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. 2019 Jun 7;10(1):2519.
doi: 10.1038/s41467-019-10409-4.

Importance of potassium ions for ribosome structure and function revealed by long-wavelength X-ray diffraction

Alexey Rozov  1   2   3   4   5 , Iskander Khusainov  6   7   8   9   10   11 , Kamel El Omari  12   13 , Ramona Duman  12   13 , Vitaliy Mykhaylyk  12 , Marat Yusupov  6   7   8   9   10 , Eric Westhof  14 , Armin Wagner  15   16 , Gulnara Yusupova  17   18   19   20
Affiliations

Importance of potassium ions for ribosome structure and function revealed by long-wavelength X-ray diffraction

Alexey Rozov et al. Nat Commun. .

Abstract

The ribosome, the largest RNA-containing macromolecular machinery in cells, requires metal ions not only to maintain its three-dimensional fold but also to perform protein synthesis. Despite the vast biochemical data regarding the importance of metal ions for efficient protein synthesis and the increasing number of ribosome structures solved by X-ray crystallography or cryo-electron microscopy, the assignment of metal ions within the ribosome remains elusive due to methodological limitations. Here we present extensive experimental data on the potassium composition and environment in two structures of functional ribosome complexes obtained by measurement of the potassium anomalous signal at the K-edge, derived from long-wavelength X-ray diffraction data. We elucidate the role of potassium ions in protein synthesis at the three-dimensional level, most notably, in the environment of the ribosome functional decoding and peptidyl transferase centers. Our data expand the fundamental knowledge of the mechanism of ribosome function and structural integrity.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Metal ions assignment in T. thermophilus 70S ribosome on the example of elongation complex. a Thermus thermophilus 70S EC represents the elongation state of the ribosome that contains poly-U mRNA with SD sequence and three cognate tRNAPhe in the A-, P- and E-sites. Parts of the central protuberance and the 30S head are omitted for clarity. Abbreviations used: PTC peptidyl transferase center, DC decoding center, SD Shine-Dalgarno. b Thermus thermophilus 70S elongation complex (PDB ID 4V6F) was used as an initial model for ions re-assignment. The same complex was crystallized and data were collected at beamline I23 (Diamond Light Source, UK) at two different wavelengths (3.542 and 3.351 Å, which correspond to energies below and above the K-absorption edge of potassium). Electron density from three independent datasets collected at different wavelengths (1.000 Å (PDB ID 4V6F), 3.542 and 3.351 Å) were used to build an atomic model, which contained 211 experimentally distinguished K+ ions, 334 Mg2+, 251 Mg(H2O)62+, 1 Zn2+ and 1 Fe4S4 cluster in place of 3255 Mg2+ and 3 Zn2+ ions assigned previously. c, d Distribution of K+ and Mg2+ ions within 70S ribosome and each individual subunit. Potassium ions are coordinated in intersubunit bridges B2a and B2c (c). One magnesium ion is coordinated in B2c bridge (in the IC magnesium ion has slightly different position and coordinated via water molecules) (d). OMC stands for 2′-O-methylcytosine. K+ ions colored in magenta, Mg2+ ions in green, solvent shell shown in red, Zn2+ in dim gray, in Fe4S4 cluster in iron colored in gray and sulfur in black. Small ribosomal subunit (30S) parts colored in yellow, large subunit (50S) components in blue. Intersubunit bridges are shown in bright yellow and bright blue. For clarity, ions within ribosome are shown in sphere representation with increased van der Waals radius. Additionally, tRNA and mRNA ligands are omitted from the interface of 30S subunit in c and d
Fig. 2
Fig. 2
Localization of potassium ions in the 70S ribosome decoding center. a Structural rearrangements of the decoding center and its stabilization by potassium ions upon binding of A-tRNA. In the initiation complex (left), only one K+ ion conserves the architecture of decoding center through coordination with C518 and G529 of h18 and amino acid residues Pro45 and Asn46 of protein uS12. The mRNA in the initiation complex in the absence of A-tRNA is shifted away from h18, while G530 adopts energetically unfavorable syn conformation. In the elongation complex (right), in contrast, mRNA is moved towards h18 in order to form base pairing with the A-tRNA. In this scenario, an additional K+ ion is involved in the stabilization of codon–anticodon interaction via coordination through C518, G530 (in anti-conformation), Pro45 and U(+6) ribose. b, c The best fitting coordination geometry was estimated to be square antiprism (coordination number 8) with an RMSD of 0.261 Å for the five identified coordinating atoms positions for the “first” K+ ion in the decoding center (b, left), and bi-capped square antiprism (coordination number 10) with RMSD of 0.319 Å for the five identified coordinating atoms positions for the “second” K+ ion in the decoding center (c, left). In silico modeling shows that Mg2+ ion does not fit into these binding pockets due to its distance and geometry constrains (b, c right). 16S rRNA elements are shown in yellow, nucleotides A/U(+6) and G530 are highlighted by light blue circles. Contacts between K+ and ribosomal components are shown in round dash, U(+6)-G34 base pair is marked by long dash lines, two K+ ions are marked with white star (“first” K+) and black star (“second” K+)
Fig. 3
Fig. 3
Metal ions in the mRNA path in elongation complex. a Along its path in the ribosome, mRNA gets distorted between A- and P- sites (A/P kink) and between P- and E-sites (P/E kink). Together with 16S rRNA nucleotides, these distortions represent negatively charged pockets, favorable for occupation by metal ions like K+ or Mg2+. b A K+ ion was identified in the P/E kink of the elongation complex. According to the distances, this K+ ion is most probably hydrated and may interact with 2-methylthio-N6-isopentenyladenosine (MIA) modification in the P-tRNAPhe and U(-1) in mRNA. c The cation identified in A/P kink was assigned as Mg2+ in the elongation complex, however, we include the possibility that it can be a different ion (probably an ammonium ion). Color code: colors are as in Fig. 2 with unidentified ion shown in gray
Fig. 4
Fig. 4
Potassium ions in the peptidyl transferase center. a Mapped K+ on secondary structure representation of PTC. The 23S rRNA secondary structure was adapted from H. Noller’s lab web site (http://rna.ucsc.edu/rnacenter/images/figs/thermus_23s_2ndry.jpg link active on 04 Sept 2018). b Interface view of the 50S (23S, 5S in light blue, proteins in blue) with three tRNAs. In close up view only PTC and K+ ions (with increased van der Waals radius) are shown. c Interface view of the 50S, with central protuberance omitted (colors are as in b)
Fig. 5
Fig. 5
Potassium ions support essential structural elements of 30S subunit. a–c decoding center forming helix 18, d neck, e, f helix h44. 16S rRNA is presented in ribbon (center). The head is colored in khaki, body in light olive green. At the interface portion of 16S rRNA, h44 and h18 are highlighted in yellow, neck between the head and the body colored in orange-red. Decoding center is marked with a blue circle. One of the potassium ions coordinated in h18 (b) was found only in one ribosome of the asymmetric unit of initiation complex and it is colored in violet and represented as small sphere in the overview panel
Fig. 6
Fig. 6
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
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