TY - JOUR A1 - Skłodowski, Kamil A1 - Riedelsberger, Janin A1 - Raddatz, Natalia A1 - Riadi, Gonzalo A1 - Caballero, Julio A1 - Chérel, Isabelle A1 - Schulze, Waltraud A1 - Graf, Alexander A1 - Dreyer, Ingo T1 - The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2 JF - Scientific reports N2 - The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a ‘potassium battery’, providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals. Y1 - 2017 U6 - https://doi.org/10.1038/srep44611 SN - 2045-2322 VL - 7 PB - Nature Publishing Group CY - London ER - TY - JOUR A1 - Navarro-Retamal, Carlos A1 - Bremer, Anne A1 - Ingolfsson, Helgi I. A1 - Alzate-Morales, Jans A1 - Caballero, Julio A1 - Thalhammer, Anja A1 - Gonzalez, Wendy A1 - Hincha, Dirk K. T1 - Folding and Lipid Composition Determine Membrane Interaction of the Disordered Protein COR15A JF - Biophysical journal N2 - Plants from temperate climates, such as the model plant Arabidopsis thaliana, are challenged with seasonal low temperatures that lead to increased freezing tolerance in fall in a process termed cold acclimation. Among other adaptations, this involves the accumulation of cold-regulated (COR) proteins, such as the intrinsically disordered chloroplast-localized protein COR15A. Together with its close homolog COR15B, it stabilizes chloroplast membranes during freezing. COR15A folds into amphipathic alpha-helices in the presence of high concentrations of low-molecular-mass crowders or upon dehydration. Under these conditions, the (partially) folded protein binds peripherally to membranes. In our study, we have used coarse-grained molecular dynamics simulations to elucidate the details of COR15A-membrane binding and its effects on membrane structure and dynamics. Simulation results indicate that at least partial folding of COR15A and the presence of highly unsaturated galactolipids in the membranes are necessary for efficient membrane binding. The bound protein is stabilized on the membrane by interactions of charged and polar amino acids with galactolipid headgroups and by interactions of hydrophobic amino acids with the upper part of the fatty acyl chains. Experimentally, the presence of liposomes made from a mixture of lipids mimicking chloroplast membranes induces additional folding in COR15A under conditions of partial dehydration, in agreement with the simulation results. Y1 - 2018 U6 - https://doi.org/10.1016/j.bpj.2018.08.014 SN - 0006-3495 SN - 1542-0086 VL - 115 IS - 6 SP - 968 EP - 980 PB - Cell Press CY - Cambridge ER - TY - JOUR A1 - Navarro-Retamal, Carlos A1 - Bremer, Anne A1 - Alzate-Morales, Jans H. A1 - Caballero, Julio A1 - Hincha, Dirk K. A1 - Gonzalez, Wendy A1 - Thalhammer, Anja T1 - Molecular dynamics simulations and CD spectroscopy reveal hydration-induced unfolding of the intrinsically disordered LEA proteins COR15A and COR15B from Arabidopsis thaliana JF - Physical chemistry, chemical physics : a journal of European Chemical Societies N2 - The LEA (late embryogenesis abundant) proteins COR15A and COR15B from Arabidopsis thaliana are intrinsically disordered under fully hydrated conditions, but obtain alpha-helical structure during dehydration, which is reversible upon rehydration. To understand this unusual structural transition, both proteins were investigated by circular dichroism (CD) and molecular dynamics (MD) approaches. MD simulations showed unfolding of the proteins in water, in agreement with CD data obtained with both HIS-tagged and untagged recombinant proteins. Mainly intramolecular hydrogen bonds (H-bonds) formed by the protein backbone were replaced by H-bonds with water molecules. As COR15 proteins function in vivo as protectants in leaves partially dehydrated by freezing, unfolding was further assessed under crowded conditions. Glycerol reduced (40%) or prevented (100%) unfolding during MD simulations, in agreement with CD spectroscopy results. H-bonding analysis indicated that preferential exclusion of glycerol from the protein backbone increased stability of the folded state. Y1 - 2016 U6 - https://doi.org/10.1039/c6cp02272c SN - 1463-9076 SN - 1463-9084 VL - 18 SP - 25806 EP - 25816 PB - Royal Society of Chemistry CY - Cambridge ER - TY - GEN A1 - Navarro-Retamal, Carlos A1 - Bremer, Anne A1 - Alzate-Morales, Jans H. A1 - Caballero, Julio A1 - Hincha, Dirk K. A1 - González, Wendy A1 - Thalhammer, Anja T1 - Molecular dynamics simulations and CD spectroscopy reveal hydration-induced unfolding of the intrinsically disordered LEA proteins COR15A and COR15B from Arabidopsis thaliana N2 - The LEA (late embryogenesis abundant) proteins COR15A and COR15B from Arabidopsis thaliana are intrinsically disordered under fully hydrated conditions, but obtain α-helical structure during dehydration, which is reversible upon rehydration. To understand this unusual structural transition, both proteins were investigated by circular dichroism (CD) and molecular dynamics (MD) approaches. MD simulations showed unfolding of the proteins in water, in agreement with CD data obtained with both HIS-tagged and untagged recombinant proteins. Mainly intramolecular hydrogen bonds (H-bonds) formed by the protein backbone were replaced by H-bonds with water molecules. As COR15 proteins function in vivo as protectants in leaves partially dehydrated by freezing, unfolding was further assessed under crowded conditions. Glycerol reduced (40%) or prevented (100%) unfolding during MD simulations, in agreement with CD spectroscopy results. H-bonding analysis indicated that preferential exclusion of glycerol from the protein backbone increased stability of the folded state. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 321 Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-394503 SP - 25806 EP - 25816 ER - TY - JOUR A1 - Gonzalez, Wendy A1 - Riedelsberger, Janin A1 - Morales-Navarro, Samuel E. A1 - Caballero, Julio A1 - Alzate-Morales, Jans H. A1 - Gonzalez-Nilo, Fernando D. A1 - Dreyer, Ingo T1 - The pH sensor of the plant K+-uptake channel KAT1 is built from a sensory cloud rather than from single key amino acids JF - The biochemical journal N2 - The uptake of potassium ions (K+) accompanied by an acidification of the apoplasm is a prerequisite for stomatal opening. The acidification (approximately 2-2.5 pH units) is perceived by voltage-gated inward potassium channels (K-in) that then can open their pores with lower energy cost. The sensory units for extracellular pH in stomatal K-in channels are proposed to be histidines exposed to the apoplasm. However, in the Arabidopsis thaliana stomatal K-in channel KAT1, mutations in the unique histidine exposed to the solvent (His(267)) do not affect the pH dependency. We demonstrate in the present study that His(267) of the KAT1 channel cannot sense pH changes since the neighbouring residue Phe(266) shifts its pK(a) to undetectable values through a cation-pi interaction. Instead, we show that Glu(240) placed in the extracellular loop between transmembrane segments S5 and S6 is involved in the extracellular acid activation mechanism. Based on structural models we propose that this region may serve as a molecular link between the pH- and the voltage-sensor. Like Glu(240), several other titratable residues could contribute to the pH-sensor of KAT1, interact with each other and even connect such residues far away from the voltage-sensor with the gating machinery of the channel. KW - Arabidopsis thaliana KW - channel protein structure KW - channel protein-proton interaction KW - KAT1 KW - pH regulation KW - potassium chanel Y1 - 2012 U6 - https://doi.org/10.1042/BJ20111498 SN - 0264-6021 VL - 442 IS - 7 SP - 57 EP - 63 PB - Portland Press CY - London ER - TY - CHAP A1 - Gonzalez, W. A1 - Riedelsberger, J. A1 - Morales-Navarro, S. E. A1 - Caballero, Julio A1 - Alzate-Morales, Jans H. A1 - Gonzalez-Nilo, F. D. A1 - Dreyer, Ingo T1 - The pH sensor of the plant K plus uptake channel KAT1 is built from a sensory cloud rather than from single key amino acids T2 - The FEBS journal Y1 - 2012 SN - 1742-464X VL - 279 SP - 455 EP - 455 PB - Wiley-Blackwell CY - Hoboken ER -