Diamond Annual Review 2019/20

18 19 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 1 9 / 2 0 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 1 9 / 2 0 Macromolecular Crystallography Group Beamline I23 There is a letter K in ribosome Related publication: Rozov A., Khusainov I., El Omari K., Duman R., Mykhaylyk V., Yusupov M.,Westhof E.,Wagner A. &Yusupova G. Importance of potassium ions for ribosome structure and function revealed by long-wavelength X-ray diffraction. Nat. Commun. 10 , 2519 (2019). DOI: 10.1038/s41467-019-10409-4 Publication keywords: X-ray crystallography; Ribosome; Translation R ibosomes are giant protein factories, found in the cells of all lifeforms. They are responsible for the accurate conversion of genetic information into proteins. The most complex RNA-protein assemblies in the cell, they require metal ions to maintain their structure and function. Until recently, the exact type and location of these metal ions had not been determined. An international team of researchers used the long wavelength macromolecular crystallography beamline, I23 (the only beamline in the world allowing access to the required X-ray wavelength), to pinpoint hundreds of potassium ions within bacterial ribosomes. Using this cutting-edge technique, they were able to demonstrate - for the first time - that potassium ions are not only involved in the overall formation of the structure of ribosomal RNA (rRNA) and ribosomal proteins, but also play an essential role in its function. These results fill a considerable gap in our knowledge and could potentially lead to therapeutic applications. Ribosomes and associated molecules, collectively known as the translational apparatus, are the primary target for more than half of current antibiotics. Problems with the translation process are also implicated in a number of human diseases. A greater understanding of the structure of ribosomes will, therefore, be a vital asset for drug discovery. Having increased the precision of our ribosomemodel, the research teamhopes to increase the efficiency of drug design and offer targets for the development of new classes of antibiotics. The ribosome is the largest (~2.5 MDa and 70 Svedberg units (70S) in bacteria and up to ~4 MDa and 80 Svedberg units (80S) in higher eukaryotes) and the most abundant RNA-containing macromolecular complex in cells. Ribosomesareconserved inallkingdomsof life;theyarecomposedofribosomal RNA (rRNA) and proteins unequally distributed among two asymmetric subunits (small and large subunits, called 30S and 50S respectively in bacteria). Generally, the folding of nucleic acid structures, especially larger ones, requires the presence of counter ions, hence, large macromolecular complexes which contain nucleic acids necessitate correspondingly large numbers of various metal ions. Overall improvement of data collection methods in X-ray crystallography and cryo-electron microscopy (cryo-EM) over the last decades has led to more detailed maps of 70S ribosomes, revealing multiple density peaks tentatively attributed to metal ions. A multitude of technical limitations has prevented empirical identification of the nature of these ions and they were generally assigned as magnesium. Magnesium was chosen since it is the best- known RNA stabilizing counter ion, and ribosomes tolerate only a very narrow concentration range during purification and in vitro translation experiments. At the same time the presence of potassium (the most abundant intracellular ion) has also been shown to be essential for these experiments, however its role might have been understated due to a wider range of concentration tolerance 1 . The universal method of localisation of metal ions in macromolecular structures is the geometrical analysis of ion coordination and solvent environment; albeit, this method is subject to severe limitations and does not provide unambiguous assignment even in the case of atomic resolution structures. Moreover, the structures of large macromolecular dynamic complexes generally have poor resolution statistics; the issue is compounded by the simultaneous presence of various ions that can be either co-purified or introduced from the solvent. Taking into account the corresponding values of coordinate errors and atomic displacement parameters, except at very high resolution, it appears almost impossible to distinguish between e.g. Mg 2+, Na + or K + , based only on average M…..O coordination distances (2.1 Å, 2.4 Å and 2.8 Å respectively) and geometry, both by means of manual inspection or automated modelling software protocols. In addition, even at high resolution, the experimentally deduced electron densitymaps are time-averages and, thus, it is not straightforward to assess the simultaneous presence of ions when in proximity. Very few experimental approaches allow tackling such problems. Anomalous X-ray diffraction is a very well-established tool to determine and localise ions in three-dimensional structures 2 . Data collection in the vicinity of the absorption edge of the specific element allows rather precise determination of the element atoms positioning in the structure. The majority of synchrotron beamlines for macromolecular crystallography are optimised for the 6–17.5 keV X-ray range 3 . However, to detect and measure the anomalous signal from potassium around its K-edge (E=3.608 keV) access to lower energies is necessary. I23 at Diamond Light Source is currently the only synchrotron beamline formacromolecular crystallography covering the energy range around the potassium K-edge. We have achieved the direct experimental assignment of K + ions in the full 70S ribosome structure by long-wavelength X-ray crystallography. Registering long-wavelength diffraction from ribosome crystals became possible thanks solely to the novel long-wavelength beamline at Diamond. Experiments at long wavelengths have a number of obstacles to overcome: mainly large diffraction angles and absorption from air in the beam path, the sample mount, solvent around the crystal and the crystal itself. Beamline I23 has been designed to address these challenges by operating in a vacuum environment with a multi-axis goniometer and a large semi-cylindrical area detector 4 . The experimental setup has allowed us to mitigate the strong absorption of X-rays from the crystals, the surrounding mother liquor and sample mounts, limiting the resolution of collected datasets. In the end, our data allowed us to unambiguously assign about 30% of the metal sites as K + . Wehavemanagedtosolvecrystalstructuresoftwo70Sribosomalfunctional complexes with bound messenger RNA (mRNA) and transfer RNAs (tRNAs), representing two distinct stages of translation: initiation and elongation. Our findings provide insights into the role of metal ions in two ribosome active sites, the decoding and peptidyl transferase centers. We demonstrate how K + (but not Mg 2+ ) coordinates mRNA within the decoding center in order to maintain correct frame position during the elongation state (Fig.1). We also localise potassium ions that are required for subunits association and stabilisation of tRNAs, rRNAs, and r-proteins (Fig. 2).These results shed light on the role of metal ions for the ribosome architecture and function, thereby expanding our view on fundamental aspects of protein synthesis. We have managed to elucidate the role of K + in protein synthesis at the three-dimensional level. The distribution of K + ions over the whole mass of the ribosome indicates that this ion is as important as Mg 2+ (Fig. 3). We show that potassium ions are involved in the stabilisation of main functional ligands such as mRNA and tRNAs, as well as ribosomal RNAs and ribosomal proteins, via the interaction with nitrogen and oxygen atoms of side chain residues, nucleotide bases, polypeptide or sugar-phosphate backbones. These observations suggest more global and general functions of K + ions in ribosomal organisation rather than its role as a stabiliser of particular regions of the ribosome or particular type of interactions. Our work adds deeper insights into the mechanism of protein synthesis and opens another dimension in understanding of ribosome organisation. We show that some regions (e.g. the decoding center) require very precise localisation, coordination and nature of metal ions. Our observations display contrasting behaviours for the interactions of potassium and magnesium ions with ribosomal complexes. While magnesium ions tend to bind in pockets around anionic phosphate oxygen atoms with tight geometrical constraints 5 , potassium ions interact with backbone carbonyl groups in protein bending folds and hydroxyl group of riboses or carbonyl groups on bases, especially guanine nucleotides, with a variable number of ligands and larger distance variations. References 1. Nierhaus K. H. Mg2 + , K + , and the ribosome. J. Bacteriol. 196 , 3817 (2014). DOI: 10.1128/JB.02297-14 JB.02297-14 2. Handing K. B. et al. Characterizing metal-binding sites in proteins with X-ray crystallography. Nat Protoc. 13 1062 (2018). DOI: 10.1038/nprot.2018.018 nprot.2018.018 3. Djinovic Carugo K. et al. Softer and soft X-rays in macromolecular crystallography. J. Synchrotron Rad. 12 , 410 (2005). DOI: 10.1107/S0909049504025762 4. Wagner A. et al. In-vacuum long-wavelength macromolecular crystallography. Acta Cryst. D72 , 430 (2016). DOI: 10.1107/S2059798316001078 5. Leonarski F. et al. Mg 2+ ions: do they bind to nucleobase nitrogens? Nucleic Acids Research 45 , 987 (2017) DOI: 10.1093/nar/gkw1175 gkw1175 Funding acknowledgement: This work was supported by the French National Research Agency grants ANR-15-CE11-0021-01, ANR-16-CE11-0007-01, a grant DBF20160635745 from“La Fondation pour la Recherche Médicale”, and the Russian Government Program of Competitive Growth of Kazan Federal University.This study was also supported by the grant ANR-10-LABX-0030-INRT under the frame program Investissements d’Avenir ANR-10-IDEX-0002-02. Corresponding authors: Dr Alexey Rozov, UraniaTherapeutics, arozov@uraniatx.com and Dr Gulnara Yusupova, Institute of Genetics, Molecular and Cellular Biology, gula@igbmc.fr Figure 1: Structural rearrangements of the decoding centre upon binding of A-tRNA. In the initiation complex (left), only one K + ion conserves the architecture of decoding centre through coordination with C518 and G529 of helix 18 (h18) and amino acids Pro45 and Asn46 of protein uS12. The mRNA in the absence of A-tRNA is shifted away from h18, while G530 adopts syn conformation. In the elongation complex (right), mRNA moves towards h18 in order to form base pairing with the A-tRNA. A second K + ion is involved in the codon–anticodon interaction via coordination through C518, G530, Pro45 and U(+6) ribose. Figure 2: (A) Potassium ions (magenta) that mediate interaction of 30S subunit r-proteins (orange) with 16S rRNA (yellow) and 50S subunit r-proteins (blue) with 23S rRNA (light blue). tRNAs and mRNA are omitted from the figure. (B) Interaction of K + ions with the ribosomal proteins from 30S subunit and from 50S subunit. K + ions are shown as magenta spheres, 30S proteins in orange, 50S proteins in blue. Figure 3: Thermus thermophilus 70S elongation complex model contains 211 experimentally distinguished K + ions, 334 Mg 2+ , 251 Mg(H2O) 6 2+ , 1 Zn 2+ and 1 Fe 4 S 4 cluster.