Crystal Structure of Pyrrolysyl-tRNA Synthetase from a Methanogenic Archaeon ISO4-G1 and Its Structure-Based Engineering for Highly-Productive Cell-Free Genetic Code Expansion with Non-Canonical Amino Acids

Abstract

Pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNA^Pyl from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). Previously, we achieved full productivity of cell-free protein synthesis for bulky non-canonical amino acids, including N^ε-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS with structure-based mutations in and around the amino acid binding pocket (first-layer and second-layer mutations, respectively). Recently, the PylRS·tRNA^Pyl pair from a methanogenic archaeon ISO4-G1 was used for genetic code expansion. In the present study, we determined the crystal structure of the methanogenic archaeon ISO4-G1 PylRS (ISO4-G1 PylRS) and compared it with those of structure-known PylRSs. Based on the ISO4-G1 PylRS structure, we attempted the site-specific incorporation of N^ε-(p-ethynylbenzyloxycarbonyl)-L-lysine (pEtZLys) into proteins, but it was much less efficient than that of TCO*Lys with M. alvus PylRS mutants. Thus, the first-layer mutations (Y125A and M128L) of ISO4-G1 PylRS, with no additional second-layer mutations, increased the protein productivity with pEtZLys up to 57 ± 8% of that with TCO*Lys at high enzyme concentrations in the cell-free protein synthesis.

Keywords: cell-free protein synthesis; crystal structure; genetic code expansion; non-canonical amino acids; tRNA.

Figure 1

Overview of the ISO4-G1 PylRS structure. (a) The ISO4-G1 PylRS dimer. The protomers of the dimer are colored gray-white and blue. The crystallographic 2-fold axis is perpendicular to the paper. (b) The α-helices, 3₁₀ helices, and β-sheets are colored wine red, olive, and green, respectively.

Figure 2

Structural comparisons of ISO4-G1 PylRS with MaPylRS, DhPylSc, and MmPylRSc. (a) The β5-β6 hairpins in the ISO4-G1 PylRS molecules A, B, C, D, E, F, G, H, I, and J are colored green, cyan, magenta, yellow, vermilion, white, lavender, orange, light green, and turquoise blue, respectively. Tyr204 is shown as stick models. (b–e) Surface models of ISO4-G1 PylRS, MaPylRS (PDB: 6JP2), DhPylSc (PDB: 2ZNI), and MmPylRSc (PDB: 2ZIM). (f–h) Superimpositions of the crystal structures of ISO4-G1 PylRS, MaPylRS (PDB: 6JP2), DhPylSc·tRNA^Pyl complex (PDB: 2ZNI), and the pyrrolysyladenylate (Pyl-AMP)-bound MmPylRSc (PDB: 2ZIM), represented by surface models. The β5-β6 hairpins in ISO4-G1 PylRS and MaPylRS and the β7-β8 hairpins in MmPylRSc are colored cyan, yellow, green, and orange, respectively. The catalytic core structures of ISO4-G1 PylRS, MaPylRS, DhPylSc, and MmPylRSc superimposed well.

Figure 3

Comparison of the amino acid binding pocket of ISO4-G1 PylRS with those of MaPylRS and MmPylRSc. (a) Ribbon models of ISO4-G1 PylRS molecule B (brown) superimposed on the MaPylRS (light green) and MmPylRSc (gray) monomers. (b,c) Close-up views of different angles of the ISO4-G1 PylRS active site. The active site residues in the ISO4-G1 PylRS apo form (brown) are superimposed on those of MaPylRS (light green) and Pyl-AMP-bound MmPylRSc (gray). Along with pyrrolysine (Pyl), the active site residues in ISO4-G1 PylRS (Tyr125, Met128, Asn164, Val166, and Tyr204), MaPylRS (Tyr126, Met129, Asn166, Val168, and Tyr206), and MmPylRSc (Tyr306, Leu309, Asn346, and Cys348) are represented as stick models. The β5-β6 and Tyr204 in ISO4-G1 PylRS, the β5-β6 and Tyr206 in MaPylRS, and the β7-β8 and Tyr384 in MmPylRSc are highlighted in cyan, yellow, and orange, respectively.

Figure 4

Open and closed conformations of the β5-β6 (ISO4-G1 PylRSs), β5-β6 (M. alvus PylRSs), and β7-β8 (M. mazei PylRScs) hairpins. Translucent surface models of the active-site pockets in the ISO4-G1 PylRS apo form (brown, a; gold, b), the ISO4-G1 PylRS mutant for cyanopyridylalanine in the apo form (light pink, c) (PDB: 7R6O), the MaPylRS apo form (yellow green, d; grass green, e) (PDB: 6JP2), the acrydonylalanine and AMPPNP-bound MaPylRS mutant (light orange, f) (PDB: 8DQG), the M. mazei PylRSc apo form (blue, g) (PDB: 2E3C), and the Pyl-AMP-bound MmPylRSc (gray, h) (PDB: 2ZIM) are shown. The β5-β6 (ISO4-G1 PylRSs), β5-β6 (M. alvus PylRSs), and β7-β8 (M. mazei PylRScs) hairpins are colored differently. Tyr204, Tyr206, and Tyr384 are highlighted in magenta. The bound TCO*Lys in the PylRS active site is shown as a stick model (PDB: AAO).

Figure 5

His225 undergoes drastic conformational changes in accordance with the β5-β6 hairpin. Superimposition of the ISO4-G1 PylRS molecules A to J (a). The superimposed Tyr204, His225, and Trp237 residues are shown as stick models (b). The Tyr204, His225, and Trp237 residues in the ten ISO4-G1 PylRS molecules are each colored green (c, molecule A), magenta (d, molecule C), vermilion (e, molecule E), white (f, molecule F), turquoise blue (g, molecule J), cyan (h, molecule B), yellow (i, molecule D), lavender (j, molecule G), orange (k, molecule H), and light green (l, molecule I). In molecules A, C, E, F, and J, a π–π stacking interaction is observed between His225 and Trp237 (c–g), while in molecules B, G, H, and I, His225 is stabilized by a π–π stacking interaction with Tyr204 (h–l).

Scheme 1

Chemical structures of pyrrolysine (Pyl) and non-canonical amino acids: BocLys, PocLys, ZLys, mAzZLys, pAzZLys, pEtZLys, and TCO*Lys are shown. The order of molecular weights from largest to smallest is as follows: mAzZZLys/pAzZLys (Mw: 321.34), pEtZLys (Mw: 304.35), TCO*Lys (Mw: 298.38), ZLys (Mw: 280.32), Pyl (Mw: 255.31), BocLys (Mw: 246.31), and PocLys (Mw: 230.26).

Figure 6

Cell-free protein synthesis for site-specific incorporation of non-canonical amino acids into the N11-GFPS1 protein by using ISO4-G1 PylRS with the first-layer mutations. The N11-GFPS1 proteins containing non-canonical amino acids were synthesized with the S30 extract from the E. coli RF1 deletion strain B-60ΔA::Z/pMINOR cells. Non-canonical amino acids were site-specifically incorporated into the N11-GFPS1 protein at position 17 in response to the UAG codon, by using the ISO4-G1 PylRS (Y125A/M128L)·tRNA^Pyl and ISO4-G1 PylRS (Y125A/M128A)·tRNA^Pyl pairs. Protein productivities with non-canonical amino acids were compared with that of the cell-free synthesis of wild-type N11-GFPS1 protein containing Ala at position 17 (WT control) and are shown above the bars. (a) The yields of the N11-GFPS1 proteins containing ZLys, mAzZLys, pAzZLys, pEtZLys, and TCO*Lys were estimated by fluorescence. The values represent the means of three independent experiments with standard deviations. (b) Cell-free synthesis of the N11-GFPS1 protein containing pEtZLys by using 10 μM of the M. mazei PylRS (Y306A/Y384F), M. alvus PylRS (Y126A/M129L), and M. alvus PylRS (Y126A/M129L/H227I/Y228P) proteins. The values represent the means of three independent experiments with standard deviations. (c) Cell-free protein synthesis of the GFPS1 protein containing pEtZLys, using increased concentrations (from 10 to 75 μM) of the ISO4-G1 PylRS (Y125A/M128L) protein. The values represent the means of three independent experiments with standard deviations.

Figure 7

Cell-free protein synthesis with non-canonical amino acids using ISO4-G1 PylRS with the H225A mutation. The N11-GFPS1 proteins synthesized with the S30 extracts from E. coli B-60ΔA::Z/pMINOR cells in the presence of non-canonical amino acids. Non-canonical amino acids were site-specifically incorporated into the N11-GFPS1 protein at position 17 in response to the UAG codon, by using the ISO4-G1 PylRS·tRNA^Pyl and ISO4-G1 PylRS (H225A)·tRNA^Pyl pairs for BocLys and PocLys. The yields of the N11-GFPS1 proteins containing non-canonical amino acids were estimated by fluorescence. Protein productivities with non-canonical amino acids were compared with those of the cell-free synthesis of wild-type N11-GFPS1 protein containing Ala at position 17 (WT control) and are shown on the bars. The values represent the means of three independent experiments with standard deviations.