title: Structure-activity relationships in flexible protein domains: regulation of rho GTPases by RhoGDI and D4 GDI.
abstract: The guanine dissociation inhibitors RhoGDI and D4GDI inhibit guanosine 5'-diphosphate dissociation from Rho GTPases, keeping these small GTPases in an inactive state. The GDIs are made up of two domains: a flexible N-terminal domain of about 70 amino acid residues and a folded 134-residue C-terminal domain. Here, we characterize the conformation of the N-terminal regions of both RhoGDI and D4GDI using a series of NMR experiments which include (15)N relaxation and amide solvent accessibility measurements. In each protein, two regions with tendencies to form helices are identified: residues 36 to 58 and 9 to 20 in RhoGDI, and residues 36 to 57 and 20 to 25 in D4GDI. To examine the functional roles of the N-terminal domain of RhoGDI, in vitro and in vivo functional assays have been carried out with N-terminally truncated proteins. These studies show that the first 30 amino acid residues are not required for inhibition of GDP dissociation but appear to be important for GTP hydrolysis, whilst removal of the first 41 residues completely abolish the ability of RhoGDI to inhibit GDP dissociation. The combination of structural and functional studies allows us to explain why RhoGDI and D4GDI are able to interact in similar ways with the guanosine 5'-diphosphate-bound GTPase, but differ in their ability to regulate GTP-bound forms; these functional differences are attributed to the conformational differences of the N-terminal domains of the guanosine 5'-diphosphate dissociation inhibitors. Therefore, the two transient helices, appear to be associated with different biological effects of RhoGDI, providing a clear example of structure-activity relationships in a flexible protein domain.
authors: Department of Biochemistry and Biological NMR Centre, University of Leicester, Leicester, LE1 7RH, UK.
authors: A P
authors: AP
authors: Golovanov
authors: T H
authors: TH
authors: Chuang
authors: C
authors: C
authors: DerMardirossian
authors: I
authors: I
authors: Barsukov
authors: D
authors: D
authors: Hawkins
authors: R
authors: R
authors: Badii
authors: G M
authors: GM
authors: Bokoch
authors: L Y
authors: LY
authors: Lian
authors: G C
authors: GC
authors: Roberts
mesh_terms: D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D018583:Pliability; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D012997:Solvents; D013329:Structure-Activity Relationship; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C569638:ARHGDIA protein, human; C569658:ARHGDIB protein, human; D000577:Amides; D020730:Guanine Nucleotide Dissociation Inhibitors; D012997:Solvents; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein