Structural insights and activating mutations in diverse pathologies define mechanisms of deregulation for phospholipase C gamma enzymes.

BACKGROUND: PLCγ enzymes are key nodes in cellular signal transduction and their mutated and rare variants have been recently implicated in development of a range of diseases with unmet need including cancer, complex immune disorders, inflammation and neurodegenerative diseases. However, molecular nature of activation and the impact and dysregulation mechanisms by mutations, remain unclear; both are critically dependent on comprehensive characterization of the intact PLCγ enzymes. METHODS: For structural studies we applied cryo-EM, cross-linking mass spectrometry and hydrogen-deuterium exchange mass spectrometry. In parallel, we compiled mutations linked to main pathologies, established their distribution and assessed their impact in cells and in vitro. FINDINGS: We define structure of a complex containing an intact, autoinhibited PLCγ1 and the intracellular part of FGFR1 and show that the interaction is centred on the nSH2 domain of PLCγ1. We define the architecture of PLCγ1 where an autoinhibitory interface involves the cSH2, spPH, TIM-barrel and C2 domains; this relative orientation occludes PLCγ1 access to its substrate. Based on this framework and functional characterization, the mechanism leading to an increase in PLCγ1 activity for the largest group of mutations is consistent with the major, direct impact on the autoinhibitory interface. INTERPRETATION: We reveal features of PLCγ enzymes that are important for determining their activation status. Targeting such features, as an alternative to targeting the PLC active site that has so far not been achieved for any PLC, could provide new routes for clinical interventions related to various pathologies driven by PLCγ deregulation. FUND: CR UK, MRC and AstaZeneca.

Cryo-EM structure of Saccharomyces cerevisiae target of rapamycin complex 2.

The target of rapamycin (TOR) kinase assembles into two distinct multiprotein complexes, conserved across eukaryote evolution. In contrast to TOR complex 1 (TORC1), TORC2 kinase activity is not inhibited by the macrolide rapamycin. Here, we present the structure of Saccharomyces cerevisiae TORC2 determined by electron cryo-microscopy. TORC2 contains six subunits assembling into a 1.4 MDa rhombohedron. Tor2 and Lst8 form the common core of both TOR complexes. Avo3/Rictor is unique to TORC2, but interacts with the same HEAT repeats of Tor2 that are engaged by Kog1/Raptor in mammalian TORC1, explaining the mutual exclusivity of these two proteins. Density, which we conclude is Avo3, occludes the FKBP12-rapamycin-binding site of Tor2's FRB domain rendering TORC2 rapamycin insensitive and recessing the kinase active site. Although mobile, Avo1/hSin1 further restricts access to the active site as its conserved-region-in-the-middle (CRIM) domain is positioned along an edge of the TORC2 active-site-cleft, consistent with a role for CRIM in substrate recruitment.

Molecular Basis of the Rapamycin Insensitivity of Target Of Rapamycin Complex 2.

Target of Rapamycin (TOR) plays central roles in the regulation of eukaryote growth as the hub of two essential multiprotein complexes: TORC1, which is rapamycin-sensitive, and the lesser characterized TORC2, which is not. TORC2 is a key regulator of lipid biosynthesis and Akt-mediated survival signaling. In spite of its importance, its structure and the molecular basis of its rapamycin insensitivity are unknown. Using crosslinking-mass spectrometry and electron microscopy, we determined the architecture of TORC2. TORC2 displays a rhomboid shape with pseudo-2-fold symmetry and a prominent central cavity. Our data indicate that the C-terminal part of Avo3, a subunit unique to TORC2, is close to the FKBP12-rapamycin-binding domain of Tor2. Removal of this sequence generated a FKBP12-rapamycin-sensitive TORC2 variant, which provides a powerful tool for deciphering TORC2 function in vivo. Using this variant, we demonstrate a role for TORC2 in G2/M cell-cycle progression.

Structural origins of AGC protein kinase inhibitor selectivities: PKA as a drug discovery tool.

The era of structure-based protein kinase inhibitor design began in the early 1990s with the determination of crystal structures of protein kinase A (PKA, or cyclic AMP-dependent kinase). Although many other protein kinases have since been extensively characterized, PKA remains a prototype for studies of protein kinase active conformations. It serves well as a model for the structural properties of AGC subfamily protein kinases, clarifying inhibitor selectivity profiles. Its reliable expression, constitutive activity, simple domain structure, and reproducible crystallizability have also made it a useful surrogate for the discovery of inhibitors of both established and emerging AGC kinase targets.

VX680 binding in Aurora A: π-π interactions involving the conserved aromatic amino acid of the flexible glycine-rich loop.

The regulation of protein kinases requires flexibility, especially near the ATP binding site. The cancer drug target Aurora A is inhibited by the ATP site inhibitor VX680, and published crystal structures show two distinct conformations. In one, a refolded glycine-rich loop creates a stacked π-π interaction between the conserved aromatic residue of the glycine-rich loop hairpin turn (F144) and the inhibitor. This refolding, associated with binding to a peptide derived from the cofactor TPX2, is absent in the other structure. We use surface plasmon resonance to measure VX680 binding to native and mutant F144A Aurora A kinase domains, with and without the TPX2 peptide. Results show that the F144 aromatic side chain contributes 2 kcal/mol to the VX680 binding energy, independent of the TPX2 peptide. This indicates that distinct VX680 bound conformations of Aurora A cannot be simply correlated with TPX2 binding and that Aurora A retains flexibility when inhibitor-bound. Molecular dynamics simulations show that alternate geometries for the π-π interactions are feasible in the absence of the rigidifying packing interactions seen in the crystal lattice.

Structural basis for both pro- and anti-inflammatory response induced by mannose-specific legume lectin from Cymbosema roseum.

Legume lectins, despite high sequence homology, express diverse biological activities that vary in potency and efficacy. In studies reported here, the mannose-specific lectin from Cymbosema roseum (CRLI), which binds N-glycoproteins, shows both pro-inflammatory effects when administered by local injection and anti-inflammatory effects when by systemic injection. Protein sequencing was obtained by Tandem Mass Spectrometry and the crystal structure was solved by X-ray crystallography using a Synchrotron radiation source. Molecular replacement and refinement were performed using CCP4 and the carbohydrate binding properties were described by affinity assays and computational docking. Biological assays were performed in order to evaluate the lectin edematogenic activity. The crystal structure of CRLI was established to a 1.8Å resolution in order to determine a structural basis for these differing activities. The structure of CRLI is closely homologous to those of other legume lectins at the monomer level and assembles into tetramers as do many of its homologues. The CRLI carbohydrate binding site was predicted by docking with a specific inhibitory trisaccharide. CRLI possesses a hydrophobic pocket for the binding of α-aminobutyric acid and that pocket is occupied in this structure as are the binding sites for calcium and manganese cations characteristic of legume lectins. CRLI route-dependent effects for acute inflammation are related to its carbohydrate binding domain (due to inhibition caused by the presence of α-methyl-mannoside), and are based on comparative analysis with ConA crystal structure. This may be due to carbohydrate binding site design, which differs at Tyr12 and Glu205 position.

Purification and molecular cloning of a new galactose-specific lectin from Bauhinia variegata seeds.

A new galactose-specific lectin was purified from seeds of a Caesalpinoideae plant, Bauhinia variegata, by affinity chromatography on lactose-agarose. Protein extracts haemagglutinated rabbit and human erythrocytes (native and treated with proteolytic enzymes), showing preference for rabbit blood treated with papain and trypsin. Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives, especially lactose. SDS-PAGE showed that the lectin, named BVL, has a pattern similar to other lectins isolated from the same genus, Bauhinia purpurea agglutinin (BPA). The molecular mass of BVL subunit is 32 871 Da, determined by MALDI-TOF spectrometry. DNA extracted from B.variegata young leaves and primers designed according to the B. purpurea lectin were used to generate specific fragments which were cloned and sequenced, revealing two distinct isoforms. The bvl gene sequence comprised an open reading frame of 876 base pairs which encodes a protein of 291 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to have 263 amino acid residues and 28 963 Da in size.

Correlation between Enterococcus faecalis biofilms development stage and quantitative surface roughness using atomic force microscopy.

Biofilms are assemblages of microorganisms and their associated extracellular products at an interface and typically with an abiotic or biotic surface. The study of the morphology of biofilms is important because they are associated with processes of biofouling, corrosion, catalysis, pollutant transformation, dental caries, drug resistance, and so forth. In the literature, biofilms have been examined by atomic force microscopy (AFM), which has proven to be a potent tool to study different aspects of the biofilm development on solid surfaces. In this work, we used AFM to investigate topographical changes during the development process of Enterococcus faecalis biofilms, which were generated on sterile cellulose nitrate membrane (CNM) filters in brain heart infusion (BHI) broth agar blood plates after 24, 36, 72, 192, and 360 h. AFM height images showed topographical changes due to biofilm development, which were used to characterize several aspects of the bacterial surface, such as the presence of extracellular polymeric substance, and the biofilm development stage. Changes in the development stage of the biofilm were shown to correlate with changes in the surface roughness as quantified through the mean roughness.

Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules.

BACKGROUND: Lectins are mainly described as simple carbohydrate-binding proteins. Previous studies have tried to identify other binding sites, which possible recognize plant hormones, secondary metabolites, and isolated amino acid residues. We report the crystal structure of a lectin isolated from Canavalia gladiata seeds (CGL), describing a new binding pocket, which may be related to pathogen resistance activity in ConA-like lectins; a site where a non-protein amino-acid, alpha-aminobutyric acid (Abu), is bound. RESULTS: The overall structure of native CGL and complexed with alpha-methyl-mannoside and Abu have been refined at 2.3 A and 2.31 A resolution, respectively. Analysis of the electron density maps of the CGL structure shows clearly the presence of Abu, which was confirmed by mass spectrometry. CONCLUSION: The presence of Abu in a plant lectin structure strongly indicates the ability of lectins on carrying secondary metabolites. Comparison of the amino acids composing the site with other legume lectins revealed that this site is conserved, providing an evidence of the biological relevance of this site. This new action of lectins strengthens their role in defense mechanisms in plants.