Molecular characterization of the GTPase-activating domain of ADP-ribosylation factor domain protein 1 (ARD1).
ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins recognized as critical components in intracellular vesicular transport and phospholipase D activation. Both guanine nucleotide-exchange proteins and GTPase-activating proteins (GAPs) for ARFs have been cloned recently. A zinc finger motif near the amino terminus of the ARF1 GAP was required for stimulation of GTP hydrolysis. ARD1 is an ARF family member that differs from other ARFs by the presence of a 46-kDa amino-terminal extension. We had reported that the ARF domain of ARD1 binds specifically GDP and GTP and that the amino-terminal extension acts as a GAP for the ARF domain of ARD1 but not for ARF proteins. The GAP domain of ARD1, synthesized in Escherichia coli, stimulated hydrolysis of GTP bound to the ARF domain of ARD1. Using ARD1 truncations, it appears that amino acids 101-190 are critical for GAP activity, whereas residues 190-333 are involved in physical interaction between the two domains of ARD1 and are required for GTP hydrolysis. The GAP function of the amino-terminal extension of ARD1 required two arginines, an intact zinc finger motif, and a group of residues which resembles a sequence present in Rho/Rac GAPs. Interaction between the two domains of ARD1 required two negatively charged residues (Asp427 and Glu428) located in the effector region of the ARF domain and two basic amino acids (Arg249 and Lys250) found in the amino-terminal extension. The GAP domain of ARD1 thus is similar to ARF GAPs but differs from other GAPs in its covalent association with the GTP-binding domain.
Vitale
N
N
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA. vitalen@fido.nhlbi.nih.gov
Moss
J
J
Vaughan
M
M
D001120:Arginine; D004252:DNA Mutational Analysis; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016335:Zinc Fingers
D020690:GTPase-Activating Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; D001120:Arginine; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins