Recognition and activation of the plant AkT1 potassium channel by the kinase CIPK23

Plant growth largely depends on the maintenance of adequate intracellular levels of potassium (K1). The families of 10 Calcineurin B-Like (CBL) calcium sensors and 26 CBL-Interacting Protein Kinases (CIPKs) of Arabidopsis (Arabidopsis thaliana) decode the calcium signals elicited by environmental inputs to regulate different ion channels and transporters involved in the control of K1 fluxes by phosphorylation-dependent and -independent events. However, the detailed molecular mechanisms governing target specificity require investigation. Here, we show that the physical interaction between CIPK23 and the noncanonical ankyrin domain in the cytosolic side of the inward-rectifier K1 channel AKT1 regulates kinase docking and channel activation. Point mutations on this domain specifically alter binding to CIPK23, enhancing or impairing the ability of CIPK23 to regulate channel activity. Our data demonstrate the relevance of this protein–protein interaction that contributes to the formation of a complex between CIPK23/CBL1 and AKT1 in the membrane for the proper regulation of K1 transport

Frq2 from Drosophila melanogaster: Cloning, expression, purification, crystallization and preliminary X-ray analysis

Drosophila melanogaster contains two calcium-binding proteins, Frq1 and Frq2, in the nervous system that control the number of synapses and the probability of release. To understand the differential function of the two proteins, whose sequence is only 5% dissimilar, the crystal structures of Frq1 and Frq2 are needed. Here, the cloning, expression, purification, crystallization and preliminary crystallographic analysis of Frq2 are presented. The full-length protein was purified using a two-step chromatographic procedure. Two different diffracting crystal forms were obtained using a progressive streak-seeding method and detergents. © 2014 International Union of Crystallography All rights reserved.Peer Reviewe

Preliminary crystallographic analysis of the ankyrin-repeat domain of Arabidopsis thaliana AKT1: Identification of the domain boundaries for protein crystallization

The Arabidopsis thaliana K+ transporter 1 (AKT1) participates in the maintenance of an adequate cell potassium (K+) concentration. The CBL-interacting protein kinase 23 (CIPK23) activates AKT1 for K+ uptake under low-K+ conditions. This process is mediated by the interaction between the cytosolic ankyrin-repeat (AR) domain of AKT1 and the kinase domain of CIPK23. However, the precise boundaries of the AR domain and the residues responsible for the interaction are still unknown. Here, the optimization procedure to obtain an AR domain construct suitable for crystallization and the preliminary crystallographic analysis of the obtained crystals are reported. The crystals belonged to space group P21212, with unit-cell parameters a = 34.83, b = 65.89, c = 85.44 Å, and diffracted to 1.98 Å resolution. © 2014 International Union of Crystallography All rights reserved.Peer Reviewe

An aminophenothiazine inhibitor of the NCS-1/Ric8a complex regulates synaptic function in Fragile X Syndrome

2 pags. -- 31st European Crystallographic Meeting, Oviedo, Spain 22-27 Augus

Structural studies on the regulation of arabidopsis thaliana ion homeostasis through the cbl-cipk pathway

Resumen del Poster presentado en Environment Workshop 2013: Genomic, Physiological and Breeding Approaches for Enhancing Drought Resistance in Crops. Baeza (Spain), 23-25 September (2013)The regulation of ionic transport in plants is essential because it establishes the key physicochemical parameters for cell function. Under abiotic stress, the intracellular levels of pH, potassium (K+) and toxic cations, such as Na+, change and this affect multiple cellular systems. Consequently, the knowledge of the molecular mechanism that regulates the ionic transport is fundamental and provides opportunities to use plants to our benefit [1]. The plant cells use calcium-signaling pathways to activate certain ion channels providing the correct response to a particular stress situation. As the most abundant cation in a living plant cell, K+ is an essential ion for processes of growth, development, maintenance of turgor pressure, and plasma membrane polarization [2]. Plants living under low K+ conditions often adapt their K+ uptake through the CBL-CIPK calcium-signaling pathway, that mobilizes K+ uptake in roots [3]. Under K+ deficiency, a CBL calcium sensor activates a CIPK kinase [3] that in turn phosphorilates and activate the K+ channel. When K+ levels are restored, a phosphatase dephosphorilates and inactivates the channel (Fig. 1) [5]. We have carried out structural studies with the kinase domain and its binding partner, the interacting region of the K+ channel, to understand at molecular level how K+ uptake is regulated under stress conditions. [1] M.J. Sánchez-Barrena, M. Martínez-Ripoll, A. Albert Int. J. Mol. Sci. 2013, 14, 5734 [2] R.E. Hirsch, B.D. Lewis, E.P. Spalding, M.R. Sussman Science. 1998, 280, 127. [3] L.Li, B.G. Kim, Y.H. Cheong, G.K. Pandey, S. Luan Proc. Natl. Acad. Sci. USA, 2006,103, 12625 [4] J. Xu, H.D. Li, L.Q. Chen Y. Wnag, L.L. Liu, L. He, W.H. Wu Cell, 2006, 125, 1347. [5] S.C. Lee, W.Z. Lan, B.G. Kim, L.Li, Y.H. Cheong, G.K. Pandey, B.B. Buchanan, S. Luan Proc. Natl. Acad. Sci. USA, 2007, 40, 15959.Peer reviewe

Structural basis of the regulatory mechanism of the plant CIPK family of protein kinases controlling ion homeostasis and abiotic stress

10 páginas.-- 6 figuras.-- 77 referencias.-- This article contains supporting information online at http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1407610111/-/DCSupplementalData deposition: The atomic coordinates have been deposited in the Protein Data Bank (PDB), www.pdb.org (PDB ID codes 4CZT, 4CZU, and 4D28).© 2014, National Academy of Sciences. All rights reserved. Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases.A.A. thanks Dr. Douglas Vinson Laurents for critical reading of the manuscript, and the European Syncrotron Radiation Facility (beamlines ID14-4 and ID23-2) and PETRAIII (beamline P13, BIOSTRUCTX_3100.5) for the access to the synchrotron radiation source. This work was funded by Ministerio de Economía y Competitividad Grants BFU2011-25384 and CSD2006-00015 (to A.A.), BIO2011-28184-C02-02 (toM.J.S.-B.), and BIO2012-36533 (to F.J.Q.), which was cofinanced by the European Regional Development Fund, and Comunidad de Madrid Grant S2010/BMD-2457 (to A.A.). A.C.-S. is supported by a Formación de Personal Investigador Predoctoral Fellowship, and M.J.S.-B. is supported by Ramón y Cajal Contract RYC-2008-03449 from MINECO.Peer Reviewe

Interference of the complex between NCS-1 & Ric8a with phenothiazines regulates synaptic function & is an approach for fragile X syndrome

The protein complex formed by the Ca2+ sensor neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor protein Ric8a coregulates synapse number and probability of neurotransmitter release, emerging as a potential therapeutic target for diseases affecting synapses, such as fragile X syndrome (FXS), the most common heritable autism disorder. Using crystallographic data and the virtual screening of a chemical library, we identified a set of heterocyclic small molecules as potential inhibitors of the NCS-1/Ric8a interaction. The aminophenothiazine FD44 interferes with NCS-1/Ric8a binding, and it restores normal synapse number and associative learning in a Drosophila FXS model. The synaptic effects elicited by FD44 feeding are consistent with the genetic manipulation of NCS-1. The crystal structure of NCS-1 bound to FD44 and the structure- function studies performed with structurally close analogs explain the FD44 specificity and the mechanism of inhibition, in which the small molecule stabilizes a mobile C-Terminal helix inside a hydrophobic crevice of NCS-1 to impede Ric8a interaction. Our study shows the drugability of the NCS-1/Ric8a interface and uncovers a suitable region in NCS-1 for development of additional drugs of potential use on FXS and related synaptic disorders.Peer Reviewe

The guanine-exchange factor Ric8a binds to the Ca2+ sensor NCS-1 to regulate synapse number and neurotransmitter release

© 2014. Published by The Company of Biologists Ltd. The conserved Ca2+-binding protein Frequenin (homolog of the mammalian NCS-1, neural calcium sensor) is involved in pathologies that result from abnormal synapse number and probability of neurotransmitter release per synapse. Both synaptic features are likely to be co-regulated but the intervening mechanisms remain poorly understood. We show here that Drosophila Ric8a (a homolog of mammalian synembryn, which is also known as Ric8a), a receptor-independent activator of G protein complexes, binds to Frq2 but not to the virtually identical homolog Frq1. Based on crystallographic data on Frq2 and site-directed mutagenesis on Frq1, the differential amino acids R94 and T138 account for this specificity. Human NCS-1 and Ric8a reproduce the binding and maintain the structural requirements at these key positions. Drosophila Ric8a and Gas regulate synapse number and neurotransmitter release, and both are functionally linked to Frq2. Frq2 negatively regulates Ric8a to control synapse number. However, the regulation of neurotransmitter release by Ric8a is independent of Frq2 binding. Thus, the antagonistic regulation of these two synaptic properties shares a common pathway, Frq2- Ric8a-Gαs, which diverges downstream. These mechanisms expose the Frq2-Ric8a interacting surface as a potential pharmacological target for NCS-1-related diseases and provide key data towards the corresponding drug design.Peer Reviewe