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.

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.

The strawberry pathogenesis-related 10 (PR-10) Fra a proteins control flavonoid biosynthesis by binding to metabolic intermediates.

Pathogenesis-related 10 (PR-10) proteins are involved in many aspects of plant biology but their molecular function is still unclear. They are related by sequence and structural homology to mammalian lipid transport and plant abscisic acid receptor proteins and are predicted to have cavities for ligand binding. Recently, three new members of the PR-10 family, the Fra a proteins, have been identified in strawberry, where they are required for the activity of the flavonoid biosynthesis pathway, which is essential for the development of color and flavor in fruits. Here, we show that Fra a proteins bind natural flavonoids with different selectivity and affinities in the low µm range. The structural analysis of Fra a 1 E and a Fra a 3-catechin complex indicates that loops L3, L5, and L7 surrounding the ligand-binding cavity show significant flexibility in the apo forms but close over the ligand in the Fra a 3-catechin complex. Our findings provide mechanistic insight on the function of Fra a proteins and suggest that PR-10 proteins, which are widespread in plants, may play a role in the control of secondary metabolic pathways by binding to metabolic intermediates.

Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic beta2-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 ß2-microglobulin (ß2m) amyloidogenesis by X-ray crystallography. For this purpose, we selected five single domain antibodies that block the fibrillogenesis of a proteolytic amyloidogenic fragment of ß2m (ΔN6ß2m). The crystal structure of ΔN6ß2m 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 ß-sheet. The ß-strands of this sheet are prone to self-associate and stack perpendicular to the direction of the strands to build large intermolecular ß-sheets that run parallel to the axis of growing oligomers, providing an elongation mechanism by self-templated growth.

X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX.

NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2's requirement for activation.

Purification, crystallization and preliminary X-ray analysis of the strawberry allergens Fra a 1E and Fra a 3 in the presence of catechin.

The strawberry Fra a proteins belong to the pathogenesis-related PR-10 protein family and share a common fold with the Bet v 1 major pollen allergen and the START/PYR/PYL proteins, which are characterized by the presence of a central cavity and are often involved in the binding of a variety of natural compounds. The Fra a proteins play a key role in the control of flavonoid biosynthesis in strawberries and are essential for pigment formation in fruits. In order to understand Fra a protein function, full-length Fra a 1E and Fra a 3 cDNAs were cloned and expressed in Escherichia coli, and the proteins were purified to homogeneity using metal-affinity chromatography. Diffraction-quality crystals of Fra a 1E and of Fra a 3 in the presence of (+)-catechin were obtained by the sitting-drop vapour-diffusion method. X-ray diffraction data from single crystals of Fra a 1E and Fra a 3 were processed to 2.2 and 3.0 Šresolution in space groups P212121 and P2221, with unit-cell parameters a = 70.02, b = 74.42, c = 84.04 Šand a = 137.91, b = 206.61, c = 174.7 Šfor Fra a 1E and Fra a 3, respectively.

Plasticity of the binding pocket in peptide transporters underpins promiscuous substrate recognition.

Proton-dependent oligopeptide transporters (POTs) are promiscuous transporters of the major facilitator superfamily that constitute the main route of entry for a wide range of dietary peptides and orally administrated peptidomimetic drugs. Given their clinical and pathophysiological relevance, several POT homologs have been studied extensively at the structural and molecular level. However, the molecular basis of recognition and transport of diverse peptide substrates has remained elusive. We present 14 X-ray structures of the bacterial POT DtpB in complex with chemically diverse di- and tripeptides, providing novel insights into the plasticity of the conserved central binding cavity. We analyzed binding affinities for more than 80 peptides and monitored uptake by a fluorescence-based transport assay. To probe whether all 8400 natural di- and tripeptides can bind to DtpB, we employed state-of-the-art molecular docking and machine learning and conclude that peptides with compact hydrophobic residues are the best DtpB binders.

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, hab1(W385A), which abolished ABA-dependent receptor-mediated PP2C inhibition without impairing basal PP2C activity. As a result, hab1(W385A) 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 hab1(W385A) qualifies as a hypermorphic mutation. Expression of hab1(W385A) 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.

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.

The Automated Crystallography Pipelines at the EMBL HTX Facility in Grenoble.

EMBL Grenoble operates the High Throughput Crystallization Laboratory (HTX Lab), a large-scale user facility offering high throughput crystallography services to users worldwide. The HTX lab has a strong focus in the development of new methods in macromolecular crystallography. Through the combination of a high throughput crystallization platform, the CrystalDirect technology for fully automated crystal mounting and cryocooling and the CRIMS software we have developed fully automated pipelines for macromolecular crystallography that can be remotely operated over the internet. These include a protein-to-structure pipeline for the determination of new structures, a pipeline for the rapid characterization of protein-ligand complexes in support of medicinal chemistry, and a large-scale, automated fragment screening pipeline enabling evaluation of libraries of over 1000 fragments. Here we describe how to access and use these resources.

An automated platform for structural analysis of membrane proteins through serial crystallography.

Membrane proteins are central to many pathophysiological processes, yet remain very difficult to analyze structurally. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins because of lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperatures. We apply this approach to human integral membrane proteins, which allowed us to identify different conformational states of intramembrane enzyme-product complexes and analyze by molecular dynamics simulations the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting, and serial crystallography, enabling screening of small-molecule libraries with membrane protein crystals grown in meso. This approach brings needed automation to this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins.

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.

Structural Bases for the Allergenicity of Fra a 1.02 in Strawberry Fruits.

Although strawberries are highly appreciated fruits, their intake can induce allergic reactions in atopic patients. These reactions can be due to the patient's previous sensitization to the major birch pollen allergen Bet v 1, by which IgE generated in response to Bet v 1 cross-reacts with the structurally related strawberry Fra a 1 protein family. Fra a 1.02 is the most expressed paralog in ripe strawberries and is highly allergenic. To better understand the molecular mechanisms regulating this allergic response, we have determined the three-dimensional structure of Fra a 1.02 and four site-directed mutants that were designed based on their positions in potential epitopes. Fra a 1.02 and mutants conform to the START fold. We show that the cross-reactivity of all the mutant variants to IgE from patients allergic to Bet v 1 was significantly reduced without altering the conserved structural fold, so that they could potentially be used as hypoallergenic Fra a 1 variants for the generation of vaccines against strawberry allergy in atopic patients.

Live-cell imaging reveals a stable cohesin-chromatin interaction after but not before DNA replication.

Cohesin is a multisubunit protein complex that links sister chromatids from replication until segregation. The lack of obvious cohesin-targeting-specific sequences on DNA, as well as cohesin's molecular arrangement as a large ring, has led to the working hypothesis that cohesin acts as a direct topological linker. To preserve the identity of sister chromatids, such a linkage would need to stably persist throughout the entire S and G2 phases of the cell cycle. Unexpectedly, cohesin binds chromatin already in telophase, and a large fraction dissociates from chromosomes during prophase in a phosphorylation-dependent manner, whereas initiation of anaphase requires proteolytic cleavage of only a small fraction of cohesin. These observations raised the question of how and when cohesin interacts with chromatin during the cell cycle. Here, we report a cell-cycle dependence in the stability of cohesin binding to chromatin. Using photobleaching and quantitative live-cell imaging, we identified several cohesin pools with different chromatin binding stabilities. Although all chromatin bound cohesin dissociated after a mean residence time of less than 25 min before replication, about one-third of cohesin was bound much more stably after S phase and persisted until metaphase, consistent with long-lived links mediating cohesion between sister chromatids.

The crystal structure of the extracellular domain of the inhibitor receptor expressed on myeloid cells IREM-1.

The immune receptors expressed on myeloid cells (IREM) are type I transmembrane proteins encoded on human chromosome 17 (17q25.1), whose function is believed to be important in controlling inflammation. To date, three IREM receptors have been identified. IREM-1 functions as an inhibitory receptor, whereas IREM-2 and IREM-3 serve an activating function. Here, we report the crystal structure of IREM-1 extracellular domain at 2.6 A resolution. The overall fold of IREM-1 resembles that of a V-type immunoglobulin domain, and reveals overall close homology with immunoglobulin domains from other immunoreceptors such as CLM-1, TREM-1, TLT-1 and NKp44. Comparing the surface electrostatic potential and hydrophobicity of IREM-1 with its murine homologous CLM-1, we observed unique structural properties for the complementary determining region of IREM-1, which suggests that they may be involved in recognition of the IREM-1 ligand. Particularly interesting is the structural conformation and physical properties of the antibody's equivalent CDR3 loop, which we show to be a structurally variable region of the molecule and therefore could be the main structural determinant for ligand discrimination and binding. In addition, the analysis of the IREM-1 structure revealed the presence of four structurally different cavities. Three of these cavities form a continuous hydrophobic groove on the IREM-1 surface, which point to a region of the molecule capable of accommodating potential ligands.

Expression, crystallization and X-ray data collection from microcrystals of the extracellular domain of the human inhibitory receptor expressed on myeloid cells IREM-1.

IREM-1 is an inhibitory receptor involved in the functional regulation of myeloid cells. The expression, in vitro folding, purification, crystallization and X-ray data collection of the Ig-V like domain of IREM-1 are reported. X-ray data were collected from a microcrystal (300 x 10 x 10 microm) at 100 K and a diffraction pattern was obtained to 2.6 A resolution on microfocus beamline ID23-2 at the ESRF. The crystal belongs to space group P3(1)21, with unit-cell parameters a = b = 54.23, c = 72.02 A, alpha = gamma = 90, beta = 120 degrees. Assuming the presence of one molecule per asymmetric unit, V(M) (the Matthews coefficient) was calculated to be 1.96 A3 Da(-1) and the solvent content was estimated to be 37.27%. Determination of the IREM-1 structure will provide insights into its structural requirements for ligand discrimination and binding.

Estimation of crystallization likelihood through a fluorimetric thermal stability assay.

Construct design and sample formulation are critical in structural biology projects. Large numbers of sample variants are often produced and analyzed for a single target and significant effort is dedicated to sample characterization in order to identify at an early stage the most promising samples to help save manpower and time. Here, we present a method based on a thermal stability assay that can help estimate the likelihood of biological samples to produce crystals. This assay is rapid, inexpensive and consumes very small amounts of sample. The results can be used to prioritize certain constructs at an early stage or as an objective test to help decide when to undertake other type of approaches addressed at improving sample properties.

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.