Conference Agenda

Heparan sulfate (HS) is a ubiquitous glycosaminoglycan component of the extracellular matrix (ECM), which facilitates important structural and signalling interactions between cells and their surroundings. The principal enzyme responsible for extracellular HS breakdown is heparanase (HPSE), an endo-glucuronidase of the CAZy GH79 family. Whilst normal HPSE activity is essential for HS processing, excessive HPSE overexpression weakens HS networks in the ECM, leading to increased cell mobility and release of growth factors stored by HS. Thus HPSE is an oncogene whose overexpression promotes metastasis in a range of cancers.

In this talk, I will give an overview of our work in this area over the last few years, covering our initial structural investigations into the molecualr basis of HPSE activity, the development of probes to visualize HPSE in tissues, and most recently, the structure guided rational design of HPSE inhibitors as anti-metastatic agents.

References

L. Wu, C. M. Viola et al (2015), Nat. Struct. Mol. Biol. (22) 1016–1022

L. Wu, J. Jiang, Y. Jin et al (2017), Nat. Chem. Biol. (13) 867–873

Fatty Acid Photodecarboxylase (FAP) is a recently discovered photoenzyme that catalyzes the conversion of fatty acids into alkane and CO₂ under light, with potential importance in green chemistry applications [1]. Its mechanism was still not fully understood and partly relied on a low-resolution crystal structure obtained from crystals with a twinning default [1]. Here, we present high-resolution crystal structures of FAP obtained in the dark and after light illumination at cryogenic temperatures (Figure 1). Combined with structural, computational, and spectroscopic techniques we are now able to provide a detailed reaction cycle of FAP. The reaction mechanism starts with an electron transfer from the fatty acid to a photoexcited oxidized flavin cofactor. Decarboxylation yields an alkyl radical, which is then reduced by back electron transfer and protonation rather than hydrogen atom transfer. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved CO₂ unexpectedly transforms in 100 ns, most likely into bicarbonate. This is orders of magnitude faster than in solution, which indicates a catalytic step. FT-IR, structural and functional studies on variants centered on two conserved active site residues (R451 and C432) showed that R451 is essential for substrate stabilization and proton transfer. Altogether this study provides a detailed characterization of this unique enzyme and reveals a striking and unanticipated mechanistic complexity [2].

[1] Sorigué D, Légeret B, Cuiné S, Blangy S, Moulin S, Billon E, Richaud P, Brugière S, Couté Y, Nurizzo D, Müller P, Brettel K, Pignol D, Arnoux P, Li-Beisson Y, Peltier G, Beisson F. (2017) Science. 357, 903.

[2] Sorigué, D., K. Hadjidemetriou, S. Blangy, G. Gotthard, A. Bonvalet, N. Coquelle, P. Samire, A. Aleksandrov, L. Antonucci, A. Benachir, S. Boutet, M. Byrdin, M. Cammarata, S. Carbajo, S. Cuiné, R. B. Doak, L. Foucar, A. Gorel, M. Grünbein, E. Hartmann, R. Hienerwadel, M. Hilpert, M. Kloos, T. J. Lane, B. Légeret, P. Legrand, Y. Li-Beisson, S. L. Y. Moulin, D. Nurizzo, G. Peltier, G. Schirò, R. L. Shoeman, M. Sliwa, X. Solinas, B. Zhuang, T. R. M. Barends, J.-P. Colletier, M. Joffre, A. Royant, C. Berthomieu, M. Weik, T. Domratcheva, K. Brettel, M. H. Vos, I. Schlichting, P. Arnoux, P. Müller, F. Beisson (2021) Science 372, 148.

Lytic polysaccharide monooxygenases (LPMOs) have been intensely studied since their first characterization in 2010 as a unique class of copper enzymes capable of oxidizing carbohydrates. LPMOs require the input of electrons and of O₂ or H₂O₂ to achieve hydroxylation of one carbon in the glycosidic bond. We focus on three aspects of the LPMO’s reaction mechanism: 1) What are the structural determinants of O₂ and H₂O₂ binding? 2) How do conserved second shell residues contribute to activity? 3) Does the O₂ based mechanism follow a superoxyl, hydroperoxyl or oxyl catalytic pathway? The ability to pinpoint hydrogen atoms to determine protonation states at and around the active site through the catalytic pathway is key to decipher the chemistry catalyzed by LPMOs. To achieve this, we combine high resolution X-ray and neutron protein crystallography to deliver precise, all atom structures of key reaction intermediates that can reveal i) the positions and interactions of all hydrogen atoms in the enzyme, ii) atomistic details of the active site without perturbing the metal oxidation state, and iii) the chemical nature of the activated dioxygen species coordinated to the active site copper. We will present our recent X-ray and neutron crystallographic studies that provide new insights into the LPMO mechanism.

The discovery of novel enzymes from antibiotic production pathways is nowadays a topic of utmost importance due to worldwide concerns with the increased resistance of pathogenic bacteria to antibiotics. In this work, we used a combination of X-ray crystallography, SAXS, and biochemical studies to identify the molecular fingerprints for a novel glucosamine kinase (GlcNK) family potentially implicated in antibiotic biosynthesis in Actinobacteria. We determined the high-resolution structure of a bacterial GlcNK in apo form and in complex with its biological substrates, providing unparalleled structural evidence of a transition state of the phosphoryl-transfer mechanism in this unique family of enzymes (PDB IDs 6HWJ, 6HWK and 6HWL; Fig. 1a-c). Conservation of glucosamine-contacting residues across a large number of uncharacterized proteins unveiled a specific glucosamine binding sequence motif. As result, a new UniProt annotation rule was created (MF_02218; Fig. 1d). The structural characterization of this enzyme provides new insights into the role of these unique GlcNKs as the missing link for the incorporation of environmental glucosamine to the metabolism of important intermediates in antibiotic production [1].

[1] Manso, J. A., Nunes-Costa, D., Macedo-Ribeiro, S., Empadinhas, N., Pereira, P. J. B. (2019). mBio. 10, e00239-19.

Mycobacteria are priority pathogens in terms of drug resistance worldwide and efforts aimed at deciphering their unique metabolic pathways and unveiling new targets for innovative drugs should be intensified. In particular, nontuberculous mycobacteria (NTM) are environmental organisms increasingly associated to opportunistic infections [1] and known to produce methylmannose polysaccharides (MMP). MMP have been implicated in the metabolism of precursors of cell envelope lipids crucial for stress resistance and pathogenesis. Although the functions of MMP remain to be confirmed experimentally, their tight interactions with fatty acids are intrinsically associated to unique and extensive methylation patterns, resulting from the action of hitherto uncharacterized methyltransferases.

In this work, we identified and characterized biochemically a novel mycobacterial methyltransferase (MeT1) that specifically blocks the non-reducing end of a MMP precursor. We crystallized and determined the first X-ray structure of the SAM-dependent MeT1 from M. hassiacum in complex with magnesium and its exhausted cofactor, SAH. In particular, the three high-resolution 3D structures (in space groups P3₂21 and C222₁; PDB entries 6H40, 6G7D and 6G80) in combination with SAXS data (SASBDB entry SASDDJ6) unveiled a dimeric arrangement of the enzyme in solution and a highly flexible lid important for its catalytic cycle. This structural information, together with molecular docking simulations, allowed the elucidation of the enzyme’s reaction mechanism, furthering our knowledge of MMP biosynthesis and providing important tools to dissect the role of MMP in NTM physiology and resilience [2].

[1] Falkinham III, J. O., (2015). Clin. Chest. Med. 36, 35.

[2] Ripoll-Rozada, J., Costa, M., Manso, J. A., Maranha, A., Miranda, V., Sequeira, A., Rita Ventura, M,. Macedo-Ribeiro, S., Pereira, P. J. B., Empadinhas, N. (2019). Proc. Natl. Acad. Sci. U.S.A. 116, 835.

We thank SOLEIL, ESRF and ALBA for provision of synchrotron radiation facilities, and their staff for help with data collection. This work was funded in part by national funds through Fundação para a Ciência e a Tecnologia (Portugal) through PhD Fellowship SFRH/BD/101191/2014 (to M.C.); the European Social Fund through Programa Operacional Capital Humano in the form of Postdoctoral Fellowship SFRH/BPD/108004/2015 (to J.R.-R.); the European Regional Development Fund (FEDER), through Centro2020 Project CENTRO-01-0145- FEDER-000012-HealthyAging2020 in the form of a postdoctoral fellowship (to A.M.); and the COMPETE 2020–Operational Programme for Competitiveness and Internationalization (POCI), PORTUGAL 2020 in the form of projects POCI-01-0145-FEDER-029221 (PTDC/BTM-TEC/29221/2017), “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), UID/NEU/04539/2013, and Research Unit MOSTMICRO (UID/CQB/04612/2013).

Time-resolved serial femtosecond crystallography (TR-SFX) at X-ray free electron lasers (XFELs) allows studying the structural dynamics of crystalline biological macromolecules down to the sub-picosecond time scale [1]. According to a pump-probe scheme, optical pump pulses initiate activity in light sensitive crystalline proteins and XFEL pulses generate diffraction patterns that allow determining intermediate-state structures. We apply TR-SFX to study light-induced dynamics in a reversibly photoswitchable fluorescent protein, rsEGFP2.

Reversibly photoswitchable fluorescent proteins are essential tools in advanced fluorescence nanoscopy of live cells. They can be repeatedly toggled back and forth between a fluorescent (on) and a non-fluorescent (off) state by irradiation with light at two different wavelengths. Our consortium (*) combines TR-SFX at XFELs, ultrafast absorption spectroscopy and simulation methods to study photoswitching intermediates in rsEGFP2 on the picosecond to nanosecond time scale. We have been able to identify the transient structure of rsEGFP2 in its excited state 1 ps after photoexcitation, and to observe the chromophore in a twisted state, midway between the stable configurations of the on and off states [2]. This observation, together with a ground-state intermediate structure determined 10 ns after photoexcitation, has allowed us to uncover details of the photo-switching mechanism of rsEGFP2 [3].

Based on the reaction intermediates determined by TR-SFX [2, 3] two rationally designed mutants of the rsEGFP2 have been generated. Pico- to nanosecond TR-SFX results experiments on these rsEGFP2 variants have been carried out at SACLA and the LCLS and provide insight into modified energy landscapes (unpublished).

[1] Colletier, J-P., Schirò, G. & Weik, M. (2018). Time-Resolved Serial Femtosecond Crystallography, Towards Molecular Movies of Biomolecules in Action in X-ray Free Electron Lasers: A Revolution in Structural Biology, edited by Fromme, P., Boutet, S., Hunter. M. Eds., Springer International Publishing, 11:331-356[2] Coquelle. N., Sliwa. M., Woodhouse. J., Schiro. G., Adam. A. … Colletier, J-P., I. Schlichting. & M. Weik. (2018). Nat. Chem. 10, 31-37 [3] Woodhouse. J., … Sliwa. M., Colletier, J-P., I. Schlichting. & M. Weik. (2020). Nat. Comm. 11, 1-11

Keywords: x-ray free electron lasers; time-resolved studies; photoswitchable fluorescent proteins

(*) the work presented involves a consortium composed of researchers from the Institut de Biologie Structurale, Grenoble, France, Institut Laue Langevin, Grenoble, France, Max-Planck-Institut for Medical Research, Heidelberg, Germany, RIKEN SPring-8 Center, Sayo, Japan, Linac Coherent Light Source, Menlo Park, USA, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, Laboratoire de Spectrochimie Infrarouge et Raman, Lille, France, Department of Physics, University of Rennes, France, Laboratoire de Chimie-Physique, CNRS/University Paris-Sud, University Paris-Saclay, Orsay, France, namely, Adam V., Andreeva E., Aquila A., Banneville A-S., Barends T., Bourgeois D., Boutet S., Byrdin M., Cammarata M., Carbajo S., Colletier J-P., Coquelle N., Demachy I., Doak B., Feliks M., Field M., Fieschi F., Foucar L., Gorel A., Grünbein M., Guillon V., Hilpert M., Hunter M., Jakobs S., Joti Y., Kloos M., Koglin J., Lane T., Liang M., Levy B., de la Mora E., Nass-Kovacs G., Owada S., Richard J., Robinson J., Roome. C., Ruckebusch C., Schirò G., Schlichting I., Seaberg M., Shoeman R., Sierra R., Sliwa M., Stricker M., Tetreau G., Thepaut M., Tono K., Uriarte L., Woodhouse J., Yabashi M., You D., Zala N. and Weik M.