Next-generation ABACUS biosensors reveal cellular ABA dynamics driving root growth at low aerial humidity.

The plant hormone abscisic acid (ABA) accumulates under abiotic stress to recast water relations and development. To overcome a lack of high-resolution sensitive reporters, we developed ABACUS2s-next-generation Förster resonance energy transfer (FRET) biosensors for ABA with high affinity, signal-to-noise ratio and orthogonality-that reveal endogenous ABA patterns in Arabidopsis thaliana. We mapped stress-induced ABA dynamics in high resolution to reveal the cellular basis for local and systemic ABA functions. At reduced foliar humidity, root cells accumulated ABA in the elongation zone, the site of phloem-transported ABA unloading. Phloem ABA and root ABA signalling were both essential to maintain root growth at low humidity. ABA coordinates a root response to foliar stresses, enabling plants to maintain foraging of deeper soil for water uptake.

Fluorescent biosensors illuminating plant hormone research.

Phytohormones act as key regulators of plant growth that coordinate developmental and physiological processes across cells, tissues and organs. As such, their levels and distribution are highly dynamic owing to changes in their biosynthesis, transport, modification and degradation that occur over space and time. Fluorescent biosensors represent ideal tools to track these dynamics with high spatiotemporal resolution in a minimally invasive manner. Substantial progress has been made in generating a diverse set of hormone sensors with recent FRET biosensors for visualising hormone concentrations complementing information provided by transcriptional, translational and degron-based reporters. In this review, we provide an update on fluorescent biosensor designs, examine the key properties that constitute an ideal hormone biosensor, discuss the use of these sensors in conjunction with in vivo hormone perturbations and highlight the latest discoveries made using these tools.

Achieving In Vivo Target Depletion through the Discovery and Optimization of Benzimidazolone BCL6 Degraders.

Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.

Affinity of a galactose-specific legume lectin from Dolichos lablab to adenine revealed by X-ray cystallography.

Crystal structure analysis of a galactose-specific lectin from a leguminous food crop Dolichos lablab (Indian lablab beans) has been carried out to obtain insights into its quaternary association and lectin-carbohydrate interactions. The analysis led to the identification of adenine binding sites at the dimeric interfaces of the heterotetrameric lectin. Structural details of similar adenine binding were reported in only one legume lectin, Dolichos biflorus, before this study. Here, we present the structure of the galactose-binding D. lablab lectin at different pH values in the native form and in complex with galactose and adenine. This first structure report on this lectin also provides a high resolution atomic view of legume lectin-adenine interactions. The tetramer has two canonical and two DB58-like interfaces. The binding of adenine, a non-carbohydrate ligand, is found to occur at four hydrophobic sites at the core of the tetramer at the DB58-like dimeric interfaces and does not interfere with the carbohydrate-binding site. To support the crystallographic observations, the adenine binding was further quantified by carrying out isothermal calorimetric titration. By this method, we not only estimated the affinity of the lectin to adenine but also showed that adenine binds with negative cooperativity in solution.

Crystal structure of a ß-prism II lectin from Remusatia vivipara.

The crystal structure of a ß-prism II (BP2) fold lectin from Remusatia vivipara, a plant of traditional medicinal value, has been determined at a resolution of 2.4 Å. This lectin (RVL, Remusatia vivipara lectin) is a dimer with each protomer having two distinct BP2 domains without a linker between them. It belongs to the "monocot mannose-binding" lectin family, which consists of proteins of high sequence and structural similarity. Though the overall tertiary structure is similar to that of lectins from snowdrop bulbs and garlic, crucial differences in the mannose-binding regions and oligomerization were observed. Unlike most of the other structurally known proteins in this family, only one of the three carbohydrate recognition sites (CRSs) per BP2 domain is found to be conserved. RVL does not recognize simple mannose moieties. RVL binds to only N-linked complex glycans like those present on the gp120 envelope glycoprotein of HIV and mannosylated blood proteins like fetuin, but not to simple mannose moieties. The molecular basis for these features and their possible functional implications to understand the different levels of carbohydrate affinities in this structural family have been investigated through structure analysis, modeling and binding studies. Apart from being the first structure of a lectin to be reported from the Araceae/Arum family, this protein also displays a novel mode of oligomerization among BP2 lectins.

Modification of the sugar specificity of a plant lectin: structural studies on a point mutant of Erythrina corallodendron lectin.

A mutant of Erythrina corallodendron lectin was generated with the aim of enhancing its affinity for N-acetylgalactosamine. A tyrosine residue close to the binding site of the lectin was mutated to a glycine in order to facilitate stronger interactions between the acetamido group of the sugar and the lectin which were prevented by the side chain of the tyrosine in the wild-type lectin. The crystal structures of this Y106G mutant lectin in complex with galactose and N-acetylgalactosamine have been determined. A structural rationale has been provided for the differences in the relative binding affinities of the wild-type and mutant lectins towards the two sugars based on the structures. A hydrogen bond between the O6 atom of the sugars and the variable loop of the carbohydrate-binding site of the lectin is lost in the mutant complexes owing to a conformational change in the loop. This loss is compensated by an additional hydrogen bond that is formed between the acetamido group of the sugar and the mutant lectin in the complex with N-acetylgalactosamine, resulting in a higher affinity of the mutant lectin for N-acetylgalactosamine compared with that for galactose, in contrast to the almost equal affinity of the wild-type lectin for the two sugars. The structure of a complex of the mutant with a citrate ion bound at the carbohydrate-binding site that was obtained while attempting to crystallize the complexes with sugars is also presented.

Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity.

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80 degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 microg/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.