the-automated-crystallography-pipelines-at-embl-htx-facility

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 ...

European Molecular Biology Laboratory

Institut de Biologie Structurale

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.

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

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.

The crystal structure of the extracellular domain of the inhibitor 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.

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

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.

Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic beta2-microglobulin variant.

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.

Modulation of abscisic acid signaling in vivo by an engineered receptor-insensitive protein phosphatase type 2C allele.

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.

A thermodynamic switch modulates abscisic acid receptor sensitivity.

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.

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

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 Å resolution in space groups P212121 and P2221, with unit-cell parameters a = 70.02, b = 74.42, c = 84.04 Å and a = 137.91, b = 206.61, c = 174.7 Å for Fra a 1E and Fra a 3, respectively.

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

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. 

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

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. 

Estimation of crystallization likelihood through a fluorimetric thermal stability assay.

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.

Florine Dupeux

European Molecular Biology Laboratory

The Automated Crystallography Pipelines at the EMBL HTX Facility in Grenoble