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Review
. 2019 Nov 8;294(45):16535-16548.
doi: 10.1074/jbc.REV119.008166. Epub 2019 Oct 7.

Chiral checkpoints during protein biosynthesis

Santosh Kumar Kuncha  1   2 , Shobha P Kruparani  1 , Rajan Sankaranarayanan  3
Affiliations
Review

Chiral checkpoints during protein biosynthesis

Santosh Kumar Kuncha et al. J Biol Chem. .

Abstract

Protein chains contain only l-amino acids, with the exception of the achiral glycine, making the chains homochiral. This homochirality is a prerequisite for proper protein folding and, hence, normal cellular function. The importance of d-amino acids as a component of the bacterial cell wall and their roles in neurotransmission in higher eukaryotes are well-established. However, the wider presence and the corresponding physiological roles of these specific amino acid stereoisomers have been appreciated only recently. Therefore, it is expected that enantiomeric fidelity has to be a key component of all of the steps in translation. Cells employ various molecular mechanisms for keeping d-amino acids away from the synthesis of nascent polypeptide chains. The major factors involved in this exclusion are aminoacyl-tRNA synthetases (aaRSs), elongation factor thermo-unstable (EF-Tu), the ribosome, and d-aminoacyl-tRNA deacylase (DTD). aaRS, EF-Tu, and the ribosome act as "chiral checkpoints" by preferentially binding to l-amino acids or l-aminoacyl-tRNAs, thereby excluding d-amino acids. Interestingly, DTD, which is conserved across all life forms, performs "chiral proofreading," as it removes d-amino acids erroneously added to tRNA. Here, we comprehensively review d-amino acids with respect to their occurrence and physiological roles, implications for chiral checkpoints required for translation fidelity, and potential use in synthetic biology.

Keywords: D-amino acids; amino acid; aminoacyl tRNA synthetase; checkpoint control; chirality; genetic code; proofreading; proteins; ribosome; stereoselectivity; transfer RNA (tRNA); translation; translation elongation factor.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.
Ramachandran plot for proteins with l- and d-chirality. Shown in blue is the structure of HIV protease (PDB entry 4HVP), and shown in red is the modeled structure of the same protein with inverted chirality. A Ramachandran plot showing the dihedral angles is color-coded according to the cartoon.
Figure 2.
Figure 2.
Models for enantioselectivity of proteins. A, l- and d-stereoisomers of amino acid; Cα represents the chiral center. B, TPA model: A, B, and C are three points on the ligands that interact with A′, B′, and C′ of the protein, and hence only ligand i can bind, and not ii. In this model, the entry/approach of the ligand is assumed to be fixed (i.e. here it is shown from above the binding plane). C, four-location model. As per this model, the fourth site is essential and decides the entry of the ligand. D′ and D″ fix the entry of the ligand. However, D′ and D″ will bind ligands with opposite chirality (adapted from Refs. and 47). This research was originally published in Nature. Ogston, A. G. Interpretation of experiments on metabolic processes, using isotopic tracer elements. Nature. 1948; 162:963. ©Springer Nature; and Nature. Mesecar, A. D., and Koshland, D. E., Jr. A new model for protein stereospecificity. Nature. 2000; 403:614–615. ©Springer Nature.
Figure 3.
Figure 3.
Amino acid selectivity of tyrosyl-tRNA synthetase. A, l-Tyr captured in the TyrRS amino acid–binding pocket; amino group, carbonyl oxygen, and the side chain hydroxyl groups help in substrate selectivity (PDB entry 1J1U). B, d-Tyr modeled in the TyrRS amino acid–binding pocket. C, overlap of d- and l-Tyr, clearly depicting the mode of amino acid binding and also explaining the weak enantioselectivity of TyrRS.
Figure 4.
Figure 4.
Elongation factor in complex with l-Phe-tRNAPhe. A, crystal structure of a ternary complex, EF-Tu (surface representation in blue), GTP (shown as spheres in the GTP-binding site), and l-Phe-tRNAPhe (tRNA shown in a wire and stick representation with amino acids as spheres) (PDB entry 1TTT). B, zoomed in view of an amino acid (of l-Phe-tRNAPhe) bound in the amino acid–binding pocket of EF-Tu. C, stick representation of the amino acid–binding pocket showing key interactions with the ligand (l-Phe).
Figure 5.
Figure 5.
Chiral discrimination at the A-site of the ribosome. Shown is the structure of the ribosome showing the A-site in complex with the aa-tRNA analog and the P-site with peptidyl-tRNA (taken from PDB entry 1VY4). The U2506 nucleotide of the 23S rRNA acts as a chiral discriminatory residue by allowing l-aa-tRNA to optimally orient for peptide bond formation but not the d-aa-tRNA. A, complex (PDB entry 6N9E) with l-aa-tRNA analog (CC-puromycin; for the sake of clarity, the hydroxy methyl of methyltyrosine is removed) shown in magenta. B, complex (PDB entry 6N9F) of d-aa-tRNA (ACCA-d-Phe) shown in yellow.
Figure 6.
Figure 6.
Enantioselectivity mechanism of DTD. A, crystal structure of DTD in complex with d-Tyr3AA (PDB entry 4NBI). The active site is at the dimeric interface (each monomer is colored differently). The ligand is shown in magenta, and the GP-motif is shown in green sticks. B, d-Tyr in the active site; the side chain projects out of the pocket, and the GP-motif forms the base. C, l-Tyr modeled in the active site, clearly showing the side chain clash with the GP-motif.
Figure 7.
Figure 7.
Chiral checkpoints for maintaining the enantiomeric fidelity of proteome. The translation apparatus, which includes aaRS, EF-Tu, and ribosome, has a preference for l-amino acids/l-aa-tRNAs but is porous to d-amino acids/d-aa-tRNAs as well. DTD specifically decouples d-aa-tRNAs and helps to recycle the tRNAs, and it also aids in maintaining the homochirality of cellular proteome.
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