A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the beta-phosphate to destabilize GDP on ARF1.
The Sec7 domain of the guanine nucleotide exchange factor ARNO (ARNO-Sec7) is responsible for the exchange activity on the small GTP-binding protein ARF1. ARNO-Sec7 forms a stable complex with the nucleotide-free form of [Delta17]ARF1, a soluble truncated form of ARF1. The crystal structure of ARNO-Sec7 has been solved recently, and a site-directed 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 Mg2+ and GDP from ARF1. The conservative mutation E156D and the charge reversal mutation E156K reduce the exchange activity of ARNO-Sec7 by several orders of magnitude. Moreover, [E156K]ARNO-Sec7 forms a complex with the Mg2+-free form of [Delta17]ARF1-GDP without inducing the release of GDP. Other mutations in ARNO-Sec7 and in [Delta17]ARF1 suggest that prominent hydrophobic residues of the switch I region of ARF1 insert into the groove of the Sec7 domain, and that Lys73 of the switch II region of ARF1 forms an ion pair with Asp183 of ARNO-Sec7.
Béraud-Dufour
S
S
CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des lucioles, 06560 Valbonne.
Robineau
S
S
Chardin
P
P
Paris
S
S
Chabre
M
M
Cherfils
J
J
Antonny
B
B
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D005656:Fungal Proteins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism*; D011487:Protein Conformation
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D010710:Phosphates; C066755:Sec7 guanine nucleotide exchange factors; C102474:cytohesin-2; D006153:Guanosine Diphosphate; D001224:Aspartic Acid; D018698:Glutamic Acid; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D008274:Magnesium