José A. Márquez

The abscisic acid receptor PYR1 in complex with abscisic acid

The plant hormone abscisic acid (ABA) has a central role in coordinating the adaptive response in situations of decreased water availability as well as the regulation of plant growth and development. Recently, a 14-member family of intracellular ABA receptors, named PYR/PYL/RCAR, has been identified. These proteins inhibit in an ABA-dependent manner the activity of a family of key negative regulators of the ABA signalling pathway: the group-A protein phosphatases type 2C (PP2Cs). Here we present the crystal structure of Arabidopsis thaliana PYR1, which consists of a dimer in which one of the subunits is bound to ABA. In the ligand-bound subunit, the loops surrounding the entry to the binding cavity fold over the ABA molecule, enclosing it inside, whereas in the empty subunit they form a channel leaving an open access to the cavity, indicating that conformational changes in these loops have a critical role in the stabilization of the hormone–receptor complex. By providing structural details on the ABA-binding pocket, this work paves the way for the development of new small molecules able to activate the plant stress response.

Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs

Abscisic acid (ABA) is a key phytohormone involved in adaption to environmental stress and regulation of plant development. Clade A protein phosphatases type 2C (PP2Cs), such as HAB1, are key negative regulators of ABA signaling in Arabidopsis. To obtain further insight into regulation of HAB1 function by ABA, we have screened for HAB1-interacting partners using a yeast two-hybrid approach. Three proteins were identified, PYL5, PYL6 and PYL8, which belong to a 14-member subfamily of the Bet v1-like superfamily. HAB1-PYL5 interaction was confirmed using BiFC and co-immunoprecipitation assays. PYL5 over-expression led to a globally enhanced response to ABA, in contrast to the opposite phenotype reported for HAB1-over-expressing plants. F(2) plants that over-expressed both HAB1 and PYL5 showed an enhanced response to ABA, indicating that PYL5 antagonizes HAB1 function. PYL5 and other members of its protein family inhibited HAB1, ABI1 and ABI2 phosphatase activity in an ABA-dependent manner. Isothermal titration calorimetry revealed saturable binding of (+)ABA to PYL5, with K(d) values of 1.1 mum or 38 nm in the absence or presence of the PP2C catalytic core of HAB1, respectively. Our work indicates that PYL5 is a cytosolic and nuclear ABA receptor that activates ABA signaling through direct inhibition of clade A PP2Cs. Moreover, we show that enhanced resistance to drought can be obtained through PYL5-mediated inhibition of clade A PP2Cs.

A thermodynamic switch modulates abscisic acid receptor sensitivity

Abscisic acid (ABA) is a key hormone regulating plant growth, development and the response to biotic and abiotic stress. ABA binding to pyrabactin resistance (PYR)/PYR1‐like (PYL)/Regulatory Component of Abscisic acid Receptor (RCAR) intracellular receptors promotes the formation of stable complexes with certain protein phosphatases type 2C (PP2Cs), leading to the activation of ABA signalling. The PYR/PYL/RCAR family contains 14 genes in Arabidopsis and is currently the largest plant hormone receptor family known; however, it is unclear what functional differentiation exists among receptors. Here, we identify two distinct classes of receptors, dimeric and monomeric, with different intrinsic affinities for ABA and whose differential properties are determined by the oligomeric state of their apo forms. Moreover, we find a residue in PYR1, H60, that is variable between family members and plays a key role in determining oligomeric state. In silico modelling of the ABA activation pathway reveals that monomeric receptors have a competitive advantage for binding to ABA and PP2Cs. This work illustrates how receptor oligomerization can modulate hormonal responses and more generally, the sensitivity of a ligand‐dependent signalling system.

Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic β2-microglobulin variant

Atomic-level structural investigation of the key conformational intermediates of amyloidogenesis remains a challenge. Here we demonstrate the utility of nanobodies to trap and characterize intermediates of β2-microglobulin (β2m) amyloidogenesis by X-ray crystallography. For this purpose, we selected five single domain antibodies that block the fibrillogenesis of a proteolytic amyloidogenic fragment of β2m (ΔN6β2m). The crystal structure of ΔN6β2m in complex with one of these nanobodies (Nb24) identifies domain swapping as a plausible mechanism of self-association of this amyloidogenic protein. In the swapped dimer, two extended hinge loops—corresponding to the heptapetide NHVTLSQ that forms amyloid in isolation—are unmasked and fold into a new two-stranded antiparallel β-sheet. The β-strands of this sheet are prone to self-associate and stack perpendicular to the direction of the strands to build large intermolecular β-sheets that run parallel to the axis of growing oligomers, providing an elongation mechanism by self-templated growth.

The Ssn6–Tup1 repressor complex of Saccharomyces cerevisiae is involved in the osmotic induction of HOG‐dependent and ‐independent genes

The response of yeast to osmotic stress has been proposed to rely on the HOG–MAP kinase signalling pathway and on transcriptional activation mediated by STRE promoter elements. However, the osmotic induction of HAL1, an important determinant of salt tolerance, is HOG independent and occurs through the release of transcriptional repression. We have identified an upstream repressing sequence in HAL1 promoter (URSHAL1) located between −231 and −156. This promoter region was able to repress transcription from a heterologous promoter and to bind proteins in non‐stressed cells, but not in salt‐treated cells. The repression conferred by URSHAL1 is mediated through the Ssn6–Tup1 protein complex and is abolished in the presence of osmotic stress. The Ssn6–Tup1 co‐repressor is also involved in the regulation of HOG‐dependent genes such as GPD1, CTT1, ALD2, ENA1 and SIP18, and its deletion can suppress the osmotic sensitivity of hog1 mutants. We propose that the Ssn6–Tup1 repressor complex might be a general component in the regulation of osmostress responses at the transcriptional level of both HOG‐dependent and ‐independent genes.

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X41–157-Cernunnos1–224 complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X41–157 and Cernunnos1–224 homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4–Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4–Cernunnos interface. We identified four X4 residues (Glu55, Asp58, Met61, and Phe106) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.

A thermal stability assay can help to estimate the crystallization likelihood of biological samples.

The identification of crystallization conditions for biological molecules largely relies on a trial-and-error process in which a number of parameters are explored in large screening experiments. Currently, construct design and sample formulation are recognized as critical variables in this process and often a number of protein variants are assayed for crystallization either sequentially or in parallel, which adds complexity to the screening process. Significant effort is dedicated to sample characterization and quality-control experiments in order to identify at an early stage and prioritize those samples which would be more likely to crystallize. However, large-scale studies relating crystallization success to sample properties are generally lacking. In this study, the thermal stability of 657 samples was estimated using a simplified Thermofluor assay. These samples were also subjected to automated vapour-diffusion crystallization screening under a constant protocol. Analysis of the data shows that samples with an apparent melting temperature (T(m)) of 318 K or higher crystallized in 49% of cases, while the crystallization success rate decreased rapidly for samples with lower T(m). Only 23% of samples with a T(m) below 316 K produced crystals. Based on this analysis, a simple method for estimation of the crystallization likelihood of biological samples is proposed. This method is easy, rapid and consumes very small amounts of sample. The results of this assay can be used to determine optimal incubation temperatures for crystallization experiments or to prioritize certain constructs. More generally, this work provides an objective test that can contribute to making decisions in both focused and structural genomics crystallography projects.

Modulation of Abscisic Acid Signaling in Vivo by an Engineered Receptor-Insensitive Protein Phosphatase Type 2C Allele

The plant hormone abscisic acid (ABA) plays a crucial role in the control of the stress response and the regulation of plant growth and development. ABA binding to PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS intracellular receptors leads to inhibition of key negative regulators of ABA signaling, i.e. clade A protein phosphatases type 2C (PP2Cs) such as ABA-INSENSITIVE1 and HYPERSENSITIVE TO ABA1 (HAB1), causing the activation of the ABA signaling pathway. To gain further understanding on the mechanism of hormone perception, PP2C inhibition, and its implications for ABA signaling, we have performed a structural and functional analysis of the PYR1-ABA-HAB1 complex. Based on structural data, we generated a gain-of-function mutation in a critical residue of the phosphatase, hab1W385A, which abolished ABA-dependent receptor-mediated PP2C inhibition without impairing basal PP2C activity. As a result, hab1W385A caused constitutive inactivation of the protein kinase OST1 even in the presence of ABA and PYR/PYL proteins, in contrast to the receptor-sensitive HAB1, and therefore hab1W385A qualifies as a hypermorphic mutation. Expression of hab1W385A in Arabidopsis (Arabidopsis thaliana) plants leads to a strong, dominant ABA insensitivity, which demonstrates that this conserved tryptophan residue can be targeted for the generation of dominant clade A PP2C alleles. Moreover, our data highlight the critical role of molecular interactions mediated by tryptophan-385 equivalent residues for clade A PP2C function in vivo and the mechanism of ABA perception and signaling.

Structural insights into PYR/PYL/RCAR ABA receptors and PP2Cs

Abscisic acid (ABA) plays an essential function in plant physiology since it is required for biotic and abiotic stress responses as well as control of plant growth and development. A new family of soluble ABA receptors, named PYR/PYL/RCAR, has emerged as ABA sensors able to inhibit the activity of specific protein phosphatases type-2C (PP2Cs) in an ABA-dependent manner. The structural and functional mechanism by which ABA is perceived by these receptors and consequently leads to inhibition of the PP2Cs has been recently elucidated. The module PYR/PYL/RCAR-ABA-PP2C offers an elegant and unprecedented mechanism to control phosphorylation signaling cascades in a ligand-dependent manner. The knowledge of their three-dimensional structures paves the way to the design of ABA agonists able to modulate the plant stress response.

Conformation of full‐length Bruton tyrosine kinase (Btk) from synchrotron X‐ray solution scattering

Brutonss tyrosine kinase (Btk) is a non‐receptor protein tyrosine kinase (nrPTK) essential for the development of B lymphocytes in humans and mice. Like Src and Abl PTKs, Btk contains a conserved cassette formed by SH3, SH2 and protein kinase domains, but differs from them by the presence of an N‐terminal PH domain and the Tec homology region. The domain structure of Btk was analysed using X‐ray synchrotron radiation scattering in solution. Low resolution shapes of the full‐length protein and several deletion mutants determined ab initio from the scattering data indicated a linear arrangement of domains. This arrangement was further confirmed by rigid body modelling using known high resolution structures of individual domains. The final model of Btk displays an extended conformation with no, or little, inter‐domain interactions. In agreement with these results, deletion of non‐catalytic domains failed to enhance the activity of Btk. Taken together, our results indicate that, contrary to Src and Abl, Btk might not require an assembled conformation for the regulation of its activity.