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Structural Investigations of the Light-Driven Sodium-Pumping Rhodopsin KR2

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Structural Investigations of the Light-Driven Sodium-Pumping Rhodopsin KR2 Structural investigations of the light-driven sodium-pumping rhodopsin KR2 Von der Fakultät für Georessourcen und Materialtechnik der Rheinisсh -Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von M. Sc. Kirill Kovalev aus Yaroslavl, Yaroslavl region, Russland Berichter: Herr Univ.-Prof. Dr. Valentin Gordeliy Herr Prof. Dr. Georg Büldt Herr Prof. Dr. Ernst Bamberg Herr Prof. Dr. Martin Engelhard Tag der mündlichen Prüfung: 07.12.2020 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar Table of contents Table of contents 2 Main results 5 Publications 6 List of Abbreviations 8 1. Introduction 9 1.1. Microbial rhodopsins 9 1.2. Identification of the light-driven Na+ pumps 12 1.3. Biological role(s) of NaRs 14 1.4. Functional and spectroscopic features of NaRs 15 1.5. NDQ motif of NaRs 17 1.6. Oligomeric state of NaRs 20 1.7. High-resolution structures of NaRs 22 1.8. Structures of the KR2 in the intermediate states 26 1.9. Mechanism of light-driven Na+ pumping 26 1.10. Functional conversions of NaRs 29 1.11. NaRs as potential optogenetic tools 31 2. Materials and Methods 33 2.1. Materials 33 2.1.1. Organisms 33 2.1.2. Vectors 33 2.1.3. Genes 33 2.1.4. Oligonucleotides 33 2.1.5. Chemicals for molecular biology 33 2.1.6. Crystallization 33 2.1.7. Crystal harvesting tools 34 2.2. Methods 35 2 2.2.1. Cloning 35 2.2.2. Protein Expression and Purification 37 2.2.3. Measurements of pumping activity in E. coli cells 37 2.2.4. Liposome preparation 38 2.2.5. Measurements of the pumping activity in liposomes 38 2.2.6. Oligomeric state analysis by size exclusion chromatography 38 2.2.7. Crystallization details and crystals preparation 39 2.2.8. Time-resolved visible absorption spectroscopy on KR2 crystals 39 2.2.9. Spectroscopic characterization and accumulation of the intermediate state in KR2 crystals 40 2.2.10. Acquisition and treatment of diffraction data 40 2.2.11. Serial millisecond crystallography data collection and processing 41 2.2.12. Structure determination and refinement 42 2.2.13. Molecular dynamics simulations 42 3. Results and Discussion 44 3.1. High-resolution structure of KR2 in the ground state 44 3.1.1. Crystallization of KR2 under physiological conditions 44 3.1.2. Crystal structure of the pentameric KR2 in the ground state 44 3.1.3. Interprotomer contacts in KR2 complex 48 3.1.4. Comparison with known KR2 structures 50 3.1.5. The second Na+ ion identified at the KR2 surface in the ground state 54 3.1.6. Structural switches in KR2 upon pH decrease 55 3.1.7. Effects of dehydration on KR2 crystals 58 3.1.8. The structure of the monomeric form of KR2 at different pH 59 3.1.9. Structures of K+-pumping mutants of KR2 60 3.1.10. Role of the KR2 pentamerization on the function of the protein 64 3.2. Crystal structure of KR2 in the O-state 68 3.2.1. Accumulation and cryo-trapping of the O intermediate state in KR2 crystals 68 3 3.2.2. Determination of the crystal structure of the O-state of KR2 71 3.2.3. The retinal binding pocket of KR2 in the O-state 73 3.2.4. Transient Na+ ion-binding site inside the KR2 protomer 75 3.2.5. Structure of the KR2 protomer in the O-state 79 3.2.6. Crystal structure of the ground and the O-states of KR2 at room temperature 83 3.2.7. Conformational switches guide Na+ uptake and release in KR2 86 3.2.8. Na+ translocation pathway 91 3.3. Molecular mechanism of light-driven Na+ pumping 99 3.4. Outlook 102 4. References 103 5. Appendix 113 Acknowledgments 119 Abstract 120 4 Main results In the framework of the present dissertation, the structure of the biologically relevant pentameric form of KR2 was obtained under physiological conditions. The structure revealed a large polar water-accessible cavity in the core of the KR2 protomer in the ground state of the protein photocycle. The results were published in Science Advances in 2019. Based on the first result, the structure of the functionally key O intermediate state of KR2 photocycle was obtained by using three alternative techniques. First, the intermediate state was accumulated and cryo-trapped in KR2 crystals upon their illumination with 532 nm laser following by flash-cooling in the cryo-stream. Second, the structure of the O-state was obtained at room temperature using data collection upon continuous light illumination of large single crystals of KR2. Third, the data on the O-state were collected using serial crystallography with the stream of KR2 microcrystals, injected into the X-ray beam of the synchrotron source with simultaneous illumination of the stream by 532 nm laser. All three structures are similar and demonstrate a transient Na+ ion-binding site in the core of KR2 protomer. Based on the structural data we suggest that the mechanism of a light-driven Na+ pumping is likely a chimera of the relay mechanism of proton translocation and passive diffusion of the Na+ ions through the cavities inside KR2. The results were published in Nature Communications in 2020. The structural studies of KR2 are still ongoing. Particularly, the next goal is to obtain the structures of the early K, L, and M states of the pentameric KR2 using time-resolved crystallography at synchrotrons and X-ray free-electron laser sources. 5 Publications T Varaksa, S Bukhdruker, I Grabovec, E Marin, A Kavaleuski, A Gusach, K Kovalev et al. (2020) Metabolic fate of human immunoactive sterols in Mycobacterium tuberculosis. bioRxiv. https://doi.org/10.1101/2020.07.07.192294. N Maliar*, K Kovalev* et al. (2020) Crystal structure of the N112A mutant of the light-driven sodium pump KR2. Crystals. Volume 10, Issue 6, Pages 1-15 K Kovalev, R Astashkin et al. (2020) Molecular mechanism of light-driven sodium pumping. Nature Communications. 11, 2137. A Remeeva, V Nazarenko, I Goncharov, A Yudenko, A Smolentseva, O Semenov, K Kovalev et al. (2020) Effects of proline substitutions on the thermostable LOV domain from Chloroflexus aggregans. Crystals. Volume 10, Issue 4 (256). K Kovalev*, D Volkov*, R Astashkin*, A Alekseev* et al. (2020) High-resolution structural insights into the heliorhodopsin family. PNAS. Volume 117, Issue 8, Pages 4131-4141. D Zabelskii*, A Alekseev*, K Kovalev* et al. (2020). Viral channelrhodopsins: calcium- dependent Na+/K+ selective light-gated channels. bioRxiv. https://doi.org/10.1101/2020.02.14.949966. A Vlasov*, K Kovalev*, S-H Marx*, E Round* et al. (2019) Unusual features of the c-ring of F1FO ATP synthases. Scientific Reports. Volume 9, Issue 1 (18547). A Gusach, A Luginina, E Marin, R Brouillette, É Besserer-Offroy, J-M Longpré, A Ishchenko, P Popov, N Patel, T Fujimoto, T Maruyama, B Stauch, M Ergasheva, D Romanovskaia, A Stepko, K Kovalev, et al. (2019) Structural basis of ligand selectivity and disease mutations in cysteinyl leukotriene receptors. Nature Communications. Volume 10, Issue 1 (5573). D Bratanov*, K Kovalev* et al. (2019) Unique structure and function of viral rhodopsins. Nature Communications. Volume 10, Issue 1 (4939). K Kovalev*, V Polovinkin* et al. (2019) Structure and mechanisms of sodium-pumping KR2 rhodopsin. Science Advances. Vol. 5, no. 4, eaav2671. V Nazarenko, A Remeeva, A Yudenko, K Kovalev et al. (2019) A thermostable flavin-based fluorescent protein from: Chloroflexus aggregans: A framework for ultra-high resolution structural studies. Photochemical and Photobiological Sciences. Volume 18, Issue 7, Pages 1793-1805. O Volkov*, K Kovalev*, V Polovinkin*, V Borshchevskiy* et al. (2017) Structural insights into ion conduction by channelrhodopsin 2. Science. Vol. 358, Issue 6366, eaan8862. 6 I Melnikov, V Polovinkin, K Kovalev et al. (2017) Fast iodide-SAD phasing for high-throughput membrane protein structure determination. Science Advances. Volume 3, Issue 5, e1602952. V Shevchenko*, T Mager*, K Kovalev*, V Polovinkin* et al. (2017) Inward H+ pump xenorhodopsin: Mechanism and alternative optogenetic approach. Science Advances, Volume 3, Issue 9, 1603187. V Borshchevskiy, E Round, Y Bertsova, V Polovinkin, I Gushchin, A Ishchenko, K Kovalev et al. (2015) Structural and functional investigation of flavin binding center of the NqrC subunit of sodium-translocating NADH: Quinone oxidoreductase from vibrio harveyi. PLoS ONE. Volume 10, Issue 3, e0118548. I Gushchin, V Shevchenko, V Polovinkin, K Kovalev et al. (2015) Crystal structure of a light- driven sodium pump. Nature Structural&Molecular Biology. Volume 22, Issue 5, Pages 390-396. *These authors contributed equally to the work 7 List of Abbreviations MR - microbial rhodopsin MP - membrane protein HsBR - bacteriorhodopsin from Halobacterium salinarum PR - proteorhodopsin RSB - retinal Schiff base PDB - protein data bank FTIR - fourier-transform infrared spectroscopy RR - resonance Raman spectroscopy NaR - light-driven sodium-pumping rhodopsin KR2 - light-driven sodium pump from Krokinobacter eikastus IPTG - isopropyl β-D-1-thiogalactopyranoside CCCP - carbonyl cyanide m-chlorophenyl hydrazone TPP+ - tetraphenylphosphonium bromide GPCR - G protein-coupled receptor NMR - nuclear magnetic resonance HS-AFM - high-speed atomic force microscopy SEC - size exclusion chromatography OD600 - optical density at 600 nm wavelength LCP - lipidic cubic phase SBC1 - Schiff base cavity 1 SBC2 - Schiff base cavity 2 IUC - ion-uptake cavity pIRC1 - putative ion-release cavity 1 pIRC2 - putative ion-release cavity 2 WT - wild type 8 1. Introduction 1.1. Microbial rhodopsins Microbial rhodopsins (MRs), also known as type-1 rhodopsins, are light-driven integral membrane proteins (MPs), found in archaea, bacteria, simple eukaryotes and also in viruses. They were shown to be major contributors to the solar energy captured in the ocean1.
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