Armando Albert

A six-stranded double-psi β barrel is shared by several protein superfamilies

Abstract Background: Six-stranded β barrels with a pseudo-twofold axis are found in several proteins. One group comprises a Greek-key structure with all strands antiparallel; an example is the N-terminal domain of ferredoxin reductase. Others involve parallel strands forming two psi structures (the double-psi β barrel). A recently discovered example of the latter class is aspartate-α-decarboxylase (ADC) from Escherichia coli , a pyruvoyl-dependent tetrameric enzyme involved in the synthesis of pantothenate. Results: Visual inspection and automated database searches identified the six-stranded double-psi β barrel in ADC, Rhodobacter sphaeroides dimethylsulfoxide (DMSO) reductase, E. coli formate dehydrogenase H (FDH H ), the plant defense protein barwin, Humicola insolens endoglucanase V (EGV) and, with a circular permutation, in the aspartic proteinases. Structure-based sequence alignments revealed several interactions including hydrophobic contacts or sidechain–mainchain hydrogen bonds that position the middle β strand under a psi loop, which may significantly contribute to stabilizing the fold. The identification of key interactions allowed the filtering of weak sequence similarities to some of these proteins, which had been detected by sequence database searches. This led to the prediction of the double-psi β-barrel domain in several families of proteins in eukaryotes and archaea. Conclusions: The structure comparison and clustering study of double-psi β barrels suggests that there could be a common homodimeric ancestor to ADC, FDH H and DMSO reductase, and also to barwin and EGV. There are other protein families with unknown structure that are likely to adopt the same fold. In the known structures, the protein active sites cluster around the psi loop, indicating that its rigidity, protrusion and free mainchain functional groups may be well suited to providing a framework for catalysis.

A single mutation in the GSTe2 gene allows tracking of metabolically based insecticide resistance in a major malaria vector

BackgroundMetabolic resistance to insecticides is the biggest threat to the continued effectiveness of malaria vector control. However, its underlying molecular basis, crucial for successful resistance management, remains poorly characterized.ResultsHere, we demonstrate that the single amino acid change L119F in an upregulated glutathione S-transferase gene, GSTe2, confers high levels of metabolic resistance to DDT in the malaria vector Anopheles funestus. Genome-wide transcription analysis revealed that GSTe2 was the most over-expressed detoxification gene in DDT and permethrin-resistant mosquitoes from Benin. Transgenic expression of GSTe2 in Drosophila melanogaster demonstrated that over-transcription of this gene alone confers DDT resistance and cross-resistance to pyrethroids. Analysis of GSTe2 polymorphism established that the point mutation is tightly associated with metabolic resistance to DDT and its geographical distribution strongly correlates with DDT resistance patterns across Africa. Functional characterization of recombinant GSTe2 further supports the role of the L119F mutation, with the resistant allele being more efficient at metabolizing DDT than the susceptible one. Importantly, we also show that GSTe2 directly metabolizes the pyrethroid permethrin. Structural analysis reveals that the mutation confers resistance by enlarging the GSTe2 DDT-binding cavity, leading to increased DDT access and metabolism. Furthermore, we show that GSTe2 is under strong directional selection in resistant populations, and a restriction of gene flow is observed between African regions, enabling the prediction of the future spread of this resistance.ConclusionsThis first DNA-based metabolic resistance marker in mosquitoes provides an essential tool to track the evolution of resistance and to design suitable resistance management strategies.

The PYL4 A194T mutant uncovers a key role of PYR1-LIKE4/PROTEIN PHOSPHATASE 2CA interaction for abscisic acid signaling and plant drought resistance.

Enhanced drought resistance through mutagenesis of an ABA receptor is associated with enhanced interaction with its protein phosphatase binding partner. Because abscisic acid (ABA) is recognized as the critical hormonal regulator of plant stress physiology, elucidating its signaling pathway has raised promise for application in agriculture, for instance through genetic engineering of ABA receptors. PYRABACTIN RESISTANCE1/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS ABA receptors interact with high affinity and inhibit clade A phosphatases type-2C (PP2Cs) in an ABA-dependent manner. We generated an allele library composed of 10,000 mutant clones of Arabidopsis (Arabidopsis thaliana) PYL4 and selected mutations that promoted ABA-independent interaction with PP2CA/ABA-HYPERSENSITIVE3. In vitro protein-protein interaction assays and size exclusion chromatography confirmed that PYL4A194T was able to form stable complexes with PP2CA in the absence of ABA, in contrast to PYL4. This interaction did not lead to significant inhibition of PP2CA in the absence of ABA; however, it improved ABA-dependent inhibition of PP2CA. As a result, 35S:PYL4A194T plants showed enhanced sensitivity to ABA-mediated inhibition of germination and seedling establishment compared with 35S:PYL4 plants. Additionally, at basal endogenous ABA levels, whole-rosette gas exchange measurements revealed reduced stomatal conductance and enhanced water use efficiency compared with nontransformed or 35S:PYL4 plants and partial up-regulation of two ABA-responsive genes. Finally, 35S:PYL4A194T plants showed enhanced drought and dehydration resistance compared with nontransformed or 35S:PYL4 plants. Thus, we describe a novel approach to enhance plant drought resistance through allele library generation and engineering of a PYL4 mutation that enhances interaction with PP2CA.

Tomato PYR/PYL/RCAR abscisic acid receptors show high expression in root, differential sensitivity to the abscisic acid agonist quinabactin, and the capability to enhance plant drought resistance.

Summary Chemical and transgenic approaches can activate ABA signalling via crop PYR/PYL ABA receptors; quinabactin can selectively activate tomato ABA receptors, and overexpression of monomeric-type receptors confers enhanced plant drought resistance.

The first asymmetric synthesis of polyfunctionalized 4H-pyrans via Michael addition of malononitrile to 2-acyl acrylates

Abstract Starting from (R)-2,3-O-isopropylideneglyceraldehyde, the first asymmetric synthesis of 4H-pyrans ( 4 ) has been developed; a detailed X-ray structural and stereochemical study has established as R the absolute configuration at the new stereocenter in compounds 4 .

The X-ray structure of the FMN-binding protein AtHal3 provides the structural basis for the activity of a regulatory subunit involved in signal transduction

BACKGROUND The Arabidopsis thaliana HAL3 gene product encodes for an FMN-binding protein (AtHal3) that is related to plant growth and salt and osmotic tolerance. AtHal3 shows sequence homology to ScHal3, a regulatory subunit of the Saccharomyces cerevisae serine/threonine phosphatase PPz1. It has been proposed that AtHal3 and ScHal3 have similar roles in cellular physiology, as Arabidopsis transgenic plants that overexpress AtHal3 and yeast cells that overexpress ScHal3 display similar phenotypes of improved salt tolerance. The enzymatic activity of AtHal3 has not been investigated. However, the AtHal3 sequence is homologous to that of EpiD, a flavoprotein from Staphylococcus epidermidis that recognizes a peptidic substrate and subsequently catalyzes the alpha, beta-dehydrogenation of its C-terminal cysteine residue. RESULTS The X-ray structure of AtHal3 at 2 A resolution reveals that the biological unit is a trimer. Each protomer adopts an alpha/beta Rossmann fold consisting of a six-stranded parallel beta sheet flanked by two layers of alpha helices. The FMN-binding site of AtHal3 contains all the structural requirements of the flavoenzymes that catalyze dehydrogenation reactions. Comparison of the amino acid sequences of AtHal3, ScHal3 and EpiD reveals that a significant number of residues involved in trimer formation, the active site, and FMN binding are conserved. This observation suggests that ScHal3 and EpiD might also be trimers, having a similar structure and function to AtHal3. CONCLUSIONS Structural comparisons of AtHal3 with other FMN-binding proteins show that AtHal3 defines a new subgroup of this protein family that is involved in signal transduction. Analysis of the structure of AtHal3 indicates that this protein is designed to interact with another cellular component and to subsequently catalyze the alpha,beta-dehydrogenation of a peptidyl cysteine. Structural data from AtHal3, together with physiological and biochemical information from ScHal3 and EpiD, allow us to propose a model for the recognition and regulation of AtHal3/ScHal3 cellular partners.

Development of methods for the synthesis of chiral, highly functionalized 2-amino-4-aryl-4H-pyrans

The development of new methods for the asymmetric synthesis of highly functionalized 2-amino-4-aryl-4 H -pyrans is described. Two alternative synthetic routes: the 1,4-conjugate addition of chiral β-ketoesters 3 or the N -acetoacetyl sultam 11 to arylidenemalononitriles 6 , and the Michael addition of malononitrile to enantiomerically pure α-acetylcinnamates 5 , have been designed. Depending upon the chiral auxiliary, the resulting 4 H -pyrans were obtained in low [(−)-menthol, (−)-borneol, (−)-ethyl lactate] to good (Oppolzers sultam) diastereomeric excesses. The absolute configuration at the new stereocenter in the minor isomer of compound 12a was determined as S by X-ray diffraction analysis. Reductive cleavage of 4 H -pyrans 12 with lithium aluminiun hydride yielded the enantiomerically pure or enriched alcohols 14 .

C2-Domain Abscisic Acid-Related Proteins Mediate the Interaction of PYR/PYL/RCAR Abscisic Acid Receptors with the Plasma Membrane and Regulate Abscisic Acid Sensitivity in Arabidopsis

Subcellular localization of abscisic acid receptors as peripheral proteins in the plasma membrane is mediated in a calcium-dependent manner by C2-domain abscisic acid-related proteins. Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calcium-dependent interactions of PYR/PYL ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL subcellular localization and positively regulates ABA signaling.

Structural Basis of the Regulatory Mechanism of the Plant Cipk Family of Protein Kinases Controlling Ion Homeostasis and Abiotic Stress

Significance The transport of ions through the plant cell membrane establishes the key physicochemical parameters for cell function. Stress situations such as those created by soil salinity or low potassium conditions alter the ion transport across the membrane producing dramatic changes in the cell turgor, the membrane potential, and the intracellular pH and concentrations of toxic cations such as sodium and lithium. As a consequence, fundamental metabolic routes are inhibited. The CIPK family of 26 protein kinases regulates the function of several ion transporters at the cell membrane to restore ion homeostasis under stress situations. Our analyses provide an explanation on how the CIPKs are differentially activated to coordinate the adequate cell response to a particular stress. 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.

The Role of Protein Denaturation Energetics and Molecular Chaperones in the Aggregation and Mistargeting of Mutants Causing Primary Hyperoxaluria Type I

Primary hyperoxaluria type I (PH1) is a conformational disease which result in the loss of alanine:glyoxylate aminotransferase (AGT) function. The study of AGT has important implications for protein folding and trafficking because PH1 mutants may cause protein aggregation and mitochondrial mistargeting. We herein describe a multidisciplinary study aimed to understand the molecular basis of protein aggregation and mistargeting in PH1 by studying twelve AGT variants. Expression studies in cell cultures reveal strong protein folding defects in PH1 causing mutants leading to enhanced aggregation, and in two cases, mitochondrial mistargeting. Immunoprecipitation studies in a cell-free system reveal that most mutants enhance the interactions with Hsc70 chaperones along their folding process, while in vitro binding experiments show no changes in the interaction of folded AGT dimers with the peroxisomal receptor Pex5p. Thermal denaturation studies by calorimetry support that PH1 causing mutants often kinetically destabilize the folded apo-protein through significant changes in the denaturation free energy barrier, whereas coenzyme binding overcomes this destabilization. Modeling of the mutations on a 1.9 Å crystal structure suggests that PH1 causing mutants perturb locally the native structure. Our work support that a misbalance between denaturation energetics and interactions with chaperones underlie aggregation and mistargeting in PH1, suggesting that native state stabilizers and protein homeostasis modulators are potential drugs to restore the complex and delicate balance of AGT protein homeostasis in PH1.