Minisymposium 2024 Structural Biology in Slovenia and Beyond Book of abstracts May 14th, 2024 Department of Molecular Biology and Nanobiotechnology National Institute of Chemistry, Ljubljana, Slovenia Minisymposium 2024 Structural Biology in Slovenia and Beyond Book of abstracts Organizer Department of Molecular Biology and Nanobiotechnology National Institute of Chemistry, Ljubljana, Slovenia @D11_KI — @kemijski — www.ki.si/en/ms2024 Editors Assoc. Prof. Dr. Marjetka Podobnik Dr. Matic Kisovec Maja Jamnik Organizing Committee Assoc. Prof. Dr. Marjetka Podobnik Maja Jamnik Issued by Department of Molecular Biology and Nanobiotechnology National Institute of Chemistry, Ljubljana, Slovenia Ljubljana, 2024 Electronic version only Kataložni zapis o publikaciji (CIP) pripravili v Narodni in univerzitetni knjižnici v Ljubljani COBISS.SI-ID 199574019 ISBN 978-961-6104-95-1 (PDF) CONTENTS CONTENTS 3 PROGRAM 4 ABSTRACTS 5 Instruct-ERIC – The Leading Research Infrastructure in Structural Biology 6 Integrated Structural Biology to unravel the impact of synonymous mutations on protein structure 7 The contribution of in-cell NMR to cellular structural biology 8 Structural snapshots of the glucose metabolism revealed by in vitro and in situ cryo-electron microscopy 9 NMR insights into unique cation dependency of DNA quadruplex structure 10 Structural insight into calcium-regulated actin crosslinking mode of non-muscle α-actinins 11 Cryo-EM studies of potyviral coat protein structural plasticity 12 Structural characterization of a de novo designed random protein walker system 13 Modular Protein Nanostructures (as markers for cryo-EM) 14 Structural insights into the interaction between actinoporin pores and lipid membranes 15 Structural insight into the enzymes involved in the synthesis of the S-layer-anchoring polysaccharide ligand of Clostridioides difficile 16 3 PROGRAM 12.15-13.15: buffet lunch Part 1: Talks by members of Instruct-ERIC Council 13.15 - 13.20: introductory words by Marjetka Podobnik 13.20 - 13.35: Claudia Alén Amaro: Instruct-ERIC – The Leading Research Infrastructure in Structural Biology 13.35 -14.00: Harald Schwalbe: Integrated Structural Biology to unravel the impact of synonymous mutations on protein structure 14.00 - 14.25: Lucia Banci: The contribution of in-cell NMR to cellular structural biology 14.25 - 14.50: Jiří Nováček: Structural snapshots of the glucose metabolism revealed by in vitro and in situ cryo-electron microscopy 14.50 - 15.05: official announcement of Slovenia as a member of Instruct-ERIC 15.05 - 15.20 coffee break Part 2: Talks by the young generation of Slovenian structural biologists 15.20 - 15.25: introductory word by Marjetka Podobnik 15.25 - 15.40: Martina Lenarčič Živković: NMR insights into unique cation dependency of DNA quadruplex structure 15.40 - 15.55: Jošt Hočevar: Structural insight into calcium-regulated actin crosslinking mode of non-muscle α-actinins 15.55 - 16.10: Neža Koritnik: Cryo-EM studies of potyviral coat protein structural plasticity 16.10 - 16.25: Ajasja Ljubetič: Structural characterization of a de novo designed random protein walker system 16.25 - 16.40: Sara Vidmar: Modular Protein Nanostructures (as markers for cryo-EM) 16.40 - 16.55: Gašper Šolinc: Structural insights into the interaction between actinoporin pores and lipid membranes 16.55 - 17.10: Nataša Lindič: Structural insight into the enzymes involved in the synthesis of the S-layer-anchoring polysaccharide ligand of Clostridioides difficile 4 ABSTRACTS 5 Instruct-ERIC – The Leading Research Infrastructure in Structural Biology Claudia Alén Amaro Head of Operation Instruct-ERIC, Oxford, United Kingdom claudia@instruct-eric.org Instruct-ERIC is a pan-European distributed research infrastructure making high-end technologies and methods in structural biology available to users. Instruct-ERIC is comprised of 17 Members, and researchers from these countries and organisations receive funded access to any of Instruct’s 11 centres across Europe, to utilise structural biology equipment and benefit from the considerable expertise of our centre teams. In addition to access to infrastructure, members can apply to host and attend a variety of training workshops and courses, apply for 3–6-month internships, receive funding for R&D and technology development projects, and get discounted tickets for the Instruct Biennial Structural Biology Conference. 6 Integrated Structural Biology to unravel the impact of synonymous mutations on protein structure Harald Schwalbe Instruct-ERIC director Centre for Biomolecular Magnetic Resonance (BMRZ), Goethe-University Frankfurt, Germany harald.schwalbe@instruct-eric.org Understanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel. Using mass spectrometry, theoretical simulations, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance and cryo-electron microscopy, we show that thiol groups of cysteine residues undergo S-glutathionylation and S-nitrosylation and form non-native disulfide bonds. Thus, covalent modification chemistry occurs already prior to nascent chain release as the ribosome exit tunnel provides sufficient space even for disulfide bond formation which can guide protein folding. Literature: Schulte et al. Nat. Commun. (2020) 11, 5569. Buhr et al. Mol Cell (2016) 61, 341-351 7 The contribution of in-cell NMR to cellular structural biology Lucia Banci CERM and Department of Chemistry, University of Florence, Sesto Fiorentino, Italy banci@cerm.unifi.it In-cell NMR, i.e. high-resolution NMR spectra of biomolecules in intact, living cells, allow the characterization of conformational and functional properties of biomolecules at atomic resolution in conditions as close as possible to the physiological ones. It also allows to monitor protein-protein interactions and to follow functional processes, as well as protein maturation and post-translational modifications. A further high- impact application is its use for drug screening in real time at cellular level, in human living cells. A few examples of the striking power of this approach will be presented. 8 Structural snapshots of the glucose metabolism revealed by in vitro and in situ cryo-electron microscopy Jiří Nováček Cryo-Electron Microscopy and Tomography Core Facility, CEITEC Masaryk University, Brno, Czech Republic 151132@muni.cz Insulin is a key hormone responsible for maintaining glucose homeostasis. It is stored in pancreatic cells in a form of dense granules. We have used FIB/SEM microscopy and correlative light-electron microscopy (CLEM) to quantify the granule presence in different cell lines and studied their structure. Insulin receptor (IR) is a receptor tyrosine kinase which upon insulin binding on the extracellular receptor domain induces autophoshorylation reaction on its cytoplasmic domains. Misregulation in the insulin signalling is a cause of Diabetes melitus I and II. We have studied the mechanism of IR inhibition with insulin non-related peptidomimetics capable to fully antagonise insulin action to prevent aberrant IR signalling. 9 NMR insights into unique cation dependency of DNA quadruplex structure Martin Gajarsky1,+, Petr Stadlbauer2, Jiri Sponer2, Anne Cucchiarini1,3, Michaela Dobrovolna2,4, Vaclav Brazda2,4, Jean-Louis Mergny2,3, Lukas Trantirek*1 and Martina Lenarčič Živković*1,5 1Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic 2Institute of Biophysics, Czech Academy of Science, Brno, Czech Republic 3Laboratoire d’Optique et Biosciences, Ecole Polytechnique, CNRS, INSERM, Institut Polytechnique de Paris, France 4Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic 5Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, Ljubljana, Slovenia +Present Address: Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany Martina.Lenarcic@ki.si In a recent study, we discovered a tetrastranded structure formed by Caenorhabditis elegans’ telomeric G-rich sequence, distinct from previously known DNA quadruplexes. Notably, stable KNa-quadruplex (KNaQ) formation requires concurrent K+ and Na+ ion coordination at two distinct binding sites. This discovery sheds light on G-rich DNA folding in intricate ionic environments relevant to eukaryotic cell function and the evolution of telomeric DNA. It highlights differences in human and nematode telomeric DNA structure, crucial for utilizing C. elegans as a model in telomere biology. Additionally, it offers opportunities for designing novel DNA recognition elements and sensors, promising advancements in DNA research. 10 Structural insight into calcium-regulated actin crosslinking mode of non-muscle α-actinins Jošt Hočevar1, Peter Sok2, Sara Drmota Prebil1, Chanchal Kumawat2, Brigita Lenarčič1, Kristina Djinović-Carugo1,2,3, Miha Pavšič1 1University of Ljubljana, Faculty of Chemistry and Chemical Technology, Chair of Biochemistry, Ljubljana, Slovenia 2University of Vienna, Max F. Perutz Laboratories, Vienna, Austria 3EMBL Grenoble, Grenoble, France Jost.Hocevar@fkkt.uni-lj.si α-actinins are actin crosslinking proteins involved in cell motility and muscle contraction. The actin crosslinking mode of non-muscle α-actinin isoforms is regulated by calcium, but the underlying molecular mechanisms have remained poorly understood for decades. We have determined the crystal structures of calcium-free and calcium-bound α-actinin 1, revealing the conformational changes induced by the binding of calcium. Notably, these changes also impact the orientation of the actin-binding domain, providing a structural basis for calcium-mediated regulation of actin crosslinking. Additionally, we have investigated calcium binding using isothermal titration calorimetry, revealing isoform-specific thermodynamic signatures. 11 Cryo-EM studies of potyviral coat protein structural plasticity Neža Koritnik1, Andreja Kežar1, Luka Kavčič1, Magda Tušek-Žnidarič2, Swarnalok De3, Maija Pollari3, Kristiina Mäkinen3, Marjetka Podobnik1 1 National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia 2 National Institute of Biology, Department of Biotechnology and Systems Biology, Ljubljana, Slovenia 3Faculty of Agriculture and Forestry, Department of Agricultural Sciences, Helsinki, Finland neza.koritnik@ki.si Potyviruses belong to the largest genus of plant viruses and pose a major threat to crops worldwide. Their virions are filamentous and flexible, with the coat protein (CP) arranged in a left-handed helical symmetry around the single-stranded RNA genome. We use cryo-EM to study the self-assembly of potyviruses and their virus-like particles (VLPs). Our recent studies have revealed an incredible intrinsic structural plasticity of the potyviral CP that enables not only the filamentous and flexible architecture of virions, but also their multitasking during the viral life cycle. Furthermore, by modifying the CP, we can manipulate the assembly of the VLP filaments and generate new structures such as rings, cubes or spheres. Our results provide the structural basis for a better understanding of viral biology to support the development of alternative antiviral strategies and for nanobiotechnological applications of structurally diverse self-assemblies of CP variants. 12 Structural characterization of a de novo designed random protein walker system Liza Ulčakar1, Tadej Satler1, Eva Rajh1, Ajasja Ljubetič1,2,3 1Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia. 2Department of Biochemistry and Institute for Protein Design, University of Washington, Seattle, WA, USA. 3EN-FIST Centre of Excellence, Ljubljana, Slovenia Ajasja.Ljubetic@ki.si Powered protein walkers are responsible for most movements within the cell. De novo design of static monomeric and oligomeric protein structures has advanced tremendously; however large dynamic protein robots have not yet been designed. I will present the design and structural characterization of a random protein walker that can diffuse along designed fibers. The scaffolds have a varying number of feet which change their mobility. We have determined the structure of the helical track using Cryo-EM and a partial brute-force search over helical parameters. We have also determined the structure of the rollers using single particle Cryo-EM. 13 Modular Protein Nanostructures (as markers for cryo-EM) Sara Vidmar†1,2, Tamara Šmidlehner†1, Jana Aupič1, Žiga Strmšek1, Ajasja Ljubetič1,2, Fei Xiao3, Guang Hu3, Chuan Liu4,5, Florian Beck5, Philipp S. Erdmann4, Roman Jerala1,2 1Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia 2EN-FIST Centre of Excellence, Ljubljana, Slovenia 3MOE Key Laboratory of Geriatric Diseases and Immunology, Suzhou Key Laboratory of Pathogen Bioscience and Anti-infective Medicine, Department of Bioinformatics, Center for Systems Biology, School of Biology and Basic Medical Sciences, Suzhou Medical College of Soochow University, Suzhou, China 4Human Technopole, Milan, Italy 5Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Sara.Vidmar@ki.si In recent years, the construction of programmable nanostructures using nucleic acids and polypeptides has noticeably advanced. Building upon DNA nanotechnology, which utilizes modular design principles, we expanded protein design, integrating tetrameric coiled coils for diverse polyhedral topologies. Experimental validation, including SAXS and cryo-EM, confirms the designed fold, structural stability, and functionality of tetrahedral nanocages. Additionally, dimeric self-assembling nanocages showcase symmetrical functionalization potential, offering regulatory avenues via external modulators like proteases and metal ions. 14 Structural insights into the interaction between actinoporin pores and lipid membranes Gašper Šolinc1, Marija Srnko1, Franci Merzel2, Ana Crnković1, Marjetka Podobnik1, Gregor Anderluh1 1Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia 2Theory Department, National Institute of Chemistry, Ljubljana, Slovenia Gasper.Solinc@ki.si Actinoporins are a group of toxins from cnidarians that form α-helical pores in lipid membranes containing sphingomyelin. We have solved the structure of the actinoporin Fav from Orbicella faveolata, using cryo-electron microscopy. Our results show that Fav forms pores with different stoichiometries, namely heptamers, octamers, and nonamers. High-resolution maps also reveal numerous lipids in the membrane surrounding the pore. In combination with atomistic simulations, it becomes clear that the lipids play a variety of roles in the pore structure. In addition to the phospholipids, we also observed cholesterol molecules and were able to describe their interactions with the Fav pore in detail, highlighting the complex interplay between the pore structure and the lipid membrane components. 15 Structural insight into the enzymes involved in the synthesis of the S-layer-anchoring polysaccharide ligand of Clostridioides difficile Nataša Lindič1, Aleksandra Usenik1,2, Jure Loboda1, Matej Novak1, Marinka Horvat1,2, Tjaša Peternel1, Tadej Uršič1, Jure Borišek3, Nikola Minovski3, Janez Mravljak4, Martina Hrast Rambaher4, Marjana Novič3, Robert Vidmar1 and Dušan Turk1,2 1Department of biochemistry, molecular and structural biology, Jožef Stefan Institute, Ljubljana, Slovenia 2Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins (CIPKeBiP), Ljubljana, Slovenia 3National Institute of Chemistry, Ljubljana, Slovenia 4Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia natasa.lindic@ijs.si The surface of Clostridioides difficile “superbug” is covered with a protein array (S-layer). S-layer is attached to bacterial cell through binding to secondary polysaccharides (PSII). Impairment of enzymes involved in PSII synthesis is often lethal. Using X-ray crystallography, we determined the structure of one representative of each group of enzymes involved in the PSII synthesis: glycosyltransferases, mannose converting enzymes and enzymes anchoring the PSII to peptidoglycan. The structures revealed domain organization similarities to their homologues and conserved ligand binding regions, but the flexible loops-rich active sites require further investigation. Our results illuminate possible ways of specifically treating C. difficile infections, and are applicable to other Gram-positive bacteria in possession of homologous proteins. 16