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.: Allosteric and uncompetitive modulations of Arf1-Arno interaction

Jad Rouhana 1 Andre Padilla 2 Sebastien Estaran 1 Sana Bakari 1 Stephane Delbecq 3 Yvan Boublik 4 Joel Chopineau 5 Martine Pugniere 6 Alain Chavanieu 1, *
* Auteur correspondant
1 IBMM - Institut des Biomolécules Max Mousseron [Pôle Chimie Balard]
2 CBS - Centre de Biochimie Structurale [Montpellier]
3 LBCM - Vaccination Antiparasitaire : Laboratoire de Biologie Cellulaire et Moléculaire
4 CRBM - Centre de recherche en Biologie Cellulaire
5 ICGM ICMMM - Institut Charles Gerhardt Montpellier - Institut de Chimie Moléculaire et des Matériaux de Montpellier
6 IRCM - U896 Inserm - UM1 - Institut de recherche en cancérologie de Montpellier
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.
Keywords : Small gtpases Guanine nucleotide exchange factor (GEF) Allosteric regulation Brefeldin A (BFA) Kinetics Surface plasmon resonance (SPR)
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Article dans une revue
Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2013, 288 (7), pp.4659-72. 〈10.1074/jbc.M112.391748〉
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ICG | VALDAURELLE | FNCLCC | INC-CNRS | UNIMES | CHIMIE | BS | UNIV-MONTPELLIER

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Jad Rouhana, Andre Padilla, Sebastien Estaran, Sana Bakari, Stephane Delbecq, et al.. 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.: Allosteric and uncompetitive modulations of Arf1-Arno interaction. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2013, 288 (7), pp.4659-72. 〈10.1074/jbc.M112.391748〉. 〈inserm-00773549〉

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