Human tryptophanyl-tRNA synthetase is switched to a tRNA-dependent mode for tryptophan activation by mutations at V85 and I311

Abstract

For most aminoacyl-tRNA synthetases (aaRS), their cognate tRNA is not obligatory to catalyze amino acid activation, with the exception of four class I (aaRS): arginyl-tRNA synthetase, glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase and class I lysyl-tRNA synthetase. Furthermore, for arginyl-, glutamyl- and glutaminyl-tRNA synthetase, the integrated 3' end of the tRNA is necessary to activate the ATP-PPi exchange reaction. Tryptophanyl-tRNA synthetase is a class I aaRS that catalyzes tryptophan activation in the absence of its cognate tRNA. Here we describe mutations located at the appended beta1-beta2 hairpin and the AIDQ sequence of human tryptophanyl-tRNA synthetase that switch this enzyme to a tRNA-dependent mode in the tryptophan activation step. For some mutant enzymes, ATP-PPi exchange activity was completely lacking in the absence of tRNA(Trp), which could be partially rescued by adding tRNA(Trp), even if it had been oxidized by sodium periodate. Therefore, these mutant enzymes have strong similarity to arginyl-tRNA synthetase, glutaminyl-tRNA synthetase and glutamyl-tRNA synthetase in their mode of amino acid activation. The results suggest that an aaRS that does not normally require tRNA for amino acid activation can be switched to a tRNA-dependent mode.

Figure 1.

Primary and tertiary structure of human TrpRS. (A) Crystal structure of full-length human TrpRS (40). The tryptophanyl–AMP intermediate is indicated by an arrowhead. The V85 and I311 interface between the β1–β2 hairpin and main body is enlarged in the boxed inset. (B) Primary sequence alignment of the region of human TrpRS containing the β1–β2 hairpin. The conserved V85 and V90 in TrpRSs are indicated by arrowheads.

Figure 2.

Enzymatic activities of the V85A, V85S, V90A and V90S mutants. (A) ATP-PPi exchange activity of the four mutants and wild-type human TrpRS. To display the whole curve of wt-hTrpRS, we enlarged the scale of the Y-axis in the boxed inset. (B) Aminoacylation activity of the four mutants and wild-type human TrpRS. The enzyme concentrations are 5 nM for wild-type human TrpRS (wt-hTrpRS), 20 nM for V85A and V90A and 50 nM for V85S and V90S. (C) PPi exchange activity of the V85S mutant in the presence of wild-type or oxidized bovine tRNA^Trp. The wt-hTrpRS curve is shown in the boxed inset.

Figure 3.

Enzymatic activities of the V85E, V85K and V85L mutants. (A) ATP-PPi exchange activity of the three mutations and wild-type human TrpRS. The boxed inset displayed the whole curve of wt-hTrpRS and the V85L mutant. (B) Aminoacylation activity of the three mutations and wild-type human TrpRS. The enzyme concentrations are 5 nM for wt-hTrpRS and V85L and 200 nM for V85E and V85K. (C) PPi exchange activity of the V85E and V85K mutants in the presence of wild-type or oxidized bovine tRNA^Trp. The enlarged scale for wt-hTrpRS and the 85L mutant are shown in the boxed inset.

Figure 4.

Structural comparisons of human T2-TrpRS and mini-TrpRS. (A) Comparison of unliganded T2-TrpRS (1O5T) and mini-TrpRS (1ULH). (B) Comparison of T2-TrpRS bound to bovine tRNA^Trp (2DR2) and mini-TrpRS (1ULH). For clarity, the tRNA molecule of 2DR2 is not displayed, and only part of the N-terminus, the AIDQ sequence, and the KMSAS loop are colored dark blue for mini-TrpRS and pink for T2-TrpRS. In both Figure 4A and B, the β1–β2 hairpin, the AIDQ sequence and the KMSAS loop are indicated with sky blue dashed ellipses. The conformational change of the AIDQ sequence is enlarged in the circled ellipse inset.

Figure 5.

Enzymatic activities of the I311E and I311V mutants. (A) ATP-PPi exchange activity of the two mutants and wild-type human TrpRS. The boxed inset displayed the whole curve of wt-hTrpRS. (B) Aminoacylation activity of the two mutants and wild-type human TrpRS. The enzyme concentrations are 5 nM for wild-type human TrpRS, 50 nM for the I311V mutant and 200 nM for the I311E mutant. (C) PPi exchange activity of the I311E mutant in the presence of wild-type or oxidized bovine tRNA^Trp and wild-type human TrpRS. The enlarged scale for wt-hTrpRS is shown in the boxed inset.

Figure 6.

Enzymatic activities of the V85A/V90A double mutants. (A) ATP-PPi exchange activity of this mutant and the wild-type human TrpRS. The enlarged scale is shown in the boxed inset for wt-hTrpRS. (B) Aminoacylation activity of the V85A/V90A double mutant and wild-type human TrpRS. The enzyme concentrations are 5 nM for wild-type human TrpRS and 200 nM for the V85A/V90A mutant. (C) PPi exchange activity of the V85A/V90A double mutant in the presence of wild-type or oxidized bovine tRNA^Trp and wild-type human TrpRS. The wt-hTrpRS curve is shown in the boxed inset.