Bacterial viruses (bacteriophages) shuttle their genomes into bacterial cells as a key initial step for the infection of the hosts. Many bacteriophages use a tail for adsorption and DNA translocation to the host’s cytosol. Tailed phages combine core tail architectures with diversified infection modules, reflecting that they encounter different envelope compositions in their wide range of bacterial hosts. Long, non-contractile tailed siphoviruses are a predominant tailed phage type. However, the nature of the infection signal these phages receive from the bacterial envelope is not well understood and needs more investigations. The goal of our French-German project is to elucidate, with cryo Electron Microscopy, the first prototype structure for a glycan-specific siphovirus tail and baseplate infecting a Gram-negative host, from Salmonella model phage 9NA. Structure in presence of the lipopolysaccharide receptor or outer membrane vesicles will allow to decipher how phage structural proteins are involved in infection initiation, opening of the tail and perforation of the cell wall upon membrane contact. We will thus gain understanding of the molecular rearrangements in the siphovirus tail leading to genome release. This will define structural differences to phages that use protein receptors for infection, for which we have determined the detailed structure, before and after interaction with the membrane receptor, of a representative: phage T5. With TIRF microscopy, and new Gram-negative model membrane set-ups, we will study siphovirus 9NA’s time-resolved genome release mechanism on a single particle level, and analyze the influence of Gram-negative membrane properties on successful infection initiation. We will moreover use these in vitro set-ups to study synergies of phage mixtures that compete for receptors on the same host and correlate this data with in vivo infection analyses on whole bacterial cells. Exploring the dynamics in bacteriophage-cell wall interactions will advance our view on phage tail structure rearrangements, leading to genome release as a key step in the phage life cycle. Elucidating the tail and baseplate structure of a model system representing a widespread type of Gram-negative, glycan-specific phages will improve structure-based genome annotation and phage receptor usage prediction from sequence data. Our work will contribute substantial molecular knowledge on how phage communities behave in a given infection ecosystem, important for the development of new antibiotic treatments that employ bacteriophages.

Glycosaminoglycans, such as heparan sulfate and chondroitin sulfate, are long, complex polysaccharide chains found on the surface of all animal cells. They are covalently attached to a core protein and mediate the interaction with diverse cellular factors, including chemokines, pathogens and signalling receptors. An immense diversity in functional and pathological roles is associated with the complex glycosaminoglycan composition. A key step of glycosaminoglycan biosynthesis, which takes place in the Golgi lumen, is the polysaccharide chain elongation. Its molecular mechanism, however, is still unknown. Moreover, the intrinsic feature of the core protein mediating the specific addition of either a heparan sulfate or chondroitin sulfate chain remains to be elucidated. The objectives of this proposal are (i) to reveal the molecular basis of heparan sulfate and chondroitin sulfate chain polymerization, (ii) to uncover the intrinsic factor in the core protein that decides the fate of the generated glycosaminoglycan chain and (iii) to study the architecture of the heparan sulfate and chondroitin sulfate polymerase complexes in their native cellular environment. To achieve these aims, in vitro glycosyltransferase assays will be combined with in cellulo functional analysis. A tetrasaccharide-peptide library will be synthesized using a chemo-enzymatic approach, and high-resolution structures of substrate-bound complexes will be determined by single-particle cryo-electron microscopy and X-ray crystallography. Golgi-localized glycosyltransferase complexes will be characterized in situ using multitask nanobodies and cryo-electron tomography. This project will provide a comprehensive understanding of glycosaminoglycan chain polymerization across the biological scales, from molecules to cells, laying the corner stone for future research on the glycosaminoglycan function in health and disease.

A complete description of biomolecular activity requires an understanding of the nature and the role of protein conformational dynamics. In recent years novel nuclear magnetic resonance (NMR) techniques have emerged that provide hitherto inaccessible detail concerning biomolecular motions occurring on physiologically important timescales. In particular residual dipolar couplings (RDCs) provide precise information about time and ensemble averaged structural and dynamic processes with correlations times up to the millisecond, and thereby encode key information for understanding biological activity. In recent years we have developed two very different approaches to the quantitative description of intrinsic protein motions on a wide range of timescales using RDCs. Application of these techniques to the study of the proteins Ubiquitin (Ub) and GB3 resulted in the convergent observation of enhanced dynamic fluctuations occurring on intermediate timescales (nano to millisecond) in the physiological interaction sites of these proteins. The motions occurring in these interaction sites were suggested to exhibit specific modes that would either optimally accommodate the interaction partner, or to intrinsically sample the conformations found in complex with diverse functional partners. In this study we will investigate, for the first time, the nature and timescale of these slower motions, not only in the isolated proteins, but also in the presence of different interaction partners. Concentrating on a specific and important cellular paradigm, we focus on characterizing the interaction between Ub and different Ub binding domains (UBDs). Ub is a versatile cellular signal, regulating a wide variety of activities ranging from protein degradation and quality control, endocytosis, transcriptional regulation to cell signaling and membrane trafficking. Ub has a large number of intracellular partners (more than 150 have been annotated), and while affinities of mono-Ub-binding interactions are very often weak, they span two orders of magnitude (Kd 3-2000µM). We will study the interaction between Ub and a range of UBDs with different affinities in order to probe the possible link between the structural dynamics of the molecular complex occurring on timescales up to the millisecond, and the kinetics of the interaction. In order to achieve this goal we will build on and extend state-of-the-art experimental, analytical, numerical and molecular simulation techniques established over the last ten years in the laboratory of the coordinator to measure and analyse RDCs, spin relaxation and relaxation dispersion from diverse Ub-UBD complexes. During the course of this project we will (a) compare, for the first time, the nature of large scale slow motions in the presence and absence of functional partners, (b) establish the dependence of interaction affinity on molecular recognition dynamics in free and bound forms of interacting partners, (c) extend the timescale over which NMR can be used to determine local entropic and enthalpic contributions to the thermodynamic equilibrium (d) more than double the number of individual proteins whose backbone dynamics have been characterised using RDCs, thereby establishing general trends concerning the nature of slow motions across different protein families. In summary, this project fully exploits the unique sensitivity of NMR to study weak protein-protein interactions at atomic resolution to address a problem of great current importance. While methods have been developed to describe slower motions in proteins, with the observation that these dynamics tend to occur in the interaction site of the proteins, the modulation of this flexibility upon interaction remains unknown. Methods developed in the group of the coordinator over the last decade will be applied in the course of this project to compare protein dynamics in the free and bound forms of Ub and UBDs participating in weak complexes.

Heparan Sulphate (HS) are sulphated polysaccharides ubiquitously present at higher eukaryotes cell surfaces and extracellular matrices. They participate in essential biological processes such as development, inflammatory responses, cell migration, blood coagulation. They are also used by pathogens during cell infection and play an important role in cancer development. The main function of this unique class of polysaccharides which belong to the glycosaminoglycan family is to interact with proteins. They are anchored to protein cores to form proteoglycans, and their localisation at cell surfaces and matrices places them in an ideal position to mediate interactions between cells and their environment. HS bind to growth factors, cytokines, chemokines, enzymes and consequently mediate their localisation, their activity or their interaction with a specific cell surface receptor. The structure/function paradigm of HS resides in the intrinsic variability generated during biosynthesis through the concerted action of several modifying enzymes. These modifications and in particular sulphations are tightly regulated during the biosynthesis of HS and depend specifically on the cell type and its environment. Cells are thus able to produce specific HS sequences that will mediate the binding of proteins and in turn modulate biological processes. While several hundreds proteins have been identified as ligands for Heparan Sulphate, identifying the determinants of the interaction specificity remains very challenging. Indeed the biosynthesis of HS is able to generate several thousands of different modifications motifs. The lack of information is largely due to the high heterogeneity of HS and consecutively the difficulty to purify defined sulphation motifs, test the interactions and characterize the protein-bound oligosaccharidic sequence. We thus propose to generate isotopically labelled (13C/15N) chemo-enzymatic HS libraries. These libraries will constitute great tools as their labelling allows at the same time to identify their chemical structures and to monitor their interactions with proteins by Nuclear Magnetic Resonance. Two proteins that have been extensively studied in our group and for which the binding to HS is critical for their function, IFN?, a pro-inflammatory cytokine and the chemokine CXCL12a will be tested in interaction with the libraries. The best-affinity HS will be selected by titration of the proteins to size-defined HS fragments, chromatographic isolation of the protein-HS complexes and NMR characterisation of the bound ligands. We will also use NMR to directly monitor interactions between mixtures of HS fragments and proteins. The oligosaccharides possessing the best affinity for the proteins will be isolated and fully characterised. Atomic resolution models of the best protein-HS complexes will be solved by Nuclear Magnetic Resonance, making full use of 13C/15N labelled ligands in conjunction with labelled or unlabelled protein. This project should permit to define of the HS motifs preferentially bound by the two proteins studied and also to characterise the structural determinants of the binding. These informations are highly invaluable in order to further study their biological function and also to design HS-based molecules that are able to modulate their function.

We propose to elucidate structure and function of the poxvirus DNA replication machinery by single particle cryo-electron microscopy (cryo-EM). The poxvirus family comprises members with a strong potential of spillover from the animal kingdom, such as cowpox and monkeypox, which leads to local epidemic outbreaks in Africa with a fatality rate of about 2 %. Furthermore poxviruses may be employed in the context of bioterrorism. They replicate without the requirement of host cell protein synthesis in cytoplasmic viral factories. The structure of the large DNA genome is only shared with asfarviruses and consists of a double-stranded DNA with circularized ends. There is no consensus model for poxvirus DNA replication. The DNA replication of vaccinia virus, a safe model system, involves the DNA polymerase holoenzyme complex, built from the polymerase E9, a structural protein A20 and an uracil-DNA glycosylase D4, which form the processivity factor. Furthermore it requires the helicase-primase D5, the ssDNA-binding protein I3 and the DNA ligase A50. We propose to determine structures of the E9-A20-D4 DNA polymerase holoenzyme in complex with different DNA substrates mimicking different steps in polymerase action and to approach in the same way the helicase-primase D5 in order to reconstitute components of the replication fork. In a second stage, the analysis may include the direct interaction between E9-A20-D4 and D5 or further proteins involved in DNA replication. The dynamic structure of the complexes with DNA precluded so far structure determination by crystallography, but with the classification techniques of cryo-EM these structures are now within reach. We can build on our X-ray structure determination and biophysical characterisations of several of the central partners, existing expression systems and our preliminary results on the cryo-EM of DNA-free forms of the complexes.