SummaryPygo and BCL9/Legless transduce the Wnt signal by promoting the transcriptional activity of β-catenin/Armadillo in normal and malignant cells. We show that human and Drosophila Pygo PHD fingers associate with their cognate HD1 domains from BCL9/Legless to bind specifically to the histone H3 tail methylated at lysine 4 (H3K4me). The crystal structures of ternary complexes between PHD, HD1, and two different H3K4me peptides reveal a unique mode of histone tail recognition: efficient histone binding requires HD1 association, and the PHD-HD1 complex binds preferentially to H3K4me2 while displaying insensitivity to methylation of H3R2. Therefore, this is a prime example of histone tail binding by a PHD finger (of Pygo) being modulated by a cofactor (BCL9/Legless). Rescue experiments in Drosophila indicate that Wnt signaling outputs depend on histone decoding. The specificity of this process provided by the Pygo-BCL9/Legless complex suggests that this complex facilitates an early step in the transition from gene silence to Wnt-induced transcription.

After the discovery of bacteriocin AS-48, a 70-residue cyclic peptide produced by Enterococcus faecalis subsp. liquefaciens, some naturally-occurring cyclic proteins from bacteria have been reported. AS-48 is encoded by the 68-kb pheromone-responsive plasmid pMB2, and the gene cluster involved in production and immunity has been identified and sequenced. This peptide exerts a bactericidal action on sensitive cells (most of the Gram-positive and some Gram-negative bacteria). Its target is the cytoplasmic membrane, in which it opens pores, leading to the dissipation of the proton motive force and cell death, a mechanism similar to that proposed for the action of defensins or, most generally, cationic antibacterial peptides. This fact, together with its remarkable stability and solubility over a wide pH range, suggest that this bacteriocin could be a good candidate as a natural food preservative. The amino acid composition of purified AS-48 shows the absence of modified or dehydrated residues, making it clearly different from lantibiotics. Bacteriocin AS-48 also differs from defensins in that it does not contain cysteines and consequently no disulfide bridges, which makes is high stability even more remarkable. Composition analysis of AS-48 shows a high proportion of basic to acidic amino acids, conferring to this peptide a strong basic character, with an isoelectric point close to 10.5. Determination of the AS-48 structural gene DNA sequence, together with the sequences of AS-48 protease digestion fragments and mass spectrometry determinations, allowed us to determine unambiguously the cyclic structure of the molecule, being the first example of a posttranslational modification in which a cyclic structure arises from a "head-to-tail" linkage. We have solved the three-dimensional structure of AS-48 in solution, and it consists of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. Interestingly, the head-to-tail peptide link between Trp-70 and Met-1 lies in the middle of alpha-helix 5, which is shown to have a pronounced effect on the stability of the three-dimensional structure. Analysis of structure-function relationship allowed us to propose models to understand the aspects of the molecular function of AS-48. The purpose of this work is to review recent developments in our understanding about the biochemical and biological characteristics and structure of this unusual type of bacteriocin.

The plant SOS2 family of protein kinases and their interacting activators, the SOS3 family of calcium-binding proteins, function together in decoding calcium signals elicited by different environmental stimuli. SOS2 is activated by Ca-SOS3 and subsequently phosphorylates the ion transporter SOS1 to bring about cellular ion homeostasis under salt stress. In addition to possessing the kinase activity, members of the SOS2 family of protein kinases can bind to protein phosphatase 2Cs. The crystal structure of the binary complex of Ca-SOS3 with the C-terminal regulatory moiety of SOS2 resolves central questions regarding the dual function of SOS2 as a kinase and a phosphatase-binding protein. A comparison with the structure of unbound SOS3 reveals the basis of the molecular function of this family of kinases and their interacting calcium sensors. Furthermore, our study suggests that the structure of the phosphatase-interaction domain of SOS2 defines a scaffold module conserved from yeast to human.

Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca 2+ are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca 2+ signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca 2+ -dependent recruitment of the pyrabactin resistance 1/PYR1-like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca 2+ sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca 2+ -dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress.signaling | ion transport | membrane biology | abiotic stress M any of the plant-adaptive responses to environmental stresses occur at the cell membrane. In particular, those related to the regulation of plant ion transporters as stress unbalance cell ion homeostasis (1, 2). The phytohormone abcisic acid (ABA) and the second messenger Ca 2+ have central roles in regulating plant stress tolerance through the control of the activity of various families of protein kinases and phosphatases that regulate the activation of different ion channels or transporters (3-7). Given the presence of the channel substrates at the cell membranes and the transient nature of their activation, the functioning of these systems relies on the regulated localization of different molecular entities in the vicinity of the channels via protein-protein (8, 9) and/or protein-membrane interactions (10, 11).The pyrabactin resistance 1/PYR1-like (PYR/PYL)/regulatory components of ABA receptors (RCAR) receptors perceive intracellular ABA levels and, as a result, form ternary complexes with clade A protein phosphatases type 2C (PP2C), thereby inactivating them (12)(13)(14)(15). This prevents the PP2C-mediated dephosphorylation of ABA-activated sucrose nonfermenting 1-related protein kinases (SnRKs) subfamily 2 (SnRK2s), which results in the activation of an SnRK2-dependent phosphorylation cascade affecting a high number of targets in the plant cell (16,17). As a result, ABA-activated SnRK2s are key players in regulating...

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The protein complex formed by the Ca 2+ 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.fragile X syndrome | synapse regulation | NCS-1 | protein-protein interaction inhibitor | X-ray crystallography

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 calciumdependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases.signaling | ion transport | abiotic stress C ell perception of extracellular stimuli is followed by a transient variation in cytosolic calcium concentration. Plants have evolved to produce the specific molecular machinery to interpret this primary information and to transmit this signal to the components that organize the cell response (1-4). The plant family of serine/threonine protein kinases PKS or CIPKs (hereinafter CIPKs) and their activators, the calcium-binding proteins SCaBPs or CBLs (hereinafter CBLs) (5, 6) function together in decoding calcium signals caused by different environmental stimuli. Available data suggest a mechanism in which calcium mediates the formation of stable CIPK-CBL complexes that regulate the phosphorylation state and activity of various ion transporters involved in the maintenance of cell ion homeostasis and abiotic stress responses in plants. Among them, the Arabidopsis thaliana CIPK24/SOS2-CBL4/SOS3 complex activates the Na + /H + antiporter SOS1 to maintain intracellular levels of the toxic Na + low under salt stress (7-9), the CIPK11-CBL2 pair regulates the plasma membrane H + -ATPase AHA2 to control the transmembrane pH gradient (10), the CIPK23-CBL1/9 (11, 12) regulates the activity of the K + transporter AKT1 to increase the plant K + uptake capability under limiting K + supply conditions (12, 13), and CIPK23-CBL1 mediates nitrate sensing and uptake by phosphorylation of the nitrate transporter CHL1 (14). Together these findings show that underst...