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

International audienceThe 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

ite-directed analyses of the C-linker domain of plant K+ Shaker channels reveal structural similarities with animal HCN channels and provide clues about the plant channel structure-function relationship

ite-directed analyses of the C-linker domain of plant K+ Shaker channels reveal structural similarities with animal HCN channels and provide clues about the plant channel structure-function relationship. Gordon Research Conferences - Ion Channel

Control of K+ channel activity and transport in plants: the C-linker of Arabidopsis Shaker inward channels plays a crucial role in both surface expression and channel activity

Control of K+ channel activity and transport in plants: the C-linker of Arabidopsis Shaker inward channels plays a crucial role in both surface expression and channel activity. Gordon Research Conferences - Salt & Water Stress in Plant

Distinct amino acids in the C-linker domain of the plant K+ channel KAT2 determine its subcellular localization and activity at the plasma membrane.

Shaker K+ channels form the major K+ conductance of the plasma membrane in plants. They are composed of four subunits arranged around a central ion-conducting pore. The intracellular carboxy-terminal region of each subunit contains several regulatory elements including a C-linker region and a cyclic-nucleotide-binding domain (CNBD). The C-linker is the first domain present downstream of the 6th transmembrane segment and connects the CNBD to the transmembrane core. With the aim of identifying the role of the C-linker in the Shaker channel properties, we performed subdomain swapping between the C-linker of two Arabidopsis Shaker subunits, KAT2 and AtKC1. These two subunits contribute to K+ transport in planta, by forming heteromeric channels with other Shaker subunits. However, they display contrasting behavior when expressed in tobacco mesophyll protoplasts: KAT2 forms homotetrameric channels active at the plasma membrane whereas AtKC1 is retained in the endoplasmic reticulum when expressed alone. The resulting chimeric/mutated constructs were analyzed for subcellular localization and functionally characterized. We identified 2 contiguous amino acids, Val381 and Ser382, located in the C-linker C-terminal end, which prevent KAT2 surface expression when mutated into the equivalent residues from AtKC1. Moreover, we demonstrated that the nine amino acid stretch 312TVRAASEFA320 which composes the first C-linker α-helix (A'-helix) located just below the pore, is a crucial determinant of KAT2 channel activity. A KAT2 C-linker/CNBD 3D model, based on animal HCN channels as structure templates, has been built and used to discuss the role of the C-linker in plant Shaker inward channel structure and function

Fragment-Based Identification of a Locus in the Sec7 Domain of Arno for the Design of Protein–Protein Interaction Inhibitors

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