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. 2022 Feb 28;50(4):2258-2269.
doi: 10.1093/nar/gkac038.

Folding of VemP into translation-arresting secondary structure is driven by the ribosome exit tunnel

Michal H Kolář  1   2 , Gabor Nagy  1 , John Kunkel  3 , Sara M Vaiana  3 , Lars V Bock  1 , Helmut Grubmüller  1
Affiliations

Folding of VemP into translation-arresting secondary structure is driven by the ribosome exit tunnel

Michal H Kolář et al. Nucleic Acids Res. .

Abstract

The ribosome is a fundamental biomolecular complex that synthesizes proteins in cells. Nascent proteins emerge from the ribosome through a tunnel, where they may interact with the tunnel walls or small molecules such as antibiotics. These interactions can cause translational arrest with notable physiological consequences. Here, we studied the arrest caused by the regulatory peptide VemP, which is known to form α-helices inside the ribosome tunnel near the peptidyl transferase center under specific conditions. We used all-atom molecular dynamics simulations of the entire ribosome and circular dichroism spectroscopy to study the driving forces of helix formation and how VemP causes the translational arrest. To that aim, we compared VemP dynamics in the ribosome tunnel with its dynamics in solution. We show that the VemP peptide has a low helical propensity in water and that the propensity is higher in mixtures of water and trifluorethanol. We propose that helix formation within the ribosome is driven by the interactions of VemP with the tunnel and that a part of VemP acts as an anchor. This anchor might slow down VemP progression through the tunnel enabling α-helix formation, which causes the elongation arrest.

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Figures

Figure 1.
Figure 1.
Overview and secondary structure (SS) characteristics of VemP constructs. (A) Relevant VemP parts with the critical nucleotides at the peptidyl trasferase center (sphere). Two substates of U2506 are denoted -A and -B. (B) VemP stalling conformation (17) in the context of two exit-tunnel proteins uL4 and uL22. (C) Primary structure and a schematic representation of the studied constructs. The VemP sequence is color coded (blue = outer helix, red = inner helix). (D) Structure-based classification by DSSP (33) for the cryo-EM model (H = α-helix, T = turn, B = β-bridge, S = bend) and sequence-based SS predictions of VemP-1 derived by JPred4 (34,35) (H = helix, E = strand) with the reliability values (JPred4-rel, larger values in green are more reliable than low values in red) and NPS@web (36) (E = strand). (E) Secondary structure (SS) content of three VemP constructs as inferred by SESCA analysis of CD spectra in three solvents differing in 2,2,2-trifluorethanol (TFE) molar fraction. For comparison, the SS content of the VemP cryo-EM model (17) in the ribosome tunnel is shown as classified by DSSP.
Figure 2.
Figure 2.
VemP helicity obtained from MD trajectories using DSSP (33). Plot titles refer to in-ribosome (R) or in-solvent (S) simulations, of VemP-1, 2 or 3, using AMBER (AM) or CHARMM (CH) force field, initiated from the helical (hel) or non-helical extended (ext) VemP conformation. (A) Helical content of the peptide as a function of simulation time. Individual trajectories are shown as thin lines, the average over the trajectories is in bold. For clarity, all lines represent running averages over 100 ns. (B) Helical content of individual AAs averaged over time. Several time blocks color-coded from yellow (beginning of simulations) to navy (end of simulations) are averaged over independent trajectories. Lines are separated in time by 500 ns; longer simulations resulted in more lines. Sequence regions classified as helical in the cryo-EM structure are highlighted in red (inner helix) and blue (outer helix) on the horizontal axis.
Figure 3.
Figure 3.
MD-generated model of ribosome-bound VemP. (A) The MD-generated model (in orange) was obtained as an ensemble average of all RNC trajectories (U2506 substates A and B). The cryo-EM model is in gray. (B) Context of the structurally conserved VemP part. W143 stacks to R92 of ribosomal protein uL22 and interacts with F131 of VemP (magenta star). The K144 side chain directs to a pocket formed by the phosphate between pseudouridine Ψ746 and 5-methylcytosine mC747 and with oxygen of C2611 (black star). (C) Residue-wise RMSD between cryo-EM and MD models. The black line represents the RMSD of non-hydrogen atoms, blue bars stand for the RMSD of the VemP backbone. The red arrow indicates structurally conserved region of VemP – the anchor. Sequence regions classified as helical in the cryo-EM structure are highlighted in red (inner helix) and blue (outer helix) on the horizontal axis. (D) Residue-wise root-mean-square fluctuation (RMSF). The mean value over eight independent RNC trajectories is shown as line, the shaded area represents the standard deviation. Sequence regions classified as helical in the cryo-EM are highlighted in red (inner helix) and blue (outer helix) on the horizontal axis.
Figure 4.
Figure 4.
Structural differences resulting from U2506 substates. (A) Residue-wise RMSD between the cryo-EM model and structures averaged over all the trajectories initiated from each substate. The mean RMSD value over the 4 independent trajectories is shown as a line, the shaded area represents the standard error of the mean. On the horizontal axis, the AAs of the inner or outer helix are highlighted in red and blue, respectively. (B) Comparison of the structures averaged over all trajectories initiated from U2506 in substate A (in teal) and the cryo-EM model (in gray). Selected ribosomal nucleotides are represented as sticks, whereas the side chains of residues 143–156 are shown as licorice. (C) Same as b) but for the substate B. (D) The detail of the VemP inner helix and its surroundings as framed in C), but depicted as licorice. The MD-derived model from trajectories initiated from the substate A is is in teal, cryo-EM in gray. Red arrow indicates a dislocation of the helix with respect to the cryo-EM model. (E) Same as d) but for the substate B.
Figure 5.
Figure 5.
Comparison of VemP-1 and VemP-3 in substates A (A), and B (B) in the ribosomal tunnel. VemP-3 substates are compared in (C). The cryo-EM model is shown in gray for reference. Each structure represents the mean conformation of one of the four independent trajectories.
Figure 6.
Figure 6.
A model for anochor-mediated VemP translational arrest. (A) Nascent peptide (NP) elongation proceeds until the anchoring amino acid (AA, green circle) reaches the anchoring point of the tunnel (green star). The anchoring interactions prevent the NP from translocating further, such that secondary structure content increases upstream until a sterical clash with the PTC (red circle) occurs, ultimately causing arrest. For simplicity, less AAs (yellow circles) than present in VemP is shown. (B) Sketch of a possible conformational free energy (G) landscapes of VemP intermediates in the tunnel with respect to a generalized conformation coordinate formula image that characterizes NP translocation. The landscapes corresponds to NP from (A) of 4, 5 and 6 AAs.
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