Structure of pyrrolysyl-tRNA synthetase, an archaeal enzyme for genetic code innovation

Abstract

Pyrrolysine (Pyl), the 22nd natural amino acid and genetically encoded by UAG, becomes attached to its cognate tRNA by pyrrolysyl-tRNA synthetase (PylRS). We have determined three crystal structures of the Methanosarcina mazei PylRS complexed with either AMP-PNP, Pyl-AMP plus pyrophosphate, or the Pyl analogue N-epsilon-[(cylopentyloxy)carbonyl]-L-lysine plus ATP. The structures reveal that PylRS utilizes a deep hydrophobic pocket for recognition of the Pyl side chain. A comparison of these structures with previously determined class II tRNA synthetase complexes illustrates that different substrate specificities derive from changes in a small number of residues that form the substrate side-chain-binding pocket. The knowledge of these structures allowed the placement of PylRS in the aminoacyl-tRNA synthetase (aaRS) tree as the last known synthetase that evolved for genetic code expansion, as well as the finding that Pyl arose before the last universal common ancestral state. The PylRS structure provides an excellent framework for designing new aaRSs with altered amino acid specificity.

Fig. 1.

PylRS substrates. (A) The solvent-flattened, experimentally phased map calculated to 2.5 Å resolution and contoured at 2σ for the PylRS structure bound to AMP–PNP. The final refined model of AMP–PNP is shown as sticks and the magnesium ions as blue spheres. (B and C) Unbiased F_o-F_c maps calculated with experimental amplitudes from data collected from PylRS crystals that were soaked with either ATP and Pyl to 2.2 Å resolution (B) or ATP and Cyc to 1.9 Å (C). Calculated amplitudes were generated from a model of PylRS that lacked nucleotide. Both maps are contoured at +1.5σ (green) and −1.5σ (maroon). The position of the side chains from the original AMP–PNP complex are shown in brown, whereas the final refined positions of the side chains for the complex with Pyl–AMP are in yellow (B) or are displayed in green for the complex with Cyc (C). The final positions of the Pyl–AMP and pyrophosphate are in pink (B), and the amino acid substrate Cyc is shown in yellow (C). The final positions of two magnesium ions (blue spheres) were confirmed by anomalous difference maps from crystals that had been soaked with manganese. Based on similarity with LysRS (19), a third metal position (red sphere) was identified, but tentatively modeled as a water due to a lack of anomalous difference density. (D) Chemical diagrams of Lys, Cyc, and Pyl.

Fig. 2.

Structure of PylRS. (A) A secondary structure diagram of PylRS with the amino acid analogue substrate Cyc shown as sticks (yellow) and the Pyl-AMP and pyrophosphate shown as sticks (pink). For clarity, the position of the ATP from the PylRS complex with Cyc was not shown. The conserved class II synthetase motifs 1, 2, and 3 are shown in blue, green, and red, respectively. (B) A stereo diagram of a surface representation of PylRS highlighting the enzyme's deep amino acid substrate-binding pocket. The surface of the enzyme has been made transparent to reveal the positions of the side chains that interact with the Pyl (pink), which is partially occluded by the enzyme.

Fig. 3.

Structures of the amino acid-binding sites of given synthetases with their amino acid substrates. Each panel is oriented similarly. (A) Side chains that interact with substrate are shown as sticks. The interactions of PylRS (yellow) with Pyl–AMP and pyrophosphate (pink) are shown. (B) The recognition of PylRS (green) with the substrate mimic Cyc (yellow) and ATP (purple) is shown. Two other synthetases are shown for comparison with PylRS. PheRS (blue) with Phe–AMP (tan) in C and LysRS (gray) with Lys–AMP (green) in D. Hydrogen bonds are indicated by solid black lines. Water molecules that mediate hydrogen bonds with substrates are shown as red spheres.

Fig. 4.

Phylogenetic trees for the subclass IIc aaRSs are shown. (A) A structural phylogeny with subclass IIa and IIb aaRS structures as outgroups. (B) A sequence-based phylogeny derived from a structure-based, multiple-sequence alignment with AlaRS sequences as the outgroup. Bootstrap support is indicated for major branches. Other bootstrap values were reported previously (16).