A GLUTAMIC FINGER IN THE GUANINE-NUCLEOTIDE EXCHANGE FACTOR ARNO DISPLACES MG2-PHOSPHATE TO DESTABILIZE GDP ON ARF1( AND THE BETA)

The Sec7 domain of the guanine nucleotide exchange factor ARNO (ARNO-S ec7) is responsible for the exchange activity on the small GTP-binding protein ARF1. ARNO-Sec7 forms a stable complex with the nucleotide-fr ee form of [Delta 17]ARF1, a soluble truncated form of ARF1. The cryst al structure of ARNO-Sec7 has been solved recently, and a site-directe d mutagenesis approach identified a hydrophobic groove and an adjacent hydrophilic loop as the ARF1-binding site. We show that Glu156 in the hydrophilic loop of ARNO-Sec7 is involved in the destabilization of M g2+ and GDP from ARF1. The conservative mutation E156D and the charge reversal mutation E156K reduce the exchange activity of ARNO-Sec7 by s everal orders of magnitude. Moreover, [E156K]ARNO-Sec7 forms a complex with the Mg2+-free form of [Delta 17]ARF1-GDP without inducing the re lease of GDP. Other mutations in ARNO-Sec7 and in [Delta 17]ARF1 sugge st that prominent hydrophobic residues of the switch I region of ARF1 insert into the groove of the Sec7 domain, and that Lys73 of the snitc h II region of ARF1 forms an ion pair with Asp183 of ARNO-Sec7.