tRNA aminoacylation by arginyl-tRNA synthetase: induced conformations during substrates binding

Abstract

The 2.2 A crystal structure of a ternary complex formed by yeast arginyl-tRNA synthetase and its cognate tRNA(Arg) in the presence of the L-arginine substrate highlights new atomic features used for specific substrate recognition. This first example of an active complex formed by a class Ia aminoacyl-tRNA synthetase and its natural cognate tRNA illustrates additional strategies used for specific tRNA selection. The enzyme specifically recognizes the D-loop and the anticodon of the tRNA, and the mutually induced fit produces a conformation of the anticodon loop never seen before. Moreover, the anticodon binding triggers conformational changes in the catalytic center of the protein. The comparison with the 2.9 A structure of a binary complex formed by yeast arginyl-tRNA synthetase and tRNA(Arg) reveals that L-arginine binding controls the correct positioning of the CCA end of the tRNA(Arg). Important structural changes induced by substrate binding are observed in the enzyme. Several key residues of the active site play multiple roles in the catalytic pathway and thus highlight the structural dynamics of the aminoacylation reaction.

Fig. 1. Overview of yArgRS–tRNA^Arg interactions. (A) The cloverleaf structure of tRNA^Arg_ICG. The one-letter code is used for the nucleotides in all figures. The following code has been used for the modified bases: ψ, pseudouridine; D, dihydrouridine; I, inosine; K, 1-methylguanosine; L, N₂-methylguanosine; R, N₂,N₂-dimethylguanosine; m⁵C, 5-methylcytidine; m¹A, 1-methyladenosine; T, 5-methyluridine. (B) Overview of one monomer of yArgRS interacting with tRNA^Arg_ICG (drawn with SETOR; Evans, 1993) showing the modular architecture of yArgRS: Add1 (residues 1–143) is colored in orange; the catalytic domain in red (residues 143–194, 266–293 and 345–410); Ins1 in green (residues 194–266); Ins2 (residues 293–345) in blue; and Add2 (residues 410–607) in yellow. The tRNA backbone is drawn with its phosphate chain traced as a thick cyan line. Numbering of strands and helices is according to the structure of the ‘tRNA-free’ yArgRS (Cavarelli et al., 1998). The water molecules are not shown. (C) A schematic representation showing the footprint of the tRNA^Arg (in pink) on the surface of yArgRS (in green) (drawn with GRASP; Nicholls and Honig, 1991). (D) The molecular surface of yArgRS showing the electrostatic potential calculated with GRASP (Nicholls and Honig, 1991): negatively charged regions are in red and positively charged areas in blue. The orientation of the yArgRS molecule is similar in all three figures. The tRNA backbone is drawn with its phosphate chain traced as a thick green line.

Fig. 1. Overview of yArgRS–tRNA^Arg interactions. (A) The cloverleaf structure of tRNA^Arg_ICG. The one-letter code is used for the nucleotides in all figures. The following code has been used for the modified bases: ψ, pseudouridine; D, dihydrouridine; I, inosine; K, 1-methylguanosine; L, N₂-methylguanosine; R, N₂,N₂-dimethylguanosine; m⁵C, 5-methylcytidine; m¹A, 1-methyladenosine; T, 5-methyluridine. (B) Overview of one monomer of yArgRS interacting with tRNA^Arg_ICG (drawn with SETOR; Evans, 1993) showing the modular architecture of yArgRS: Add1 (residues 1–143) is colored in orange; the catalytic domain in red (residues 143–194, 266–293 and 345–410); Ins1 in green (residues 194–266); Ins2 (residues 293–345) in blue; and Add2 (residues 410–607) in yellow. The tRNA backbone is drawn with its phosphate chain traced as a thick cyan line. Numbering of strands and helices is according to the structure of the ‘tRNA-free’ yArgRS (Cavarelli et al., 1998). The water molecules are not shown. (C) A schematic representation showing the footprint of the tRNA^Arg (in pink) on the surface of yArgRS (in green) (drawn with GRASP; Nicholls and Honig, 1991). (D) The molecular surface of yArgRS showing the electrostatic potential calculated with GRASP (Nicholls and Honig, 1991): negatively charged regions are in red and positively charged areas in blue. The orientation of the yArgRS molecule is similar in all three figures. The tRNA backbone is drawn with its phosphate chain traced as a thick green line.

Fig. 2. Recognition of the anticodon loop of tRNA^Arg_ICG by yArgRS. (A) Stereo view of a final (2F_obs – F_calc) cross-validated σ_A-weighted omit map, contoured at 1.5σ, showing the nucleotides of the anticodon loop (resolution limits 15–2.2 Å, all data used, calculated with CNS; Brünger et al., 1998). The protein residues are not shown for reasons of clarity. (B) Stereo view of the anticodon-binding site. yArgRS approaches the tRNA^Arg from the minor groove side of the anticodon stem, and the anticodon loop binds in a pocket delimited by five helices of Add2 (shown in yellow). The conformation of the anticodon loop is characterized by: (i) the formation of a bulge at the level of A38; (ii) the intercalation of A37 between the base pair (G31–C39) and nucleotide C32 and; (iii) the splaying out of three bases (U33, I34 and C35). (C) Recognition of the identity determinant C35 by yArgRS. C35, the strongest identity determinant for tRNA^Arg, is recognized mainly by main chain atoms of the protein belonging to the loop between helices H22 and H23 and by a stacking interaction with Trp569. (D) Interactions of Met607 with A38 and G36. Met607, the last residue of yArgRS, interacts, via its main chain atoms, with G36 and A38, and stabilizes the conformation of the anticodon loop, therefore explaining the strong evolutionary pressure on the C-terminal end of ArgRS. The side chain atoms of Met607 are not shown for reasons of clarity. Figures 2–5 were drawn with SETOR (Evans, 1993). The water molecules are shown as red spheres.

Fig. 2. Recognition of the anticodon loop of tRNA^Arg_ICG by yArgRS. (A) Stereo view of a final (2F_obs – F_calc) cross-validated σ_A-weighted omit map, contoured at 1.5σ, showing the nucleotides of the anticodon loop (resolution limits 15–2.2 Å, all data used, calculated with CNS; Brünger et al., 1998). The protein residues are not shown for reasons of clarity. (B) Stereo view of the anticodon-binding site. yArgRS approaches the tRNA^Arg from the minor groove side of the anticodon stem, and the anticodon loop binds in a pocket delimited by five helices of Add2 (shown in yellow). The conformation of the anticodon loop is characterized by: (i) the formation of a bulge at the level of A38; (ii) the intercalation of A37 between the base pair (G31–C39) and nucleotide C32 and; (iii) the splaying out of three bases (U33, I34 and C35). (C) Recognition of the identity determinant C35 by yArgRS. C35, the strongest identity determinant for tRNA^Arg, is recognized mainly by main chain atoms of the protein belonging to the loop between helices H22 and H23 and by a stacking interaction with Trp569. (D) Interactions of Met607 with A38 and G36. Met607, the last residue of yArgRS, interacts, via its main chain atoms, with G36 and A38, and stabilizes the conformation of the anticodon loop, therefore explaining the strong evolutionary pressure on the C-terminal end of ArgRS. The side chain atoms of Met607 are not shown for reasons of clarity. Figures 2–5 were drawn with SETOR (Evans, 1993). The water molecules are shown as red spheres.

Fig. 2. Recognition of the anticodon loop of tRNA^Arg_ICG by yArgRS. (A) Stereo view of a final (2F_obs – F_calc) cross-validated σ_A-weighted omit map, contoured at 1.5σ, showing the nucleotides of the anticodon loop (resolution limits 15–2.2 Å, all data used, calculated with CNS; Brünger et al., 1998). The protein residues are not shown for reasons of clarity. (B) Stereo view of the anticodon-binding site. yArgRS approaches the tRNA^Arg from the minor groove side of the anticodon stem, and the anticodon loop binds in a pocket delimited by five helices of Add2 (shown in yellow). The conformation of the anticodon loop is characterized by: (i) the formation of a bulge at the level of A38; (ii) the intercalation of A37 between the base pair (G31–C39) and nucleotide C32 and; (iii) the splaying out of three bases (U33, I34 and C35). (C) Recognition of the identity determinant C35 by yArgRS. C35, the strongest identity determinant for tRNA^Arg, is recognized mainly by main chain atoms of the protein belonging to the loop between helices H22 and H23 and by a stacking interaction with Trp569. (D) Interactions of Met607 with A38 and G36. Met607, the last residue of yArgRS, interacts, via its main chain atoms, with G36 and A38, and stabilizes the conformation of the anticodon loop, therefore explaining the strong evolutionary pressure on the C-terminal end of ArgRS. The side chain atoms of Met607 are not shown for reasons of clarity. Figures 2–5 were drawn with SETOR (Evans, 1993). The water molecules are shown as red spheres.

Fig. 3. Interaction of the D-loop of the tRNA^Arg with yArgRS. (A) Overview. yArgRS recognizes the sugar backbone conformation and interacts specifically with nucleotides D16 and D20. D16 binds in a pocket formed by strand S1 of Add1 and helices H21–H22 of Add2, while D20 interacts mainly with β-hairpin S3–S4. The tRNA backbone is drawn with its phosphate chain traced as a thick light green line. (B) Recognition of D20 by yArgRS, illustrating the co-evolution of aaRS and tRNAs sequences. D20 is recognized mainly by Asn106, Phe109 and Gln111. Phe109, a highly conserved residue in ArgRS sequences, is involved in a stacking-type interaction with D20. The D20–Gln111 interaction is specific for the arginine system in S.cerevisiae. All other ArgRS–tRNA^Arg complexes from other species use an A20–Asn interaction at this position.

Fig. 4. Conformation of the acceptor arm of tRNA^Arg and l-Arg recognition. Comparison of the CCA hairpin conformation in tRNA^Arg and tRNA^Gln. The similar conformation found for the nucleotides C75 and A76 is stabilized by two different molecular mechanisms involving a different intramolecular interaction within the tRNA. (A) In tRNA^Arg, the bending of the 3′-terminal CCA is stabilized by a hydrogen bond involving the 4-amino group of C75 and the phosphate oxygen atom of residue 72. The water molecules are not shown. (B) In tRNA^Gln, nucleotide G73 stabilizes the bending by a hydrogen bond involving its 2-amino group and the phosphate oxygen atom of nucleotide 72, and is also involved in a stacking interaction with C75 and A76. l-Arg recognition. Comparison of the recognition mode of the l-Arg substrate (C and D) in the ternary complex with tRNA^Arg and (E) in the absence of the tRNA^Arg molecule. The two structures show a similar scheme of interactions for the guanidinium moiety, involving amino acids strictly conserved in all ArgRS sequences. The recognition of A76 in the ternary complex illustrates the role of Asn153, Glu294, Gln375 and Tyr347. The water molecules that occupy the putative AMP-binding site are shown as red spheres. tRNA binding produces structural changes of the conformation of the two histidines of the first signature motif characteristic of class I aaRSs; moreover, Asn153 and Tyr347 play a multiple role.

Fig. 4. Conformation of the acceptor arm of tRNA^Arg and l-Arg recognition. Comparison of the CCA hairpin conformation in tRNA^Arg and tRNA^Gln. The similar conformation found for the nucleotides C75 and A76 is stabilized by two different molecular mechanisms involving a different intramolecular interaction within the tRNA. (A) In tRNA^Arg, the bending of the 3′-terminal CCA is stabilized by a hydrogen bond involving the 4-amino group of C75 and the phosphate oxygen atom of residue 72. The water molecules are not shown. (B) In tRNA^Gln, nucleotide G73 stabilizes the bending by a hydrogen bond involving its 2-amino group and the phosphate oxygen atom of nucleotide 72, and is also involved in a stacking interaction with C75 and A76. l-Arg recognition. Comparison of the recognition mode of the l-Arg substrate (C and D) in the ternary complex with tRNA^Arg and (E) in the absence of the tRNA^Arg molecule. The two structures show a similar scheme of interactions for the guanidinium moiety, involving amino acids strictly conserved in all ArgRS sequences. The recognition of A76 in the ternary complex illustrates the role of Asn153, Glu294, Gln375 and Tyr347. The water molecules that occupy the putative AMP-binding site are shown as red spheres. tRNA binding produces structural changes of the conformation of the two histidines of the first signature motif characteristic of class I aaRSs; moreover, Asn153 and Tyr347 play a multiple role.

Fig. 4. Conformation of the acceptor arm of tRNA^Arg and l-Arg recognition. Comparison of the CCA hairpin conformation in tRNA^Arg and tRNA^Gln. The similar conformation found for the nucleotides C75 and A76 is stabilized by two different molecular mechanisms involving a different intramolecular interaction within the tRNA. (A) In tRNA^Arg, the bending of the 3′-terminal CCA is stabilized by a hydrogen bond involving the 4-amino group of C75 and the phosphate oxygen atom of residue 72. The water molecules are not shown. (B) In tRNA^Gln, nucleotide G73 stabilizes the bending by a hydrogen bond involving its 2-amino group and the phosphate oxygen atom of nucleotide 72, and is also involved in a stacking interaction with C75 and A76. l-Arg recognition. Comparison of the recognition mode of the l-Arg substrate (C and D) in the ternary complex with tRNA^Arg and (E) in the absence of the tRNA^Arg molecule. The two structures show a similar scheme of interactions for the guanidinium moiety, involving amino acids strictly conserved in all ArgRS sequences. The recognition of A76 in the ternary complex illustrates the role of Asn153, Glu294, Gln375 and Tyr347. The water molecules that occupy the putative AMP-binding site are shown as red spheres. tRNA binding produces structural changes of the conformation of the two histidines of the first signature motif characteristic of class I aaRSs; moreover, Asn153 and Tyr347 play a multiple role.

Fig. 5. Structural changes on yArgRS upon substrate binding. The yArgRS backbone (in orange, red, green, heavy blue and yellow) corresponds to the structure found in the ternary complex. The tRNA backbone is drawn with its phosphate chain traced as a thick purple line. Superpositions were carried out by superimposing the entire protein. (A) Comparison of the structure of the ternary complex with the ‘tRNA-free’ structure of yArgRS shows the structural movements due to the tRNA binding. Structural elements colored in light blue correspond to the ‘tRNA-free’ yArgRS structure. Only large movements are displayed. The conformations of two peptides are particularly altered: the first goes from strand S13 to helix H15 and the second involves strand S14, helix H17 and the Ω loop. Structural changes of the conformation of helix H15 induce the modification of the structure of the two signature motifs characteristic of class I aaRSs; the ‘H₁₅₉A₁₆₀G₁₆₁H₁₆₂’ loop is located between strand S5 and helix H6, while the ‘M₄₀₈S₄₀₉T₄₁₀R₄₁₁’ loop is located between strand S13 and helix H15. (B) Comparison of the structure of the ternary complex with the binary complex shows the structural movements due to the l-Arg binding. Structural elements colored in light blue correspond to the conformation found in the binary complex. Conformational changes are located mainly in the two insertion modules (Ins1 and Ins2) and helices H13 and H14 of the second moiety of the Rossmann fold. The overall conformation of the tRNA is the same; however, the absence of l-Arg substrate in the active site strongly affects the conformation of the CCA end (see below). Active site of yArgRS: (C) in the ternary complex and (D) in the binary complex, illustrating the molecular switch control by Tyr347 and l-Arg. In the absence of l-Arg substrate (D), G73 extends the helical conformation of the acceptor stem, and the last three nucleotides C₇₄C₇₅A₇₆ are not visible in the electron density map and are therefore certainly disordered. The water molecules are not shown.

Fig. 5. Structural changes on yArgRS upon substrate binding. The yArgRS backbone (in orange, red, green, heavy blue and yellow) corresponds to the structure found in the ternary complex. The tRNA backbone is drawn with its phosphate chain traced as a thick purple line. Superpositions were carried out by superimposing the entire protein. (A) Comparison of the structure of the ternary complex with the ‘tRNA-free’ structure of yArgRS shows the structural movements due to the tRNA binding. Structural elements colored in light blue correspond to the ‘tRNA-free’ yArgRS structure. Only large movements are displayed. The conformations of two peptides are particularly altered: the first goes from strand S13 to helix H15 and the second involves strand S14, helix H17 and the Ω loop. Structural changes of the conformation of helix H15 induce the modification of the structure of the two signature motifs characteristic of class I aaRSs; the ‘H₁₅₉A₁₆₀G₁₆₁H₁₆₂’ loop is located between strand S5 and helix H6, while the ‘M₄₀₈S₄₀₉T₄₁₀R₄₁₁’ loop is located between strand S13 and helix H15. (B) Comparison of the structure of the ternary complex with the binary complex shows the structural movements due to the l-Arg binding. Structural elements colored in light blue correspond to the conformation found in the binary complex. Conformational changes are located mainly in the two insertion modules (Ins1 and Ins2) and helices H13 and H14 of the second moiety of the Rossmann fold. The overall conformation of the tRNA is the same; however, the absence of l-Arg substrate in the active site strongly affects the conformation of the CCA end (see below). Active site of yArgRS: (C) in the ternary complex and (D) in the binary complex, illustrating the molecular switch control by Tyr347 and l-Arg. In the absence of l-Arg substrate (D), G73 extends the helical conformation of the acceptor stem, and the last three nucleotides C₇₄C₇₅A₇₆ are not visible in the electron density map and are therefore certainly disordered. The water molecules are not shown.