S B September 28th, 2022 Department of Molecular Biology and Nanobiotechnology National Institute of Chemistry, Ljubljana, Slovenia Minisymposium 2022 Give structural biology to young people Book of abstracts Organizer Department of Molecular Biology and Nanobiotechnology National Institute of Chemistry, Ljubljana, Slovenia @NanoSciNIC — @kemijski — www.ki.si/en/ms2022 Editor Dr. Marjetka Podobnik Technical editors Maja Jamnik, Dr. Andreja Kežar and Dr. Matic Kisovec Issued by Department of Molecular Biology and Nanobiotechnology National Institute of Chemistry, Slovenia Ljubljana, 2022 Electronic version only Kataložni zapis o publikaciji (CIP) pripravili v Narodni in univerzitetni knjižnici v Ljubljani COBISS.SI-ID 121862659 ISBN 978-961-6104-80-7 (PDF) ii Dobrodošli Welcome na 9. Mini simpoziju Odseka za to the 9th Minisymposium organized Molekularno Biologijo in Nanobioteh- by the Department of Molecular Bi- nologijo Kemijskega inštituta! Po ology and Nanobiotechnology at Na- premoru zaradi pandemije smo se tional Institute of Chemistry! After zopet vrnili, tokrat v hibridnem a hiatus due to the pandemics, we načinu, da omogočimo udeležbo čim are finally back, this time in a hybrid večjemu številu poslušalcev. mode, to enable as many attendees as Cilj naših mini simpozijev je deliti possible. vznemirljive znanstvene rezultate, iz- The goal of our minisymposium is to kušnje in strokovno znanje ter raz- share exciting scientific results, ex- pravljati o najsodobnejših pristopih perience and expertise, as well as za proučevanje struktur bioloških mo- to discuss state-of-the-art approaches lekul in njihovih interakcij. Letošnji in studying structures of biologi- simpozij nosi naslov Podarimo struk- cal molecules and their interactions. ” turno biologijo mladim“ in v skladu This years’ minisymposium is entitled s tem smo pripravili program, ki “Give structural biology to young vključuje predavanja nadobudnih mla- people” and we have prepared a pro- dih raziskovalcev. Za odlične razisko- gram including talks from enthusias- valne rezultate je potrebno trdo delo tic young researchers. As much as in sodelovanje znanstvenikov vseh successful research is based on hand- generacij, vsekakor pa moramo še po- in-hand hard work of scientists of all sebej spodbujati in podpirati mlade generations, we need to nourish and raziskovalce, da nadaljujejo in nad- support young researches to continue grajujejo dediščino svojih mentor- and upgrade the legacy of their men- jev. Vsem želim uspešno in prijetno tors. I wish everybody a fruitful and srečanje! Zelo se ga veselim in tudi enjoyable meeting! I am very much vseh, ki bodo še sledila! looking forward to it and to many more to come! izr. prof. dr. Marjetka Podobnik Assoc. Prof. Dr. Marjetka Podobnik iii Contents Dobrodošli / Welcome iii Contents v Sponsors of the Minisymposium vii Schedule ix Abstracts 1 Structural studies of cnidarian pore-forming toxins, actinoporins, using cryo-EM (Gaˇ sper ˇ Solinc et al.) . . . . . . . . . . . . . . 2 Cryo-EM structure of a track for a completely de novo designed random protein roller (Ajasja Ljubetiˇ c et al.) . . . . . . . . . 3 NMR insights into a G-quadruplex formation in the 5’-UTR region of the RANKL gene (Martina Lenarˇ ciˇ c ˇ Zivkoviˇ c et al.) . . . . 4 Rigidity and flexibility of structure govern specificity and promis- cuity of cysteine cathepsins (Jure Loboda et al.) . . . . . . . . 5 Measurements of (sub)cellular Ca2+ and cAMP dynamics in single astrocytes using confocal microscopy (Anemari Horvat et al.) 6 Using small-angle X-ray scatering to determine the shape of deco- rated coiled-coil protein origami ( ˇ Ziga Strmˇ sek et al.) . . . . . 7 The cryo-transmission electron microscopy facility at the National Institute of Chemistry (Matic Kisovec et al.) . . . . . . . . . 8 Regulation of L-lactate metabolism via GPCR (Dorian Dolanc et al.) 9 Cryo-EM studies of filamentous potyviruses and their virus-like par- ticles (Neˇ za Koritnik et al.) . . . . . . . . . . . . . . . . . . . 10 Stalk domain of SARS-CoV-2 spike protein and its structural poly- morphism (Zala ˇ Ziviˇ c et al.) . . . . . . . . . . . . . . . . . . . 11 Structural aspects of the mutations associated to Type B Kufs dis- ease (CLN13) (Tea Sinoˇ zi´ c et al.) . . . . . . . . . . . . . . . . 12 v Structural and functional aspects of VaaMPIII-3: a disintegrin- like/cysteine-rich protein from the venom of the nose-horned viper (Kity Poˇ zek et al.) . . . . . . . . . . . . . . . . . . . . . 13 The unique story of OlyA6: From specific lipid binding to potential biopesticide (Anastasija Panevska et al.) . . . . . . . . . . . . 14 vi Sponsors of the Minisymposium Thank you for your support. vii Schedule 9:00 - 9:05 Marjetka Podobnik Welcome address by the head of the Department for Molecular Biology and Nanobiotechnology 9:05 - 9:30 Gašper Šolinc Structural studies of cnidarian pore-forming toxins, actinoporins, using cryo-EM 9:30 - 9:55 Ajasja Ljubetič Cryo-EM structure of a track for a completely de novo designed random protein roller 9:55 - 10:20 Martina Lenarčič Živkovič NMR insights into a G-quadruplex formation in the 5’-UTR region of the RANKL gene 10:20 - 10:55 tea/coffee, refreshments 10:55 - 11:20 Jure Loboda Rigidity and flexibility of structure govern specificity and promiscuity of cysteine cathepsins 11:20 - 11:45 Anemari Horvat Measurements of (sub)cellular Ca2+ and cAMP dynamics in single astrocytes using confocal microscopy 11:45 - 13:00 lunch 13:00 - 13:15 Žiga Strmšek Using small-angle X-ray scatering to determine the shape of decorated coiled-coil protein origami ix 13:15 - 13:30 Matic Kisovec The cryo-transmission electron microscopy facility at the National Institute of Chemistry 13:30 - 13:45 Dorian Dolanc Regulation of L-lactate metabolism via GPCR 13:45 - 14:00 Neža Koritnik Cryo-EM studies of filamentous potyviruses and their virus-like particles 14:00 - 14:30 tea/coffee, refreshments 14:30 - 14:45 Zala Živič Stalk domain of SARS-CoV-2 spike protein and its structural polymorphism 14:45 - 15:00 Tea Sinožić Structural aspects of the mutations associated to Type B Kufs disease (CLN13) 15:00 - 15:15 Kity Požek Structural and functional aspects of VaaMPIII-3: a disintegrin-like/cysteine-rich protein from the venom of the nose-horned viper 15:15 - 15:30 Anastasija Panevska The unique story of OlyA6: From specific lipid binding to potential biopesticide 15:30 - 15:45 Marjetka Podobnik Closing remarks by the head of the Department for Molecular Biology and Nanobiotechnology x Abstracts Structural studies of cnidarian pore-forming toxins, 9:05 actinoporins, using cryo-EM Gašper Šolinc1, Marija Srnko1, Ana Crnković1, Gregor Anderluh1, Marjetka Podobnik1 1Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry (Slovenia) Pore-forming toxins are a diverse group of proteins that form pores in lipid membranes. The final pore complex usually consists of circularly arranged protomers of one or more protein species, crossing the membrane with their hydrophobic regions. In some cases, as in actinoporins, lipids are not only the medium in which the pores float but also a building block of the pore. These relatively large protein-lipid complexes are ideally suited for cryo-EM, a powerful and versatile tool in structural biology. In our work, we use the single particle analysis approach to study both solubilized actinoporin pores and pores embedded in lipid membranes. 2 Cryo-EM structure of a track for a completely de novo designed random protein roller 9:30 Ajasja Ljubetič*1,2, Hao Shen*1, Eric Lynch*1, David Baker1,3 1Department of Biochemistry and Institute for Protein Design, University of Washington (USA); 2Department of Synthetic Biology and Immunology, National Institute of Chemistry (Slovenia); 3Howard Hughes Medical Institute, University of Washington (USA) Powered protein walkers such as kinesin, dynein or myosin 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 characterization of a random protein walker that can diffuse along micro-meter long fibers. This represents a scaffold for future powered molecular robots. In particular, I will focus on the track. We have determined the structure of the helical track using Cryo-EM and a partial brute-force search over helical parameters. 3 NMR insights into a G-quadruplex formation in 9:55 the 5’-UTR region of the RANKL gene Martina Lenarčič Živković1, Lara Rems*2, Janez Plavec*1,2,3 1Slovenian NMR Centre, National Institute of Chemistry (Slovenia); 2Faculty of chemistry and chemical technology, University of Ljubljana (Slovenia); 3EN-FIST Centre of Excellence (Slovenia) NMR is one of the most powerful spectroscopic techniques in structural biology, allowing the structures of nucleic acids to be determined at atomic resolution under near-physiological conditions. In addition to the well-known double helix, nucleic acids can adopt various non-canonical structures, such as G-quadruplexes formed by G-rich sequences, which may play an important role in cell regulation. Our work focuses on the G-rich sequence from the 5’-UTR region of the RANKL gene, whose excessive activity may influence the occurrence of osteoporosis. NMR study reveals the formation of G-quadruplexes at the DNA and RNA levels and suggests their influence on the regulation of the RANKL gene. 4 Rigidity and flexibility of structure govern specificity and promiscuity of cysteine cathepsins 10:55 Jure Loboda*1,3, Livija Tušar*1,2, Francis Impens*4, Piotr Sosnowski2, Emmy Van Quickelberghe4, Robert Vidmar1, Hans Demol4, Koen Sedeyn5, Xavier Saelens5, Matej Vizovišek1, Marko Mihelič1, Marko Fonović1, Jaka Horvat6, Gregor Kosec6, Boris Turk1,7, Kris Gevaert**4, Dušan Turk**1,2 1Department for Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute (Slovenia); 2Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins (CIPKeBiP) (Slovenia); 3The Jožef Stefan International Postgraduate School (Slovenia); 4VIB-UGent Center for Medical Biotechnology and UGent Department of Biomolecular Medicine, Technologiepark-Zwijnaarde (Belgium); 5VIB-UGent Center for Medical Biotechnology, Department for Biochemistry and Microbiology, Ghent University (Belgium); 6Acies Bio d.o.o. (Slovenia); 7Faculty of Chemistry, University of Ljubljana (Slovenia) Statistical analysis of large-scale proteomics data prompted us to address the elusive specificity of cysteine cathepsins. Thirty peptidyl sequences, representing a variety of all seven clusters of cathepsin V substrates, were syn-thesized. Structural analysis showed that the heterogeneous positions (substrate positions with non-normal residue distribution) bind to structurally restraint regions, whereas homogeneous positions (normal distribution) ex-ploit structural variability of the protease. Taken together, the combination of cathepsin specificity and promiscuity is explained by the restraining of specific substrate-binding interactions resembling the lock and key mechanism, complemented by the induced fit and conformational variability of promiscuous parts of the binding region. 5 Measurements of (sub)cellular Ca2+ and cAMP dynamics in single astrocytes using confocal 11:20 microscopy Anemari Horvat1,2, Samo Pirnat1,2, Dorian Dolanc1, Matjaž Stenovec1,2, Robert Zorec1,2, Nina Vardjan1,2 1Laboratory of Neuroendocrinology - Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana (Slovenia); 2Laboratory of Cell Engineering, Celica Biomedical (Slovenia) Advances in optical microscopy, together with constantly evolving tools and applications are making important contributions to our understanding of cell physiology including the dynamics of intracellular signalling mechanisms. Ca2+ and cAMP are important intracellular signals regulating cellular processes. Using real-time confocal microscopy in combination with genetically encoded fluorescent nanosensors and fluorescent dyes, we studied (sub)cellular dynamics of Ca2+ and cAMP signalling in single astrocytes, abundant and heterogeneous neuroglial cells. We found distinct temporal properties of Ca2+ and cAMP signalling (phasic Ca2+ and v10-fold faster tonic cAMP response) and subcellular distribution of cAMP levels in astrocytes, which may explain why Ca2+ and cAMP signals regulate astroglial cellular processes with distinct temporal dynamics. 6 Using small-angle X-ray scatering to determine the shape of decorated coiled-coil protein origami 13:00 Žiga Strmšek1,2, Anja Tušar1, Neža Pavko1, Jana Aupič1, Fabio Lapenta1, Roman Jerala1,2 1Department of Synthetic Biology and Immunology, National Institute of Chemistry (Slovenia); 2EN-FIST Centre of Excellence (Slovenia) Coiled-coil protein origami (CCPO) is a de novo, rationally designed type of protein folds, composed by concatenating basic building modules, coiled-coils (CC) into a single polypeptide chain, that form characteristic polyhead-ral shapes. CCPO have unique characteristics, such as lack of traditional hydrophobic core, which has been substituted by a hydrophilic cavity; therefore structural determination by methods, such as X-ray crystalography, or cryoEM prove challenging, therefore alternative approaches had to be investigated. Small-angle X-ray scattering (SAXS) has been proven to be a good method for CCPO shape determination, which coupled with in-house SAXS at NIC offers high throughput and robust pipeline, thus aiding further CCPO development. 7 The cryo-transmission electron microscopy facility 13:15 at the National Institute of Chemistry Matic Kisovec1, Marjetka Podobnik1 1Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry (Slovenia) The cryo-EM facility at the National Institute of Chemistry was estab-lished in 2019 and remains the only one in the region. It includes the 200 kV Glacios cryo-transmission electron microscope, which can perform single particle analysis (SPA), tomography (cryo-ET) and microcrystal electron diffraction (MicroED). Samples are vitrified by plunge-freezing (Vitrobot), and the Falcon 3 detector enables acquisition of high-quality data. High performance computing (HPC) infrastructure is available for data storage and analysis. The cryo-EM facility is part of the Centre for Molecular Interactions and Structural Biology within the department D11 and is open to internal and external users. 8 Regulation of L-lactate metabolism via GPCR 13:30 D. Dolanc1, T. M. Zorec2,3, Z. Smole1, A. Maver1, A. Horvat1,3, T. Pillaiyar4, S. Trkov Bobnar3, N. Vardjan1,3, M. Kreft2,3,5, H. H. Chowdhury1,3, R. Zorec1,3 1Laboratory of Neuroendocrinology, Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana (Slovenia); 2Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana (Slovenia); 3Laboratory of Cell Engineering, Celica Biomedical (Slovenia); 4Pharmaceutical/Medicinal Chemistry and Tübingen Center for Academic Drug Discovery, Institute of Pharmacy, Eberhard Karls University Tübingen (Germany); 5Department of Biology, Biotechnical Faculty, University of Ljubljana (Slovenia) Aerobic glycolysis represents the conversion of D-glucose to L-lactate de-spite the presence of oxygen. We investigated how L-lactate, acting as a fuel and a signal, regulates aerobic glycolysis. L-lactate signals via the activation of L-lactate-sensitive receptors such as the G-protein coupled receptor GPR81 or yet-unidentified plasma membrane receptor(s). We used fluorescence microscopy and FRET (fluorescence resonance energy transfer) nanosensors to see if agonists known to target GPR81 and other receptors determined by bioinformatics cause changes in cytosolic L-lactate in cells with knocked-out GPCR genes and wild type cells. The results revealed a new orphan GPCR that mediates L-lactate synthesis. 9 Cryo-EM studies of filamentous potyviruses and 13:45 their virus-like particles Neža Koritnik1, Andreja Kežar1, Luka Kavčič1, Magda Tušek-Žnidarič2, Swarnalok De3, Maija Pollari3, Kristiina Mäkinen3, Marjetka Podobnik1 1Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry (Slovenia); 2Department of Biotechnology and Systems Biology, National Institute of Biology (Slovenia); 3Department of Microbiology and Viikki Plant Science Centre, University of Helsinki (Finland) Potyvirus is the largest genus of plant RNA-viruses that infect a wide range of crops, causing significant economic losses worldwide. There are 183 potyviral species known to date and their virions are filamentous and flexible particles, consisting of an RNA-genome encapsidated by multiple copies of helically arranged coat protein (CP). Our research group uses cryo-EM to study potato virus Y (PVY), potato virus A (PVA) and their filamentous virus-like particles (VLPs). VLPs are produced upon expression and self-assembly of potyviral CP in bacterial cells. Despite the high similarity of the amino acid sequences of CPPVY and CPPVA, we found important differences in the quaternary structure of both, viral particles and VLPs. 10 Stalk domain of SARS-CoV-2 spike protein and its structural polymorphism 14:30 Zala Živič1, Žiga Strmšek2,3, Marko Novinec1, Jurij Lah1, San Hadži1,2 1Faculty of Chemistry and Chemical Technology, University of Ljubljana (Slovenia); 2Department of Synthetic Biology and Immunology, National Institute of Chemistry (Slovenia); 3EN-FIST Centre of Excellence (Slovenia) Spike trimer plays a key role in SARS-CoV-2 infection and vaccine development. It consists of a globular head and a flexible stalk domain. While the head has been extensively studied, properties of the adjoining stalk are poorly understood. We characterized the coiled-coil formation and thermodynamic stability of the stalk domain and its segments. We found that the N-terminal segment of stalk remains disordered in solution, while the C-terminal stalk segment forms a trimeric coiled-coil in solution, which becomes significantly stabilized in the context of full-length stalk. Its crystal structure reveals a novel antiparallel tetramer coiled-coil with unusual hydrophobic core packing. 11 Structural aspects of the mutations associated to 14:45 Type B Kufs disease (CLN13) Tea Sinožić1,2, Veronika Stoka1,3 1Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute (Slovenia); 2PhD Program in Biomedicine, Faculty of Medicine, University of Ljubljana (Slovenia); 3Jožef Stefan International Postgraduate School (Slovenia) Cathepsin F is a lysosomal cysteine protease with some unique features. However, due to the difficulties of producing a recombinant protein in sufficient quantities, the enzyme is still poorly characterized. Therefore, a sequence-based approach was crucial to assess its suitability from cloning to 3D structure determination. On the available 3D structure of the human cathepsin F mature form, we showed CLN13 mutations have a destabilizing effect on the structure, confirming their detrimental effect on the enzyme’s function. Furthermore, using a novel cell-free protein expression system, we expressed wild type human cathepsin F in sufficient amounts for its further characterization. 12 Structural and functional aspects of VaaMPIII-3: a disintegrin-like/cysteine-rich protein from the venom of the nose-horned viper 15:00 Kity Požek1, Adrijana Leonardi1, Igor Križaj1 1Department of Molecular and Biomedical Sciences, Jožef Stefan Institute (Slovenia) Disintegrin-like/cysteine-rich (DC) proteins, haemostatically active toxins found in viperid venoms, were originally interpreted as proteolytic prod-ucts of the P- III class of snake venom metalloproteinases (SVMPs). However, the recently discovered DC protein VaaMPIII-3 from the venom of the nose-horned viper (Vipera a. ammodytes, Vaa) was found to be encoded per se by a P- III SVMP-like gene lacking the entire MP-coding region and part of the disintegrin-like domain. This defined a new subclass of SVMPs called P-IIIe. We purified VaaMPIII-3 from the venom and determined its biochemical and some functional properties. We have constructed a 3D ho-mology model of VaaMPIII-3 to predict its additional traits. We will present our latest findings. 13 The unique story of OlyA6: From specific lipid 15:15 binding to potential biopesticide Anastasija Panevska1, Matej Milijaš Jotić1, Ioan Iacovache2, Špela Modic3, Jaka Razinger3, Rok Kostanjšek1, Kristina Sepčić1 1Department of Biology, Biotechnical Faculty, University of Ljubljana (Slovenia); 2Institute of Anatomy, University of Bern (Switzerland); 3Agricultural Institute of Slovenia (Slovenia) Ostreolysin A6 (OlyA6), an aegerolysin from the fungal genus Pleu-rotus, interacts strongly with membranes containing ceramide phospho-ethanolamine (CPE), the major lipid component in insects. Together with a 59-kDa protein partner with a MACPF domain, pleurotolysin B (PlyB), OlyA6 forms transmembrane pores and has potent insecticidal activity against selected pests. Using cryo-electron microscopy, we investigated the molecular mechanism of insecticidal activity of the OlyA6/PlyB complex in insects. The results strongly suggest that the molecular mechanism of OlyA6/PlyB is based on specific interactions of OlyA6 with CPE and the formation of transmembrane pores in the presence of PlyB in the insect midgut. 14 September 28th, 2022 Department of Molecular Biology and Nanobiotechnology National Institute of Chemistry, Ljubljana, Slovenia Document Outline Welcome Dobrodošli Welcome Contents Sponsors of the Minisymposium Schedule Abstracts Structural studies of cnidarian pore-forming toxins, actinoporins, using cryo-EM (Gašper Šolinc, Marija Srnko, Ana Crnkovic, Gregor Anderluh, Marjetka Podobnik) Cryo-EM structure of a track for a completely de novo designed random protein roller (Ajasja Ljubetic*, Hao Shen*, Eric Lynch*, David Baker) NMR insights into a G-quadruplex formation in the 5'-UTR region of the RANKL gene (Martina Lenarcic Živkovic, Lara Rems*, Janez Plavec*) Rigidity and flexibility of structure govern specificity and promiscuity of cysteine cathepsins (Jure Loboda*, Livija Tušar*, Francis Impens*, Piotr Sosnowski, Emmy Van Quickelberghe, Robert Vidmar, Hans Demol, Koen Sedeyn, Xavier Saelens, Matej Vizovišek, Marko Mihelic, Marko Fonovic, Jaka Horvat, Gregor Kosec, Boris Turk, Kris Gevaert**, Dušan Turk**) Measurements of (sub)cellular Ca and cAMP dynamics in single astrocytes using confocal microscopy (Anemari Horvat, Samo Pirnat, Dorian Dolanc, Matjaž Stenovec, Robert Zorec, Nina Vardjan) Using small-angle X-ray scatering to determine the shape of decorated coiled-coil protein origami (Žiga Strmšek, Anja Tušar, Neža Pavko, Jana Aupic, Fabio Lapenta, Roman Jerala) The cryo-transmission electron microscopy facility at the National Institute of Chemistry (Matic Kisovec, Marjetka Podobnik) Regulation of L-lactate metabolism via GPCR (D. Dolanc, T. M. Zorec, Z. Smole, A. Maver, A. Horvat, T. Pillaiyar, S. Trkov Bobnar, N. Vardjan, M. Kreft, H. H. Chowdhury, R. Zorec) Cryo-EM studies of filamentous potyviruses and their virus-like particles (Neža Koritnik, Andreja Kežar, Luka Kavcic, Magda Tušek-Žnidaric, Swarnalok De, Maija Pollari, Kristiina Mäkinen, Marjetka Podobnik) Stalk domain of SARS-CoV-2 spike protein and its structural polymorphism (Zala Živic, Žiga Strmšek, Marko Novinec, Jurij Lah, San Hadži) Structural aspects of the mutations associated to Type B Kufs disease (CLN13) (Tea Sinožic, Veronika Stoka) Structural and functional aspects of VaaMPIII-3: a disintegrin-like/cysteine-rich protein from the venom of the nose-horned viper (Kity Požek, Adrijana Leonardi, Igor Križaj) The unique story of OlyA6: From specific lipid binding to potential biopesticide (Anastasija Panevska, Matej Milijaš Jotic, Ioan Iacovache, Špela Modic, Jaka Razinger, Rok Kostanjšek, Kristina Sepcic)