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, R ecent studies indicated that brefeldin A (BFA) inhibits Sec7d-catalyzed nuc leotide exchange on ARF1 in an uncompetitive manner by trapping an early in termediate of the reaction: a complex between GDP-bound ARF1 and Sec7d, Usi ng H-3-labeled BFA, we show that BFA binds to neither isolated Sec7d nor is olated ARF1-GDP, but binds to the transitory Sec7d-ARF1-GDP complex and sta bilizes it. Two pairs of residues at positions 190-191 and 198-208 (Arno nu mbering) 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 AR F1-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 swi tch 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.