title: Kinetics of interaction between ADP-ribosylation factor-1 (Arf1) and the Sec7 domain of Arno guanine nucleotide exchange factor, modulation by allosteric factors, and the uncompetitive inhibitor brefeldin A.
abstract: The GDP/GTP nucleotide exchange of Arf1 is catalyzed by nucleotide exchange factors (GEF), such as Arno, which act through their catalytic Sec7 domain. This exchange is a complex mechanism that undergoes conformational changes and intermediate complex species involving several allosteric partners such as nucleotides, Mg(2+), and Sec7 domains. Using a surface plasmon resonance approach, we characterized the kinetic binding parameters for various intermediate complexes. We first confirmed that both GDP and GTP counteract equivalently to the free-nucleotide binary Arf1-Arno complex stability and revealed that Mg(2+) potentiates by a factor of 2 the allosteric effect of GDP. Then we explored the uncompetitive inhibitory mechanism of brefeldin A (BFA) that conducts to an abortive pentameric Arf1-Mg(2+)-GDP-BFA-Sec7 complex. With BFA, the association rate of the abortive complex is drastically reduced by a factor of 42, and by contrast, the 15-fold decrease of the dissociation rate concurs to stabilize the pentameric complex. These specific kinetic signatures have allowed distinguishing the level and nature as well as the fate in real time of formed complexes according to experimental conditions. Thus, we showed that in the presence of GDP, the BFA-resistant Sec7 domain of Arno can also associate to form a pentameric complex, which suggests that the uncompetitive inhibition by BFA and the nucleotide allosteric effect combine to stabilize such abortive complex.
authors.affiliation: Institut des Biomolécules Max Mousseron, IBMM, UMR 5247 CNRS-Universités Montpellier 1 et 2 Faculté de Pharmacie, 15 avenue Charles Flahault BP14491, 34093 Montpellier cedex 5, France.
authors.forename: Jad
authors.initials: J
authors.lastname: Rouhana
authors.forename: André
authors.initials: A
authors.lastname: Padilla
authors.forename: Sébastien
authors.initials: S
authors.lastname: Estaran
authors.forename: Sana
authors.initials: S
authors.lastname: Bakari
authors.forename: Stephan
authors.initials: S
authors.lastname: Delbecq
authors.forename: Yvan
authors.initials: Y
authors.lastname: Boublik
authors.forename: Joel
authors.initials: J
authors.lastname: Chopineau
authors.forename: Martine
authors.initials: M
authors.lastname: Pugnière
authors.forename: Alain
authors.initials: A
authors.lastname: Chavanieu
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry* / Q000378:metabolism; D000495:Allosteric Site; D001667:Binding, Competitive; D019921:Biotinylation; D020126:Brefeldin A / Q000737:chemistry*; D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020349:Surface Plasmon Resonance
chemical_list: D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; C066755:Sec7 guanine nucleotide exchange factors; C102474:cytohesin-2; D006153:Guanosine Diphosphate; D020126:Brefeldin A; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1