Tina Izard

Frequent in-frame somatic deletions activate gp130 in inflammatory hepatocellular tumours.

Inflammatory hepatocellular adenomas are benign liver tumours defined by the presence of inflammatory infiltrates and by the increased expression of inflammatory proteins in tumour hepatocytes. Here we show a marked activation of the interleukin (IL)-6 signalling pathway in this tumour type; sequencing candidate genes pinpointed this response to somatic gain-of-function mutations in the IL6ST gene, which encodes the signalling co-receptor gp130. Indeed, 60% of inflammatory hepatocellular adenomas harbour small in-frame deletions that target the binding site of gp130 for IL-6, and expression of four different gp130 mutants in hepatocellular cells activates signal transducer and activator of transcription 3 (STAT3) in the absence of ligand. Furthermore, analysis of hepatocellular carcinomas revealed that rare gp130 alterations are always accompanied by β-catenin-activating mutations, suggesting a cooperative effect of these signalling pathways in the malignant conversion of hepatocytes. The recurrent gain-of-function gp130 mutations in these human hepatocellular adenomas fully explains activation of the acute inflammatory phase observed in tumourous hepatocytes, and suggests that similar alterations may occur in other inflammatory epithelial tumours with STAT3 activation.

Vinculin activation by talin through helical bundle conversion

Vinculin is a conserved component and an essential regulator of both cell–cell (cadherin-mediated) and cell–matrix (integrin–talin-mediated focal adhesions) junctions, and it anchors these adhesion complexes to the actin cytoskeleton by binding to talin in integrin complexes or to α-actinin in cadherin junctions. In its resting state, vinculin is held in a closed conformation through interactions between its head (Vh) and tail (Vt) domains. The binding of vinculin to focal adhesions requires its association with talin. Here we report the crystal structures of human vinculin in its inactive and talin-activated states. Talin binding induces marked conformational changes in Vh, creating a novel helical bundle structure, and this alteration actively displaces Vt from Vh. These results, as well as the ability of α-actinin to also bind to Vh and displace Vt from pre-existing Vh–Vt complexes, support a model whereby Vh functions as a domain that undergoes marked structural changes that allow vinculin to direct cytoskeletal assembly in focal adhesions and adherens junctions. Notably, talins effects on Vh structure establish helical bundle conversion as a signalling mechanism by which proteins direct cellular responses.

Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas

Somatic STAT3 mutations present in a subset of inflammatory hepatocellular adenomas result in the generation of constitutively active STAT3 proteins that homodimerize independently of IL-6 stimulation.

Mapping the interaction surface of linker histone H1 0 with the nucleosome of native chromatin in vivo

H1 linker histones stabilize the nucleosome, limit nucleosome mobility and facilitate the condensation of metazoan chromatin. Here, we have combined systematic mutagenesis, measurement of in vivo binding by photobleaching microscopy, and structural modeling to determine the binding geometry of the globular domain of the H10 linker histone variant within the nucleosome in unperturbed, native chromatin in vivo. We demonstrate the existence of two distinct DNA-binding sites within the globular domain that are formed by spatial clustering of multiple residues. The globular domain is positioned via interaction of one binding site with the major groove near the nucleosome dyad. The second site interacts with linker DNA adjacent to the nucleosome core. Multiple residues bind cooperatively to form a highly specific chromatosome structure that provides a mechanism by which individual domains of linker histones interact to facilitate chromatin condensation.

The Vinculin Binding Sites of Talin and α-Actinin Are Sufficient to Activate Vinculin

Vinculin regulates both cell-cell and cell-matrix junctions and anchors adhesion complexes to the actin cytoskeleton through its interactions with the vinculin binding sites of α-actinin or talin. Activation of vinculin requires a severing of the intramolecular interactions between its N- and C-terminal domains, which is necessary for vinculin to bind to F-actin; yet how this occurs in cells is not resolved. We tested the hypothesis that talin and α-actinin activate vinculin through their vinculin binding sites. Indeed, we show that these vinculin binding sites have a high affinity for full-length vinculin, are sufficient to sever the head-tail interactions of vinculin, and they induce conformational changes that allow vinculin to bind to F-actin. Finally, microinjection of these vinculin binding sites specifically targets vinculin in cells, disrupting its interactions with talin and α-actinin and disassembling focal adhesions. In their native (inactive) states the vinculin binding sites of talin and α-actinin are buried within helical bundles present in their central rod domains. Collectively, these results support a model where the engagement of adhesion receptors first activates talin or α-actinin, by provoking structural changes that allow their vinculin binding sites to swing out, which are then sufficient to bind to and activate vinculin.

The crystal structure of a novel bacterial adenylyltransferase reveals half of sites reactivity

Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in bacteria that catalyses a rate‐limiting step in coenzyme A (CoA) biosynthesis, by transferring an adenylyl group from ATP to 4′‐phosphopantetheine, yielding dephospho‐CoA (dPCoA). Each phosphopantetheine adenylyltransferase (PPAT) subunit displays a dinucleotide‐binding fold that is structurally similar to that in class I aminoacyl‐tRNA synthetases. Superposition of bound adenylyl moieties from dPCoA in PPAT and ATP in aminoacyl‐tRNA synthetases suggests nucleophilic attack by the 4′‐phosphopantetheine on the α‐phosphate of ATP. The proposed catalytic mechanism implicates transition state stabilization by PPAT without involving functional groups of the enzyme in a chemical sense in the reaction. The crystal structure of the enzyme from Escherichia coli in complex with dPCoA shows that binding at one site causes a vice‐like movement of active site residues lining the active site surface. The mode of enzyme product formation is highly concerted, with only one trimer of the PPAT hexamer showing evidence of dPCoA binding. The homologous active site attachment of ATP and the structural distribution of predicted sequence‐binding motifs in PPAT classify the enzyme as belonging to the nucleotidyltransferase superfamily.

The three-dimensional structure of N -acetylneuraminate lyase from Escherichia coli

BACKGROUND N-acetylneuraminate lyase catalyzes the cleavage of N-acetylneuraminic acid (sialic acid) to form pyruvate and N-acetyl-D-mannosamine. The enzyme plays an important role in the regulation of sialic acid metabolism in bacteria. The reverse reaction can be exploited for the synthesis of sialic acid and some of its derivatives. RESULTS The structure of the enzyme from Escherichia coli has been determined to 2.2 A resolution by X-ray crystallography. The enzyme is shown to be a tetramer, in which each subunit consists of an alpha/beta-barrel domain followed by a carboxy-terminal extension of three alpha-helices. CONCLUSIONS The active site of the enzyme is tentatively identified as a pocket at the carboxy-terminal end of the eight-stranded beta-barrel. Lys165 lies within this pocket and is probably the reactive residue which forms a Schiff base intermediate with the substrate. The sequence of N-acetylneuraminate lyase has similarities to those of dihydrodipicolinate synthase and MosA (an enzyme implicated in rhizopine synthesis) suggesting that these last two enzymes share a similar structure to N-acetylneuraminate lyase.

Structural Dynamics of α-Actinin-Vinculin Interactions

ABSTRACT α-Actinin and vinculin orchestrate reorganization of the actin cytoskeleton following the formation of adhesion junctions. α-Actinin interacts with vinculin through the binding of an α-helix (αVBS) present within the R4 spectrin repeat of its central rod domain to vinculins N-terminal seven-helical bundle domain (Vh1). The Vh1:αVBS structure suggests that αVBS first unravels from its buried location in the triple-helical R4 repeat to allow it to bind to vinculin. αVBS binding then induces novel conformational changes in the N-terminal helical bundle of Vh1, which disrupt its intramolecular association with vinculins tail domain and which differ from the alterations in Vh1 provoked by the binding of talin. Surprisingly, αVBS binds to Vh1 in an inverted orientation compared to the binding of talins VBSs to vinculin. Importantly, the binding of αVBS and talins VBSs to vinculins Vh1 domain appear to also trigger distinct conformational changes in full-length vinculin, opening up distant regions that are buried in the inactive molecule. The data suggest a model where vinculins Vh1 domain acts as a molecular switch that undergoes distinct structural changes provoked by talin and α-actinin binding in focal adhesions versus adherens junctions, respectively.

Crystal structures of the metal-dependent 2-dehydro-3-deoxy-galactarate aldolase suggest a novel reaction mechanism

Carbon–carbon bond formation is an essential reaction in organic chemistry and the use of aldolase enzymes for the stereochemical control of such reactions is an attractive alternative to conventional chemical methods. Here we describe the crystal structures of a novel class II enzyme, 2‐dehydro‐3‐deoxy‐galactarate (DDG) aldolase from Escherichia coli, in the presence and absence of substrate. The crystal structure was determined by locating only four Se sites to obtain phases for 506 protein residues. The protomer displays a modified (α/β)8 barrel fold, in which the eighth α‐helix points away from the β‐barrel instead of packing against it. Analysis of the DDG aldolase crystal structures suggests a novel aldolase mechanism in which a phosphate anion accepts the proton from the methyl group of pyruvate.

The Cytoskeletal Protein α-Catenin Unfurls upon Binding to Vinculin

Background: α-Catenin provides links for cadherin receptors to the actin cytoskeleton at cell-cell adherens junctions. Results: Extensive α-catenin interactions with vinculin are displaced by the vinculin tail domain. Conclusion: α-Catenin-vinculin interactions are stabilized by F-actin. Significance: The data support a new model whereby vinculin activation at adherens junctions is sufficient to stabilize connections of α-catenin with the actin network. Adherens junctions (AJs) are essential for cell-cell contacts, morphogenesis, and the development of all higher eukaryotes. AJs are formed by calcium-dependent homotypic interactions of the ectodomains of single membrane-pass cadherin family receptors. These homotypic interactions in turn promote binding of the intracellular cytoplasmic tail domains of cadherin receptors with β-catenin, a multifunctional protein that plays roles in both transcription and AJs. The cadherin receptor-β-catenin complex binds to the cytoskeletal protein α-catenin, which is essential for both the formation and the stabilization of these junctions. Precisely how α-catenin contributes to the formation and stabilization of AJs is hotly debated, although the latter is thought to involve its interactions with the cytoskeletal protein vinculin. Here we report the crystal structure of the vinculin binding domain (VBD) of α-catenin in complex with the vinculin head domain (Vh1). This structure reveals that α-catenin is in a unique unfurled mode allowing dimer formation when bound to vinculin. Finally, binding studies suggest that vinculin must be in an activated state to bind to α-catenin and that this interaction is stabilized by the formation of a ternary α-catenin-vinculin-F-actin complex, which can be formed via the F-actin binding domain of either protein. We propose a feed-forward model whereby α-catenin-vinculin interactions promote their binding to the actin cytoskeleton to stabilize AJs.