Articles Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX
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Kinetics of D‑Amino Acid Incorporation in Translation Josefine Liljeruhm,‡ Jinfan Wang,†,‡ Marek Kwiatkowski, Samudra Sabari, and Anthony C. Forster* Department of Cell and Molecular Biology, Uppsala University, Husargatan 3, Box 596, 751 24 Uppsala, Sweden
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S Supporting Information *
ABSTRACT: Despite the stereospecificity of translation for L-amino acids (L-AAs) in vivo, synthetic biologists have enabled ribosomal incorporation of D-AAs in vitro toward encoding polypeptides with pharmacologically desirable properties. However, the steps in translation limiting D-AA incorporation need clarification. In this work, we compared D- and L-Phe incorporation in translation by quench-flow kinetics, measuring 250-fold slower incorporation into the dipeptide for the D isomer from a tRNAPhe-based adaptor (tRNAPheB). Incorporation was moderately hastened by tRNA body swaps and higher EF-Tu concentrations, indicating that binding by EF-Tu can be rate-limiting. However, from tRNAAlaB with a saturating concentration of EF-Tu, the slow D-Phe incorporation was unexpectedly very efficient in competition with incorporation of the L isomer, indicating fast binding to EF-Tu, fast binding of the resulting complex to the ribosome, and rate-limiting accommodation/peptide bond formation. Subsequent elongation with an L-AA was confirmed to be very slow and inefficient. This understanding helps rationalize incorporation efficiencies in vitro and stereospecific mechanisms in vivo and suggests approaches for improving incorporation.
used highly minimized substrate analogues with 50S ribosomal subunits,11,12 but such reactions have since been found to be poor models for full-length substrates in translation.13−15 Even experiments with full-length substrates face the challenge that commercial suppliers of D-AAs state ∼1% contamination by the corresponding L-AAs, which is compounded by general preferences for L-AAs by (i) AA-tRNA synthetases (which charge the tRNAs), (ii) elongation factor Tu (EF-Tu, which delivers the AA-tRNA to the ribosomal A/T site, hydrolyses GTP upon cognate codon recognition, and then releases the AAtRNA for accommodation into the A site), and (iii) the ribosomal peptidyl transferase center.16 Contamination issues can be exacerbated by incomplete deacylation in commercially available, native, full-length, deacylated tRNA preparations,7 by typically low ratios of products to substrates, and in crude translation systems by the universal presence of D-AA-tRNA deacylase,17 AA-tRNA synthetases, and L-AAs. Some of these challenges have been addressed by comparison with uncharged tRNA controls and pretreatment of D-AAs with L-AA oxidase,17 by the use of in vitro-synthesized tRNA transcripts,18 and by the use of purified translation systems.10,16 Nevertheless, we found that the translation product from substrates prepared by standard chemical conjugations between D-AAs and dinucleotides was largely from contaminating L-AA10 and that the standard chemistry of these conjugations resulted in significant racemization of the AA due to the basic conditions.19 Basic conditions can be circumvented by using AA-tRNA synthetases17 or ribozymes8 to catalyze aminoacylation (see refs 20−22 for more discussion of the merits of different amino-
D-Amino
acids (D-AAs) are widespread in nature. Apart from their important role in bacterial cell wall synthesis, D-AAs in Amphibia and invertebrates not only contribute to peptide sequence diversity but also provide unique three-dimensional structures for biological activities.1 Although D-AAs sometimes occur at high (millimolar) concentrations in cells, protein synthesis (translation) is extremely selective for incorporation of the L isomers of the 19 chiral, standard, proteinogenic AAs. This specificity is obviously crucial for accurate translation of the genetic code into biologically functional proteins. D-AA chirality also affects peptide degradation and recognition by the immune system, as peptides containing D-AAs are resistant to these processes.2,3 Such resistance is desirable pharmacologically, motivating use of D-AAs for drug discovery and explaining why 3 D-AAs are found in many natural antibiotics. Even a single D-AA per peptide chain can dramatically change the conformation and activity.4 D-AAs are only incorporated efficiently and specifically into polypeptides by nonribosomal synthesis or post-translational isomerization in nature1 or by chemical synthesis in the laboratory. However, there is growing interest in engineering translation in vitro for this purpose because this approach has the advantage of direct encoding for genetic selections of drug candidates.5,6 Thus, investigating the mechanisms governing specificity of translation for L- over D-AAs is important not only for understanding fundamental biological selectivity but also for rationally circumventing limitations on D-AA incorporation in translation for applications. The field of in vitro translation with D-AAs has a long history complicated by low-efficiency incorporations, variabilty in yields, and the potential for contamination by L-AAs. Variabilty was presumably the result of different AAs, tRNA bodies, translation systems, and reaction conditions.7−10 Early studies © XXXX American Chemical Society
Received: October 5, 2016 Accepted: December 26, 2018
A
DOI: 10.1021/acschembio.8b00952 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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ACS Chemical Biology
method was used in part to avoid potential artifacts due to incomplete deacylation in commercially available native tRNA preparations (see the Introduction). The D,L-Phe pair had the advantages of forming diastereoisomeric peptides that separate well on HPLC (see below), of having been used in many translation studies, and of being the only mirror-image AA pair computationally modeled in crystal structures of EF-Tu and the ribosomal A and P sites.7,23 Mindful of known contamination of commercial D-AAs by the L isomers and the potential for partial racemization under the basic conditions in three of the steps during AA-dinucleotide synthesis (see the Introduction, Methods, and the Supporting Appendix), we first needed to ascertain the extent of contamination/racemization during substrate preparation. We used Marfey’s analysis31 to determine that our conditions for conjugation of commercial D-Phe to dinucleotide resulted in an optical purity of ∼98.5% (Supporting Appendix and Supporting Figure 1), similar to our previously determined optical purity of 97.9% of L-Ala conjugated to dinucleotide under exactly the same conditions.19 This gave us confidence in the method, but it should be noted that increasing the percentage of triethylamine/ temperature/aminoacylation time increased racemization,19 so chiral purities from this synthetic method likely differ among laboratories depending on the exact conditions used. Dipeptide formation reactions from the initiator [3H]fMettRNAfMet and D-Phe- or L-Phe-tRNAAlaB (tRNAAla-based adaptor; see Figure 1b) elongator substrates were analyzed by C18 reversed-phase HPLC after incorporation of all active input elongator substrate (Figure 1c,d; see Methods). The [3H]fMetD-Phe dipeptide product (retention time of ∼12.5 min) was clearly synthesized, as it migrated differently from [3H]fMet-LPhe dipeptide product (∼8.5 min) and unreacted fMet (∼4.5 min), and the yields of the reactions of the D and L isomers were comparable. Only near-background contaminations of the dipeptide formed from the other isomer were detected (Figure 1d). Thus, D-AA-tRNA can be prepared by the standard chemoenzymatic ligation method with high chiral purity and is capable of efficient translation incorporation. 250-Fold Slower D-Phe Incorporation Is Moderately Hastened by a Body Swap from tRNAPheB to tRNAAlaB or tRNAProB. Previous individual studies had not accurately measured rates of both D- and L-AA incorporation at physiological temperature, and the estimated rate effects of altered AA chirality differed widely (see the Introduction). Here we charged L-Phe and D-Phe on tRNAPheB and compared the kinetics of dipeptide formation from the initiator [3H]fMettRNAfMet (Figure 2a,b). In these reactions, the final concentration of the initiated 70S ribosomes was in large molar excess over the AA-tRNAs to ensure that the measured rates were independent of the exact tRNA concentration in the factor mix. A concentration of EF-Tu that was high in in vitro terms (10 μM final) was used to facilitate D-Phe-tRNAPheB:EF-Tu:GTP ternary complex formation, with the measured τdip of 11 s (Figure 2b, black triangles; Table 1) being 250× longer than that for L-PhetRNAPheB (Figure 2a and Table 1). The kdip of 0.092 s−1 is 5× the value of 0.020 s−1 reported for natural tRNAPhe charged with DPhe by a ribozyme.7 Given our earlier finding that tRNAAlaB can improve the incorporation rates of non-N-alkyl-L-AAs because of higher EFTu binding affinity compared with tRNAPheB, whereas tRNAProB can increase the incorporation rates of N-methyl-L-AAs by another mechanism, we next tested for effects of swapping the tRNA body. Indeed, swapping tRNAPheB to tRNAProB or
acylation methods). Another challenge is proving that the product contains the expected D-AA rather than the contaminating L-AA, given that the two incorporations lead to products with the same mass and, in the case of incorporation into a long peptide or protein, the same electrophoretic mobility (the same mobility may also result from read-through by a noncognate AAtRNA that can be competitive with a poor substrate). An early way of addressing this was digestion of the product with carboxypeptidase A,11 but the gold standard for measuring D-AA incorporation has become incorporation by a purified translation system into short peptides that have altered mobilities for the D- versus L-AA-containing diastereoisomeric products. This was achieved by reversed-phase HPLC10 (also used here), tricine polyacrylamide gel electrophoresis,8,23 or thin-layer chromatography.7 Kinetic comparisons between D- and L-AAs in translation have been limited. An early study that avoided the need for time points at
