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. 2019 Feb 28;47(4):2089-2100.
doi: 10.1093/nar/gky1211.

Mechanistic insights into the slow peptide bond formation with D-amino acids in the ribosomal active site

Sergey V Melnikov  1 , Nelli F Khabibullina  2 , Elisabeth Mairhofer  3 , Oscar Vargas-Rodriguez  1 , Noah M Reynolds  1 , Ronald Micura  3 , Dieter Söll  1   4 , Yury S Polikanov  2   5
Affiliations

Mechanistic insights into the slow peptide bond formation with D-amino acids in the ribosomal active site

Sergey V Melnikov et al. Nucleic Acids Res. .

Abstract

During protein synthesis, ribosomes discriminate chirality of amino acids and prevent incorporation of D-amino acids into nascent proteins by slowing down the rate of peptide bond formation. Despite this phenomenon being known for nearly forty years, no structures have ever been reported that would explain the poor reactivity of D-amino acids. Here we report a 3.7Å-resolution crystal structure of a bacterial ribosome in complex with a D-aminoacyl-tRNA analog bound to the A site. Although at this resolution we could not observe individual chemical groups, we could unambiguously define the positions of the D-amino acid side chain and the amino group based on chemical restraints. The structure reveals that similarly to L-amino acids, the D-amino acid binds the ribosome by inserting its side chain into the ribosomal A-site cleft. This binding mode does not allow optimal nucleophilic attack of the peptidyl-tRNA by the reactive α-amino group of a D-amino acid. Also, our structure suggests that the D-amino acid cannot participate in hydrogen-bonding with the P-site tRNA that is required for the efficient proton transfer during peptide bond formation. Overall, our work provides the first mechanistic insight into the ancient mechanism that helps living cells ensure the stereochemistry of protein synthesis.

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Figures

Figure 1.
Figure 1.
Chemical synthesis of a short hydrolysis-resistant D-phenylalanyl-tRNA analog. (A) Synthetic route. Letters indicate specific reaction conditions as follows: (a) 1.1 equivalent of Fmoc-Cl, Na2CO3, in 1,4-dioxane/H2O, room temperature, 7 h, yield 86% for compound 2; (b) 1.3 equivalent of Fmoc-D-Phe, 1.3 equivalent of O-(Benzotriazole-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate, 1.3 equivalent of 1-hydroxybenzotriazole hydrate, 1.5 equivalent of N,N-diisopropylethylamine in DMF, room temperature, 14 h, yield 70% for compound 4; (c) i. 2.5 equivalent of adipic acid bis(pentafluorophenyl)ester, 1 equivalent of 4-(N,N-dimethylamino)pyridine in N,N-dimethylformamide/pyridine (1/1, v/v), room temperature, 1 h, yield 52% of crude ester; ii. ∼1 equivalent of amino-functionalized polystyrene support (GE Healthcare, Custom Primer SupportTM 200 Amino), pyridine, N,N-dimethylformamide, room temperature, one day, loading: 37 μmol/g for solid-support 5. (d) RNA solid-phase synthesis, deprotection, and purification. DMT — 4,4′-dimethoxytrityl, Fmoc — N-(9- fluorenyl)methoxycarbonyl. (B) Anion-exchange HPLC profiles of crude (top) and purified (bottom) ACCA-D-Phe conjugate. Anion-exchange chromatography conditions: Dionex DNAPac PA-100 (4 × 250 mm) column; temperature: 60°C; flow rate: 1 ml/min; eluant A: 25 mM Tris–HCl (pH 8.0), 6 M urea; eluant B: 25 mM Tris–HCl (pH 8.0), 6 M urea, 500 mM NaClO4; gradient: 0–40% B in A within 25 min; UV detection at 260 nm. (C) LC-ESI mass spectra of the purified product ACCA-D-Phe (compound 6).
Figure 2.
Figure 2.
The electron density maps allow to unambiguously position L- and D-amino acid side chains bound to the ribosomal active site. (A, B) Chemical structures of the L-aminoacyl-tRNA mimic CC-Pmn (A), and of the D-aminoacyl-tRNA mimic ACCA-D-Phe (B). The amino acid moieties of L-methyl-tyrosine and D-phenylalanine are highlighted in blue and red, respectively. (C, D) Unbiased Fo-Fc electron difference Fourier maps of CC-Pmn (C), and ACCA-D-Phe (D). The refined model of each compound is displayed in its respective electron density map before the refinement (green mesh). Carbon atoms are colored yellow for ACCA-D-Phe, and magenta for CC-Pmn. Nitrogens are colored blue; oxygens are red; phosphorus atoms are orange. Each of the difference electron density maps is contoured at 2.7σ. Note that, due to the presence of an additional 5′-terminal adenine nucleotide in the ACCA-D-Phe in comparison to the CC-Pmn, each of these compounds can be unambiguously distinguished in the electron density maps. (E, F) Comparison of the current structures with the previously reported structures of the A-site-bound short and full-length tRNA substrates. Shown are superimposed ribosome-bound (E) CC-Pmn (magenta, current model) or (F) ACCA-D-Phe (yellow, current model) and CC-hPmn (blue, PDB entry 1VQN (48)), CC-Pmn (red, PDB entry 1VY7 (37)), and Phe-NH-tRNAPhe(green, PDB entry 1VY4 (37)). All structures were aligned based on domain V of the 23S rRNA. Note that differences between the compared structures of the A-site substrates are within experimental error.
Figure 3.
Figure 3.
D-aminoacyl-tRNA analog establishes canonical A-loop interactions. Watson-Crick base-pairing between the penultimate cytidine of the (A) L-Phe-tRNAPhe (green, PDB entry 1VY4 (37)) or (B) D-aminoacyl-tRNA analog ACCA-D-Phe (yellow) and the nucleotide G2553 in the A-loop (Helix 92) of the 23S rRNA (light blue). Note that these A-loop interactions play a key functional role in accommodation and proper positioning of the substrate in the A site of the ribosome.
Figure 4.
Figure 4.
Side chains of both L- and D-amino acids occupy the A-site cleft of the ribosome. (A, B) Overview of the CC-Pmn (magenta) and ACCA-D-Phe (yellow) binding sites in the T. thermophilus 70S ribosome viewed from the PTC down the tunnel as indicated by the inset (A), or as a cross-cut section through the ribosome (B). The 30S subunit is shown in light yellow, the 50S subunit is in light blue, the mRNA is in green, and the P-site tRNA is in dark blue. (C–F) Close-up views of the CC-Pmn (C, D) and ACCA-D-Phe (E, F) bound in the A-site cleft of the PTC. The E. coli nucleotide numbering is used throughout. In (C, D), H-bond between the α-amino group and the 2′-OH of the A76 of the P-site tRNA is shown with the black dotted line. This H-bond is pivotal to optimally orient α-amine for an in-line nucleophilic attack onto the carbonyl carbon of the P-site substrate (red arrow). Note that the formation of the same H-bond is not plausible for ACCA-D-Phe because its α-amino group is located further away and oriented towards the nucleotide U2506 (red dotted line). The ability of this group to attack the P-site substrate from this remote location is expected to be reduced due to the non-optimal geometry (curved red arrow). In (D, F), the aromatic side chains of the CC-Pmn and ACCA-D-Phe are highlighted by semi-transparent spheres to illustrate their tight binding in the A-site cleft. Also in (D, F), the observed deacylated P-site tRNAiMet (dark blue) is superimposed with the aminoacylated fMet-tRNAiMet (light blue, PDB entry 1VY4 (37)) based on alignment of the 23S rRNA. Note that the superimposed tRNAs structures are nearly identical even though one is determined at 3.7Å (observed) and the other – at 2.55Å (modeled).
Figure 5.
Figure 5.
Reactive conformation of the D-amino acid is likely prevented by the conserved rRNA residues in the peptidyl-transferase center. (A) Comparison of the observed structures of the L- and D-aminoacyl-tRNA analogs bound to the ribosome. Shown are the 23S-rRNA-aligned energy-minimized conformations of CC-Pmn (magenta) and ACCA-D-Phe (yellow) bound to the ribosomal A site. Note that, due to the opposite chirality of the Cα-atoms, the α-amino group of the D-phenylalanine is positioned further away from the carbonyl carbon of the P-site substrate resulting in reduced reactivity. (B) The conformation of the D-phenylalanine, in which its α-amino group is aligned for the optimal nucleophilic attack onto the carbonyl carbon of the P-site substrate. In this state, the side chain of D-amino acid (especially Cβ-atom) severely clashes with the key functional nucleotide U2506 of the PTC. (C) Mutations in the 23S rRNA that improve ribosomal usage of D-amino acids. Relative locations of the 23S rRNA residues A2451 and C2452 forming the A-site cleft (light blue spheres) and the residues G2447, A2448, U2449 and A2450, whose mutations improve utilization of the D-amino acids by the ribosome (blue). Shown is the close-up view of the PTC with bound P-site tRNA (dark blue) and A-site short substrate ACCA-D-Phe (yellow). Note that residues G2447 and A2450 (blue spheres) are located near the A-site cleft. Mutations of these purine residues to smaller pyrimidines might lead to either an increased size of the A-site cleft or increased flexibility of the adjacent residues forming the A-site cleft.
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