Binding site of brefeldin A at the interface between the small G protein ADP-ribosylation factor 1 (ARF1) and the nucleotide-exchange factor Sec7 domain.
Sec7 domains (Sec7d) catalyze the exchange of guanine nucleotide on ARFs. Recent studies indicated that brefeldin A (BFA) inhibits Sec7d-catalyzed nucleotide exchange on ARF1 in an uncompetitive manner by trapping an early intermediate of the reaction: a complex between GDP-bound ARF1 and Sec7d. Using (3)H-labeled BFA, we show that BFA binds to neither isolated Sec7d nor isolated ARF1-GDP, but binds to the transitory Sec7d-ARF1-GDP complex and stabilizes it. Two pairs of residues at positions 190-191 and 198-208 (Arno numbering) in Sec7d contribute equally to the stability of BFA binding, which is also sensitive to mutation of H80 in ARF1. The catalytic glutamic (E156) residue of Sec7d is not necessary for BFA binding. In contrast, BFA does not bind to the intermediate catalytic complex between nucleotide-free ARF1 and Sec7d. These results suggest that, on initial docking steps between ARF1-GDP and Sec7d, BFA inserts like a wedge between the switch II region of ARF1-GDP and a surface encompassing residues 190-208, at the border of the characteristic hydrophobic groove of Sec7d. Bound BFA would prevent the switch regions of ARF1-GDP from reorganizing and forming tighter contacts with Sec7d and thereby would maintain the bound GDP of ARF1 at a distance from the catalytic glutamic finger of Sec7d.
Robineau
S
S
Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des lucioles, 06560 Valbonne, France.
Chabre
M
M
Antonny
B
B
D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D020126:Brefeldin A / Q000493:pharmacokinetics*; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion
D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D011994:Recombinant Proteins; C066755:Sec7 guanine nucleotide exchange factors; D006153:Guanosine Diphosphate; D020126:Brefeldin A; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1