Armelle Vigouroux

Conformational change and protein–protein interactions of the fusion protein of Semliki Forest virus

Fusion of biological membranes is mediated by specific lipid-interacting proteins that induce the formation and expansion of an initial fusion pore. Here we report the crystal structure of the ectodomain of the Semliki Forest virus fusion glycoprotein E1 in its low-pH-induced trimeric form. E1 adopts a folded-back conformation that, in the final post-fusion form of the full-length protein, would bring the fusion peptide loop and the transmembrane anchor to the same end of a stable protein rod. The observed conformation of the fusion peptide loop is compatible with interactions only with the outer leaflet of the lipid bilayer. Crystal contacts between fusion peptide loops of adjacent E1 trimers, together with electron microscopy observations, suggest that in an early step of membrane fusion, an intermediate assembly of five trimers creates two opposing nipple-like deformations in the viral and target membranes, leading to formation of the fusion pore.

The Pih1-Tah1 Cochaperone Complex Inhibits Hsp90 Molecular Chaperone ATPase Activity

Hsp90 (heat shock protein 90) is an ATP-dependent molecular chaperone regulated by collaborating proteins called cochaperones. This machinery is involved in the conformational activation of client proteins like signaling kinases, transcription factors, or ribonucleoproteins (RNP) such as telomerase. TPR (TetratricoPeptide Repeat)-containing protein associated with Hsp90 (Tah1) and protein interacting with Hsp90 (Pih1) have been identified in Saccharomyces cerevisiae as two Hsp90 cochaperones involved in chromatin remodeling complexes and small nucleolar RNP maturation. Tah1 possesses a minimal TPR domain and binds specifically to the Hsp90 C terminus, whereas Pih1 displays no homology to other protein motifs and has been involved in core RNP protein interaction. While Pih1 alone was unstable and was degraded from its N terminus, we showed that Pih1 and Tah1 form a stable heterodimeric complex that regulates Hsp90 ATPase activity. We used different biophysical approaches such as analytical ultracentrifugation, microcalorimetry, and noncovalent mass spectrometry to characterize the Pih1-Tah1 complex and its interaction with Hsp90. We showed that the Pih1-Tah1 heterodimer binds to Hsp90 with a similar affinity and the same stoichiometry as Tah1 alone. However, the Pih1-Tah1 complex antagonizes Tah1 activity on Hsp90 and inhibits the chaperone ATPase activity. We further identified the region within Pih1 responsible for interaction with Tah1 and inhibition of Hsp90, allowing us to suggest an interaction model for the Pih1-Tah1/Hsp90 complex. These results, together with previous reports, suggest a role for the Pih1-Tah1 cochaperone complex in the recruitment of client proteins such as core RNP proteins to Hsp90.

Central ions and lateral asparagine/glutamine zippers stabilize the post-fusion hairpin conformation of the SARS coronavirus spike glycoprotein☆

Abstract The coronavirus spike glycoprotein is a class I membrane fusion protein with two characteristic heptad repeat regions (HR1 and HR2) in its ectodomain. Here, we report the X-ray structure of a previously characterized HR1/HR2 complex of the severe acute respiratory syndrome coronavirus spike protein. As expected, the HR1 and HR2 segments are organized in antiparallel orientations within a rod-like molecule. The HR1 helices form an exceptionally long (120 Å) internal coiled coil stabilized by hydrophobic and polar interactions. A striking arrangement of conserved asparagine and glutamine residues of HR1 propagates from two central chloride ions, providing hydrogen-bonding “zippers” that strongly constrain the path of the HR2 main chain, forcing it to adopt an extended conformation at either end of a short HR2 α-helix.

Identification of structural and molecular determinants of the tyrosine-kinase Wzc and implications in capsular polysaccharide export

Capsular polysaccharides are well‐established virulence factors of pathogenic bacteria. Their biosynthesis and export are regulated within the transmembrane polysaccharide assembly machinery by the autophosphorylation of atypical tyrosine‐kinases, named BY‐kinases. However, the accurate functioning of these tyrosine‐kinases remains unknown. Here, we report the crystal structure of the non‐phosphorylated cytoplasmic domain of the tyrosine‐kinase Wzc from Escherichia coli in complex with ADP showing that it forms a ring‐shaped octamer. Mutational analysis demonstrates that a conserved EX2RX2R motif involved in subunit interactions is essential for polysaccharide export. We also elucidate the role of a putative internal regulatory tyrosine and we show that BY‐kinases from proteobacteria autophosphorylate on their C‐terminal tyrosine cluster via a single‐step intermolecular mechanism. This structure‐function analysis also allows us to demonstrate that two different parts of a conserved basic region called the RK‐cluster are essential for polysaccharide export and for kinase activity respectively. Based on these data, we revisit the dichotomy made between BY‐kinases from proteobacteria and firmicutes and we propose a unique process of oligomerization and phosphorylation. We also reassess the function of BY‐kinases in the capsular polysaccharide assembly machinery.

Compounds Triggering ER Stress Exert Anti-Melanoma Effects and Overcome BRAF Inhibitor Resistance

We have discovered and developed a series of molecules (thiazole benzenesulfonamides). HA15, the lead compound of this series, displayed anti-cancerous activity on all melanoma cells tested, including cells isolated from patients and cells that developed resistance to BRAF inhibitors. Our molecule displayed activity against other liquid and solid tumors. HA15 also exhibited strong efficacy in xenograft mouse models with melanoma cells either sensitive or resistant to BRAF inhibitors. Transcriptomic, proteomic, and biochemical studies identified the chaperone BiP/GRP78/HSPA5 as the specific target of HA15 and demonstrated that the interaction increases ER stress, leading to melanoma cell death by concomitant induction of autophagic and apoptotic mechanisms.

Purification and crystallization reveal two types of interactions of the fusion protein homotrimer of Semliki Forest virus

ABSTRACT The fusion proteins of the alphaviruses and flaviviruses have a similar native structure and convert to a highly stable homotrimer conformation during the fusion of the viral and target membranes. The properties of the alpha- and flavivirus fusion proteins distinguish them from the class I viral fusion proteins, such as influenza virus hemagglutinin, and establish them as the first members of the class II fusion proteins. Understanding how this new class carries out membrane fusion will require analysis of the structural basis for both the interaction of the protein subunits within the homotrimer and their interaction with the viral and target membranes. To this end we report a purification method for the E1 ectodomain homotrimer from the alphavirus Semliki Forest virus. The purified protein is trimeric, detergent soluble, retains the characteristic stability of the starting homotrimer, and is free of lipid and other contaminants. In contrast to the postfusion structures that have been determined for the class I proteins, the E1 homotrimer contains the fusion peptide region responsible for interaction with target membranes. This E1 trimer preparation is an excellent candidate for structural studies of the class II viral fusion proteins, and we report conditions that generate three-dimensional crystals suitable for analysis by X-ray diffraction. Determination of the structure will provide our first high-resolution views of both the low-pH-induced trimeric conformation and the target membrane-interacting region of the alphavirus fusion protein.

A conserved mechanism of GABA binding and antagonism is revealed by structure-function analysis of the periplasmic binding protein Atu2422 in Agrobacterium tumefaciens.

Bacterial periplasmic binding proteins (PBPs) and eukaryotic PBP-like domains (also called as Venus flytrap modules) of G-protein-coupled receptors are involved in extracellular GABA perception. We investigated the structural and functional basis of ligand specificity of the PBP Atu2422, which is implicated in virulence and transport of GABA in the plant pathogen Agrobacterium tumefaciens. Five high-resolution x-ray structures of Atu2422 liganded to GABA, Pro, Ala, and Val and of point mutant Atu2422-F77A liganded to Leu were determined. Structural analysis of the ligand-binding site revealed two essential residues, Phe77 and Tyr275, the implication of which in GABA signaling and virulence was confirmed using A. tumefaciens cells expressing corresponding Atu2422 mutants. Phe77 restricts ligand specificity to α-amino acids with a short lateral chain, which act as antagonists of GABA signaling in A. tumefaciens. Tyr275 specifically interacts with the GABA γ-amino group. Conservation of these two key residues in proteins phylogenetically related to Atu2422 brought to light a subfamily of PBPs in which all members could bind GABA and short α-amino acids. This work led to the identification of a fingerprint sequence and structural features for defining PBPs that bind GABA and its competitors and revealed their occurrence among host-interacting proteobacteria.

Crystal structures of A. acidocaldarius endoglucanase Cel9A in complex with cello-oligosaccharides: strong -1 and -2 subsites mimic cellobiohydrolase activity.

Alicyclobacillus acidocaldarius endoglucanase Cel9A (AaCel9A) is an inverting glycoside hydrolase with beta-1,4-glucanase activity on soluble polymeric substrates. Here, we report three X-ray structures of AaCel9A: a ligand-free structure at 1.8 A resolution and two complexes at 2.66 and 2.1 A resolution, respectively, with cellobiose obtained by co-crystallization and with cellotetraose obtained by the soaking method. AaCel9A forms an (alpha/alpha)(6)-barrel like other glycoside hydrolase family 9 enzymes. When cellobiose is used as a ligand, three glucosyl binding subsites are occupied, including two on the glycone side, while with cellotetraose as a ligand, five subsites, including four on the glycone side, are occupied. A structural comparison showed no conformational rearrangement of AaCel9A upon ligand binding. The structural analysis demonstrates that of the four minus subsites identified, subsites -1 and -2 show the strongest interaction with bound glucose. In conjunction with the open active-site cleft of AaCel9A, this is able to reconcile the previously observed cleavage of short-chain oligosaccharides with cellobiose as main product with the endo mode of action on larger polysaccharides.

Plant ALDH10 family: identifying critical residues for substrate specificity and trapping a thiohemiacetal intermediate.

Background: Plant aminoaldehyde dehydrogenases (AMADHs) detoxify ω-aminoaldehydes from several metabolic pathways. Results: Two of five new AMADHs exhibit unusual kinetic properties. A thiohemiacetal intermediate was trapped in a crystal structure. Conclusion: Five critical residues can modulate substrate specificity, and a new substrate was identified. Significance: The present findings allow sequence-based predictions of AMADH substrate specificity linked with the production of individual osmoprotectants in plants. Plant ALDH10 family members are aminoaldehyde dehydrogenases (AMADHs), which oxidize ω-aminoaldehydes to the corresponding acids. They have been linked to polyamine catabolism, osmoprotection, secondary metabolism (fragrance), and carnitine biosynthesis. Plants commonly contain two AMADH isoenzymes. We previously studied the substrate specificity of two AMADH isoforms from peas (PsAMADHs). Here, two isoenzymes from tomato (Solanum lycopersicum), SlAMADHs, and three AMADHs from maize (Zea mays), ZmAMADHs, were kinetically investigated to obtain further clues to the catalytic mechanism and the substrate specificity. We also solved the high resolution crystal structures of SlAMADH1 and ZmAMADH1a because these enzymes stand out from the others regarding their activity. From the structural and kinetic analysis, we can state that five residues at positions 163, 288, 289, 444, and 454 (PsAMADHs numbering) can, directly or not, significantly modulate AMADH substrate specificity. In the SlAMADH1 structure, a PEG aldehyde derived from the precipitant forms a thiohemiacetal intermediate, never observed so far. Its absence in the SlAMADH1-E260A structure suggests that Glu-260 can activate the catalytic cysteine as a nucleophile. We show that the five AMADHs studied here are capable of oxidizing 3-dimethylsulfoniopropionaldehyde to the cryo- and osmoprotectant 3-dimethylsulfoniopropionate. For the first time, we also show that 3-acetamidopropionaldehyde, the third aminoaldehyde besides 3-aminopropionaldehyde and 4-aminobutyraldehyde, is generally oxidized by AMADHs, meaning that these enzymes are unique in metabolizing and detoxifying aldehyde products of polyamine degradation to nontoxic amino acids. Finally, gene expression profiles in maize indicate that AMADHs might be important for controlling ω-aminoaldehyde levels during early stages of the seed development.

Role and structural characterization of plant aldehyde dehydrogenases from family 2 and family 7.

Aldehyde dehydrogenases (ALDHs) are responsible for oxidation of biogenic aldehyde intermediates as well as for cell detoxification of aldehydes generated during lipid peroxidation. So far, 13 ALDH families have been described in plants. In the present study, we provide a detailed biochemical characterization of plant ALDH2 and ALDH7 families by analysing maize and pea ALDH7 (ZmALDH7 and PsALDH7) and four maize cytosolic ALDH(cALDH)2 isoforms RF2C, RF2D, RF2E and RF2F [the first maize ALDH2 was discovered as a fertility restorer (RF2A)]. We report the crystal structures of ZmALDH7, RF2C and RF2F at high resolution. The ZmALDH7 structure shows that the three conserved residues Glu(120), Arg(300) and Thr(302) in the ALDH7 family are located in the substrate-binding site and are specific to this family. Our kinetic analysis demonstrates that α-aminoadipic semialdehyde, a lysine catabolism intermediate, is the preferred substrate for plant ALDH7. In contrast, aromatic aldehydes including benzaldehyde, anisaldehyde, cinnamaldehyde, coniferaldehyde and sinapaldehyde are the best substrates for cALDH2. In line with these results, the crystal structures of RF2C and RF2F reveal that their substrate-binding sites are similar and are formed by an aromatic cluster mainly composed of phenylalanine residues and several nonpolar residues. Gene expression studies indicate that the RF2C gene, which is strongly expressed in all organs, appears essential, suggesting that the crucial role of the enzyme would certainly be linked to the cell wall formation using aldehydes from phenylpropanoid pathway as substrates. Finally, plant ALDH7 may significantly contribute to osmoprotection because it oxidizes several aminoaldehydes leading to products known as osmolytes.