore, the N-terminus of RhoGDI (the region involved in inhibition of nucleotide exchange) can be identified in the electron-density map: it is bound to the switch I and switch II regions of RhoA, occluding an epitope which binds Dbl-like nucleotide-exchange factors. The entrance of the hydrophobic pocket of RhoGDI is 25 A from the last residue in the RhoA model, with its C-terminus oriented to accommodate the geranylgeranyl group without conformational change in RhoA.
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, VA 22906, USA.
authors.forename: K
authors.initials: K
authors.lastname: Longenecker
authors.forename: P
authors.initials: P
authors.lastname: Read
authors.forename: U
authors.initials: U
authors.lastname: Derewenda
authors.forename: Z
authors.initials: Z
authors.lastname: Dauter
authors.forename: X
authors.initials: X
authors.lastname: Liu
authors.forename: S
authors.initials: S
authors.lastname: Garrard
authors.forename: L
authors.initials: L
authors.lastname: Walker
authors.forename: A V
authors.initials: AV
authors.lastname: Somlyo
authors.forename: R K
authors.initials: RK
authors.lastname: Nakamoto
authors.forename: A P
authors.initials: AP
authors.lastname: Somlyo
authors.forename: Z S
authors.initials: ZS
authors.lastname: Derewenda
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors; D006801:Humans; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020742:rhoA GTP-Binding Protein
chemical_list: C569638:ARHGDIA protein, human; D020730:Guanine Nucleotide Dissociation Inhibitors; D009842:Oligopeptides; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein