Stéphanie Blangy

Modular Structure of the Receptor Binding Proteins of Lactococcus lactis Phages THE RBP STRUCTURE OF THE TEMPERATE PHAGE TP901-1

Lactococcus lactis is a Gram-positive bacterium widely used by the dairy industry. Several industrial L. lactis strains are sensitive to various distinct bacteriophages. Most of them belong to the Siphoviridae family and comprise several species, among which the 936 and P335 are prominent. Members of these two phage species recognize their hosts through the interaction of their receptor-binding protein (RBP) with external cell wall saccharidices of the host, the “receptors.” We report here the 1.65 Å resolution crystal structure of the RBP from phage TP901-1, a member of the P335 species. This RBP of 163 amino acids is a homotrimer comprising three domains: a helical N terminus, an interlaced β-prism, and a β-barrel, the head domain (residues 64-163), which binds a glycerol molecule. Fluorescence quenching experiments indicated that the RBP exhibits high affinity for glycerol, muramyl-dipeptide, and other saccharides in solution. The structural comparison of this RBP with that of lactococcal phage p2 RBP, a member of the 936 species (Spinelli, S., Desmyter, A., Verrips, C. T., de Haard, J. W., Moineau, S., and Cambillau, C. (2006) Nat. Struct. Mol. Biol. 13, 85-89) suggests a large extent of modularity in RBPs of lactococcal phages.

Receptor-Binding Protein of Lactococcus lactis Phages: Identification and Characterization of the Saccharide Receptor-Binding Site

Phage p2, a member of the lactococcal 936 phage species, infects Lactococcus lactis strains by binding initially to specific carbohydrate receptors using its receptor-binding protein (RBP). The structures of p2 RBP, a homotrimeric protein composed of three domains, and of its complex with a neutralizing llama VH domain (VHH5) have been determined (S. Spinelli, A. Desmyter, C. T. Verrips, H. J. de Haard, S. Moineau, and C. Cambillau, Nat. Struct. Mol. Biol. 13:85-89, 2006). Here, we show that VHH5 was able to neutralize 12 of 50 lactococcal phages belonging to the 936 species. Moreover, escape phage mutants no longer neutralized by VHH5 were isolated from 11 of these phages. All of the mutations (but one) cluster in the RBP/VHH5 interaction surface that delineates the receptor-binding area. A glycerol molecule, observed in the 1.7-A resolution structure of RBP, was found to bind tightly (Kd= 0.26 microM) in a crevice located in this area. Other saccharides bind RBP with comparable high affinity. These data prove the saccharidic nature of the bacterial receptor recognized by phage p2 and identify the position of its binding site in the RBP head domain.

Towards a structural comprehension of bacterial type VI secretion systems: characterization of the TssJ-TssM complex of an Escherichia coli pathovar.

Type VI secretion systems (T6SS) are trans-envelope machines dedicated to the secretion of virulence factors into eukaryotic or prokaryotic cells, therefore required for pathogenesis and/or for competition towards neighboring bacteria. The T6SS apparatus resembles the injection device of bacteriophage T4, and is anchored to the cell envelope through a membrane complex. This membrane complex is composed of the TssL, TssM and TagL inner membrane anchored proteins and of the TssJ outer membrane lipoprotein. Here, we report the crystal structure of the enteroaggregative Escherichia coli Sci1 TssJ lipoprotein, a two four-stranded β-sheets protein that exhibits a transthyretin fold with an additional α-helical domain and a protruding loop. We showed that TssJ contacts TssM through this loop since a loop depleted mutant failed to interact with TssM in vitro or in vivo. Biophysical analysis of TssM and TssJ-TssM interaction suggest a structural model of the membrane-anchored outer shell of T6SS. Collectively, our results provide an improved understanding of T6SS assembly and encourage structure-aided drug design of novel antimicrobials targeting T6SS.

Structure of the phage TP901-1 1.8 MDa baseplate suggests an alternative host adhesion mechanism

Phages of the Caudovirales order possess a tail that recognizes the host and ensures genome delivery upon infection. The X-ray structure of the approximately 1.8 MDa host adsorption device (baseplate) from the lactococcal phage TP901-1 shows that the receptor-binding proteins are pointing in the direction of the host, suggesting that this organelle is in a conformation ready for host adhesion. This result is in marked contrast with the lactococcal phage p2 situation, whose baseplate is known to undergo huge conformational changes in the presence of Ca2+ to reach its active state. In vivo infection experiments confirmed these structural observations by demonstrating that Ca2+ ions are required for host adhesion among p2-like phages (936-species) but have no influence on TP901-1-like phages (P335-species). These data suggest that these two families rely on diverse adhesion strategies which may lead to different signaling for genome release.

A Topological Model of the Baseplate of Lactococcal Phage Tuc2009

Phages infecting Lactococcus lactis, a Gram-positive bacterium, are a recurrent problem in the dairy industry. Despite their economical importance, the knowledge on these phages, belonging mostly to Siphoviridae, lags behind that accumulated for members of Myoviridae. The three-dimensional structures of the receptor-binding proteins (RBP) of three lactococcal phages have been determined recently, illustrating their modular assembly and assigning the nature of their bacterial receptor. These RBPs are attached to the baseplate, a large phage organelle, located at the tip of the tail. Tuc2009 baseplate is formed by the products of 6 open read frames, including the RBP. Because phage binding to its receptor induces DNA release, it has been postulated that the baseplate might be the trigger for DNA injection. We embarked on a structural study of the lactococcal phages baseplate, ultimately to gain insight into the triggering mechanism following receptor binding. Structural features of the Tuc2009 baseplate were established using size exclusion chromatography coupled to on-line UV-visible absorbance, light scattering, and refractive index detection (MALS/UV/RI). Combining the results of this approach with literature data led us to propose a “low resolution” model of Tuc2009 baseplate. This model will serve as a knowledge base to submit relevant complexes to crystallization trials.

Crystal Structure of the Receptor-Binding Protein Head Domain from Lactococcus lactis Phage bIL170

ABSTRACT Lactococcus lactis, a gram-positive bacterium widely used by the dairy industry, is subject to lytic phage infections. In the first step of infection, phages recognize the host saccharidic receptor using their receptor binding protein (RBP). Here, we report the 2.30-Å-resolution crystal structure of the RBP head domain from phage bIL170. The structure of the head monomer is remarkably close to those of other lactococcal phages, p2 and TP901-1, despite any sequence identity with them. The knowledge of the three-dimensional structures of three RBPs gives a better insight into the module exchanges which have occurred among phages.

Crystal structure and function of a DARPin neutralizing inhibitor of lactococcal phage TP901-1: Comparison of DARPin and camelid VHH binding mode

Combinatorial libraries of designed ankyrin repeat proteins (DARPins) have been proven to be a valuable source of specific binding proteins, as they can be expressed at very high levels and are very stable. We report here the selection of DARPins directed against a macromolecular multiprotein complex, the baseplate BppU·BppL complex of the lactococcal phage TP901-1. Using ribosome display, we selected several DARPins that bound specifically to the tip of the receptor-binding protein (RBP, the BppL trimer). The three selected DARPins display high specificity and affinity in the low nanomolar range and bind with a stoichiometry of one DARPin per BppL trimer. The crystal structure of a DARPin complexed with the RBP was solved at 2.1 Å resolution. The DARPin·RBP interface is of the concave (DARPin)-convex (RBP) type, typical of other DARPin protein complexes and different from what is observed with a camelid VHH domain, which penetrates the phage p2 RBP inter-monomer interface. Finally, phage infection assays demonstrated that TP901-1 infection of Lactococcus lactis cells was inhibited by each of the three selected DARPins. This study provides proof of concept for the possible use of DARPins to circumvent viral infection. It also provides support for the use of DARPins in co-crystallization, due to their rigidity and their ability to provide multiple crystal contacts.

Identification and characterization of a triacylglycerol lipase in Arabidopsis homologous to mammalian acid lipases

Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms. By contrast, very little is known on plant TAG lipases. An Arabidopsis cDNA called AtLip1 (At2g15230), which exhibits strong homology to lysosomal acid lipase, was found to drive the synthesis of an active TAG lipase when expressed in the baculovirus system. The lipase had a maximal activity at pH 6 and the specific activity was estimated to be about 45 μmol min−1 mg−1 protein using triolein as a substrate. Knock‐out mutant analysis showed no phenotype during germination indicating that this enzyme is fully dispensable for TAG storage breakdown during germination. Northern blot analyses indicated that the transcript is present in all tissues tested.

Characterization of the Interactions between the Nucleoprotein and the Phosphoprotein of Henipavirus

The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (NTAIL) are both intrinsically disordered. Here we show that Henipavirus NTAIL domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (PXD) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that NTAIL and PXD form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 m and has a KD in the μm range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that PXD triggers an increase in the α-helical content of NTAIL. Using fluorescence spectroscopy, we show that PXD has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus NTAIL domain, thus arguing for the lack of stable contacts between the C termini of NTAIL and PXD. Finally, we present a tentative structural model of the NTAIL-PXD interaction in which a short, order-prone region of NTAIL (α-MoRE; amino acids 473–493) adopts an α-helical conformation and is embedded between helices α2 and α3 of PXD, leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the NTAIL-PXD interaction as a valuable target for rational antiviral approaches.

Acidianus filamentous virus 1 coat proteins display a helical fold spanning the filamentous archaeal viruses lineage

Acidianus filamentous virus 1 (AFV1), a member of the Lipothrixviridae family, infects the hyperthermophilic, acidophilic crenarchaeaon Acidianus hospitalis. The virion, covered with a lipidic outer shell, is 9,100-Å long and contains a 20.8-kb linear dsDNA genome. We have identified the two major coat proteins of the virion (MCPs; 132 and 140 amino acids). They bind DNA and form filaments when incubated with linear dsDNA. A C-terminal domain is identified in their crystal structure with a four-helix-bundle fold. In the topological model of the virion filament core, the genomic dsDNA superhelix wraps around the AFV1–132 basic protein, and the AFV1–140 basic N terminus binds genomic DNA, while its lipophilic C-terminal domain is imbedded in the lipidic outer shell. The four-helix bundle fold of the MCPs from AFV1 is identical to that of the coat protein (CP) of Sulfolobus islandicus rod-shaped virus (SIRV), a member of the Rudiviridae family. Despite low sequence identity between these proteins, their high degree of structural similarity suggests that they could have derived from a common ancestor and could thus define an yet undescribed viral lineage.