Misactivated amino acids translocate at similar

Misactivated amino acids translocate at similar rates across surface of a tRNA synthetase Tyzoon K. Nomanbhoy and Paul R. Schimmel* The Skaggs Institute for Chemical Biology, The Scripps Research Institute, Beckman Center, 10550 North Torrey Pines Road, La Jolla, CA 92037 Contributed by Paul R. Schimmel, March 8, 2000

A

minoacyl-tRNA synthetases catalyze the esterification reaction between an amino acid and its cognate tRNA (1). In this way, they link the anticodon triplet of the genetic code to a specific amino acid. The esterification reaction occurs in two steps. In the first step (termed amino acid activation), the amino acid is condensed with ATP to yield an activated aminoacyl adenylate. In the second step (termed acyl transfer), the activated aminoacyl moiety is transferred to the 3⬘ end of its cognate tRNA. During amino acid activation, discrimination by synthetases of their cognate amino acid from other, structurally similar, amino acids is critical (refs. 2–5, and ref. 6, pp. 384–399). Some of these enzymes have evolved tRNA-dependent editing mechanisms to ensure that misactivated noncognate amino acids are hydrolyzed before they can be incorporated into a polypeptide chain during ribosomal protein synthesis. A prominent example is isoleucyltRNA synthetase (IleRS), which misactivates valine only 180 times less efficiently than it activates isoleucine (7). To ensure that misactivated valine is hydrolyzed, the enzyme has a discrete editing domain that is separated from the active site by ⬎25 Å (8, 9). In a tRNAIle-dependent manner, the misactivated valine is translocated from the active site to the editing site, where it is hydrolyzed. Valyl-tRNA synthetase (ValRS) is a close homolog of IleRS (10, 11). Both enzymes have an insertion of ⬇300 amino acids that splits in half the nucleotide binding fold of the active site domain (12). This insertion is known as connective polypeptide 1 (CP1), because it connects one half of the active site with the other (13). The active site for editing is located within CP1 (14). According to current models for the editing reaction, access to the CP1 site for editing is obtained by translocation of misactivated aminoacyl adenylate (pretransfer editing) or the mischarged aminoacyl group on the tRNA substrate (posttransfer editing) from the active site to the editing site. The misactivated amino acid moiety must translocate a distance of ⬎25 Å across the surface of the enzyme (8, 9). The mechanism of this translocation event is not understood. In a previous study with IleRS, the f luorescent nucleotide Nmethylanthraniloyl-dATP (dATP†) was developed to monitor the translocation of misactivated valine during editing (15). To

expand on these studies and possibly to learn more about the mechanisms of translocation, we turned to ValRS. This enzyme misactivates threonine (isosteric with valine), ␣-aminobutyrate, and cysteine. We thought that the differences in the side chains of these amino acids might affect the rate of translocation, especially if contacts were made between the side chain and enzyme during translocation. Thus, we studied the kinetics of the translocation of misactivated threonine, ␣-aminobutyrate, and cysteine from the active site to the editing site of ValRS to see whether the nature of the side chain affected translocation. Materials and Methods Protein Expression and Purification. The ORF of the Escherichia coli ValRS gene was cloned into the E. coli expression vector pET-21b (Novagen, Madison, WI) to generate a fusion protein that has a C-terminal His6 tag. Standard protocols were used to express and purify this fusion protein. Fluorescence Spectroscopy and Data Analysis. Fluorescence mea-

surements were made by using a Perkin–Elmer LS 50 Luminescence Spectrometer. Samples were stirred continuously at room temperature (⬇20°C) in 150 mM Tris䡠Cl (pH 7.5) and 10 mM MgCl2. To determine the translocation rate constants for the editing of the different amino acids, we first determined the dissociation constant (Kd) for the binding of tRNAVal to the ValRS䡠aa‡ (where aa‡ represents the activated amino acid) complex. The amplitude of the rapid phase of dATP† fluorescence increase (i.e., the fluorescence increase caused by translocation) was estimated and plotted versus the total tRNAVal concentration (15). The points were then fit to the following equation: F ⫽ F i ⫹ F f 兵共共K d ⫹ 关tRNAVal兴 T ⫹ 关ValRS兴 T兲 ⫺ 共共K d ⫹ 关tRNAVal兴 T ⫹ 关ValRS兴 T兲 2 1 ⫺ 4关tRNAVal兴 T关ValRS兴 T兲 ⁄2兲兾2关ValRS兴 T其 ,

where F is the amplitude of the initial change in fluorescence caused by the addition of tRNAVal, Fi is the initial fluorescence before addition of tRNAVal, Ff is the amplitude of the maximum fluorescence change at saturating tRNAVal, [ValRS]T is the total concentration of ValRS, and [tRNAVal]T is the total concentration of tRNAVal. The data were fit with the IGOR WaveMetrics (Lake Oswego, OR) application. The observed rate constants (kobs) for the rapid phase of the fluorescence increase were determined for each tRNAVal concentration by fitting to a single exponential the first 5 s of Abbreviations: IleRS, isoleucyl-tRNA synthetase; ValRS, valyl-tRNA synthetase; dATP†, Nmethylanthraniloyl-dATP. *To whom reprint requests should be addressed. E-mail: [email protected]. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073兾pnas.090102197. Article and publication date are at www.pnas.org兾cgi兾doi兾10.1073兾pnas.090102197

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Certain aminoacyl-tRNA synthetases have a second active site that destroys (by hydrolysis) errors of amino acid activation. For example, isoleucyl-tRNA synthetase misactivates valine (to produce valyl adenylate or Val-tRNAIle) at its active site. The misactivated amino acid is then translocated to an editing site located >25 Å away. The role of the misactivated amino acid in determining the rate of translocation is not known. Valyl-tRNA synthetase, a close homolog of isoleucyl-tRNA synthetase, misactivates threonine, ␣-aminobutyrate, and cysteine. In this paper, we use a recently developed fluorescence-energy-transfer assay to study translocation of misactivated threonine, ␣-aminobutyrate, and cysteine. Although their rates of misactivation are clearly distinct, their rates of translocation are similar. Thus, the rate of translocation is independent of the nature of the misactivated amino acid. This result suggests that the misactivated amino acid per se has little or no role in directing translocation.

transient change in fluorescence after the addition of tRNAVal (15). The 1兾kobs values were plotted versus 1兾[tRNAVal] (the concentration of free tRNAVal was computed from the total concentration of tRNAVal by using the Kd value obtained above). The y intercept of a linear fit to this plot was used to determine the translocation rate constant (kT). The IGOR WaveMetrics application was used to fit the data. Editing Assays. The rate constant for the overall editing reaction

was determined by following the tRNAVal-dependent consumption of [␥-32P]ATP in the presence of threonine, ␣-aminobutyrate, or cysteine, by using a protocol that has been described in detail for IleRS (16). A reaction mixture contained 500 nM ValRS in 150 mM Tris䡠Cl (pH 7.5), 10 mM MgCl2, 0.5 mM CaCl2, 3 mM [␥-32P]ATP (25 ␮Ci兾␮mol), 4 nM pyrophosphatase, 10 ␮M E. coli tRNAVal [1300 pmol兾A260 (Sigma)], and threonine (20 mM), ␣-aminobutyrate (20 mM), or cysteine (30 mM). Results Fluorescence Energy Transfer Assay to Monitor the Translocation of Amino Acids from the Active Site to the Editing Site. The rationale

for the fluorescence assay that monitors the translocation of misactivated amino acids during editing is illustrated in Fig. 1A. The fluorescent nucleotide dATP† competitively inhibits the binding of aminoacyl adenylates to the active site of ValRS. The nucleotide has an excitation maximum at 350 nm and an emission peak at 440 nm (17). The excitation maximum overlaps with the emission peak of tryptophan (330 nm), which is normally excited at 295 nm. Thus, when bound, dATP† is close to one or more tryptophans in the protein, and excitation at 295 nm leads to energy-transfer-dependent emission at 440 nm. The dATP† binds with a Kd of ⬇1 ␮M as compared with a Kd for the adenylate of ⬍10 nM. In these studies, we used a concentration of dATP† (10 ␮M) that was not sufficient to bind to the active site except when it was vacated by an adenylate. The interaction between dATP† and the active site was monitored by the aforementioned fluorescence resonance energy transfer between the tryptophan residues of ValRS and dATP†. However, the displacement of dATP† from the active site by the more tightly bound aminoacyl adenylates leads to a loss of energy transfer that results in a decrease in emission at 440 nm. Thus, the energy-transfer-dependent emission at 440 nm measures the fractional occupation of the active site with dATP† versus with aminoacyl adenylates. If a noncognate aminoacyl adenylate is used to displace dATP† from the active site, then the addition of tRNAVal leads to hydrolytic editing of the misactivated amino acid. The misactivated substrate first is translocated from the active site to the editing site, where it is hydrolyzed (Fig. 1B). The translocation of the misactivated amino acid results in the emptying of the active site, thus allowing ValRS to rebind dATP†. This occurrence, in turn, results in energy-transfer-dependent dATP† fluorescence at 440 nm. The rate of this fluorescence increase measures the kinetics of the translocation of the misactivated amino acid from the active site to the editing site (15). The fluorescent dATP† nucleotide was displaced from the active site by threonyl adenylate (formed enzymatically by the addition of threonine and ATP in the presence of pyrophosphatase). The concentration of ATP used was greater than the concentration of ValRS (25 ␮M versus 550 nM, respectively) to allow for multiple rounds of adenylate formation. Under these conditions, the first round of translocation of misactivated threonine during editing can be observed as a distinct phase in the overall increase of dATP† fluorescence (see below and Fig. 2). Because of the inner-filter effect of the UV-absorbing tRNA nucleotides, we were prevented from using tRNAVal concentrations that were significantly greater than the ValRS concentration; hence, the concentrations of tRNAVal added to stimulate 5120 兩 www.pnas.org

Fig. 1. Design of the assay for fluorescence energy-transfer-dependent editing. (A) When dATP† is bound to the active site, excitation of the ValRS䡠dATP† complex leads to energy-transfer-dependent emission at 440 nm. The displacement of dATP† from the active site by an aminoacyl adenylate leads to a loss of energy transfer that results in a decrease in emission at 440 nm. The positions of the active site and the editing site are indicated, and the activated amino acid is designated aa‡. In this example, aa‡ refers to the aminoacyl adenylate. The different shades of the enzyme active site are used to indicate different intensities of emission at 440 nm. (B) The binding of tRNAVal to ValRS complexed to a misactivated amino acid first results in the translocation of the misactivated amino acid from the active site to the editing site, allowing for the rapid rebinding of the fluorescent dATP† to the active site. (In this example, Thr‡ refers to either Thr-AMP or Thr-tRNAVal). This translocation is followed by the hydrolysis of the misactivated amino acid. The forward and reverse rate constants (k1 and k⫺1, respectively) for the binding of tRNAVal to ValRS, and the rate constant kT for translocation are indicated.

editing were substoichiometric relative to the concentration of ValRS (70–470 nM versus 550 nM, respectively). The addition of tRNAVal resulted in an increase in dATP† fluorescence (Fig. 2), because of the rebinding of dATP† to the active site after the translocation of misactivated threonine from the active site to the editing site and the continuous recycling of tRNA until all of the ATP was hydrolyzed. In addition, the fluorescence increase seen for threonine depended on the addition of tRNAVal, specifically; this increase was not observed on addition of either tRNAPhe or tRNAIle (data not shown). As a control, dATP† also was displaced by activated valine (Fig. 2). Because valine is the cognate amino acid, the addition of tRNAVal did not result in any dATP† fluorescence increase, because the tRNA is charged with valine and the activated Val-AMP or Val-tRNAVal is not hydrolyzed. After charging, a new Val-AMP is formed so that dATP† is blocked from binding the active site. Under our experimental conditions, the dATP† fluorescence increase seen for the editing of threonine consisted of two phases. There was an initial rapid increase in dATP† fluoresNomanbhoy and Schimmel

cence occurring immediately after tRNAVal addition (Fig. 2 Inset), followed by a slower recovery of dATP† fluorescence. We previously had established for IleRS that the rapid phase of fluorescence increase consisted of one round of translocation. Because IleRS and ValRS are close homologs having similar connective polypeptide 1 insertions, we assumed that the rapid phase of fluorescence increase seen for editing of threonine by ValRS also consisted of one round of translocation of misactivated threonine. Consistent with this assumption, we observed that the amplitude of the fluorescence change during the rapid phase increased with (substoichiometric) tRNAVal concentration (data not shown). Determination of the Translocation Rate Constant (kT). To determine the rate constant for the translocation of misactivated threonine (kT), the kinetics of the rapid phase of the fluorescence increase was examined over a range of concentrations of tRNAVal. The profiles for the fluorescence increase of the rapid phase for each concentration of tRNAVal first were normalized so that the amplitude of the fluorescence change was the same in all cases (Fig. 3). The normalized profiles were fit to single exponentials to determine the rates of fluorescence increase during the rapid phase. The observed rate constants (kobs) showed a hyperbolic dependence on the tRNAVal concentration (data not shown). This dependence is consistent with the rapid phase of fluorescence increase consisting of a unimolecular event (i.e., the translocation of misactivated threonine) after a rapid bimolecular event (i.e., the binding of tRNAVal to the ValRS䡠Thr-AMP complex) (Fig. 1B). For this mechanism, the relationship between kobs and kT is (ref. 6, p. 148, and ref. 15):

k obs ⫽

Nomanbhoy and Schimmel

kT , Kd 1⫹ 关tRNAVal兴

Fig. 3. Determination of the rate constant for the translocation of misactivated threonine at pH 7.5, 20°C. ValRS (550 nM) was incubated with dATP† (10 ␮M), pyrophosphatase (4 nM), and threonine (1.2 mM) for 4 min to ensure that all of the ValRS was complexed to Thr-AMP. The energy-transfer-dependent fluorescence of dATP† was monitored (excitation ⫽ 295 nm; emission ⫽ 440 nm), and tRNAVal was added to stimulate the editing of misactivated threonine. The fluorescence profiles corresponding to the rapid phase of dATP† fluorescence increase were normalized so that the amplitude of the fluorescence increase during this phase was approximately the same for the different tRNAVal concentrations. The observed rate constants (kobs) for the fluorescence increase for each concentration of tRNAVal were determined by fitting to a single exponential the first 5 s of transient fluorescence change after the addition of tRNA. (Inset) A linear fit of 1兾kobs versus 1兾[tRNAVal] was used to estimate the rate constant for translocation.

where [tRNAVal] is the free tRNAVal concentration and Kd is the dissociation constant for the interaction between tRNAVal and the ValRS䡠Thr-AMP complex. The kT value for translocation of threonine was determined from the y intercept of a linear fit of a plot of 1兾kobs versus 1兾[tRNAVal], for which we obtained a value of kT ⫽ 3.3 s⫺1 (Fig. 3 Inset, and see Materials and Methods). This value is in good agreement with the rate constant of 3.1 s⫺1 that was measured for the overall editing reaction of threonine by ValRS by us and by others (18) using assays based on the overall rate of ATP hydrolysis during editing (data not shown). Thus, translocation is rate-limiting for editing by ValRS, as it is for editing IleRS. Examination of Misactivated Amino Acid Translocation During Editing of ␣-Aminobutyrate and Cysteine. The fluorescence editing assay

also was used to examine the translocation of misactivated ␣-aminobutyrate and cysteine during the editing reaction. In the initial amino acid activation reaction, kcat兾Km for ␣-aminobutyrate was 3800 M⫺1䡠s⫺1, whereas the kcat兾Km for cysteine was 700 M⫺1䡠s⫺1. These values compare with a value of 2600 M⫺1䡠s⫺1 found for threonine (18, 19). At a particular tRNAVal concentration, the kinetics for the translocation of misactivated threonine, ␣-aminobutyrate, and cysteine were similar (Fig. 4). In fact, the analysis of the kinetics of ␣-aminobutyrate and cysteine translocation over a range of tRNAVal concentrations gave kT values of 3.5 s⫺1 and 2.7 s⫺1, respectively (data not shown). These values are in good agreement with the rate constants of about 3.7 s⫺1 and 2.2 s⫺1 that were measured by us for the overall editing reaction (after tRNAVal-dependent ATP hydrolysis) with ␣-aminobutyrate and cysteine, respectively (data not shown). Discussion In this study, we characterize the translocation of threonine, ␣-aminobutyrate, and cysteine during editing by ValRS. For all PNAS 兩 May 9, 2000 兩 vol. 97 兩 no. 10 兩 5121

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Fig. 2. Time-dependent tRNAVal-dependent editing at pH 7.5, 20°C. ValRS (550 nM) was incubated with dATP† (10 ␮M), pyrophosphatase (4 nM), and either valine (1.2 mM) or threonine (1.2 mM) for 4 min to ensure that all of the ValRS was complexed to aminoacyl adenylate. The energy-transferdependent fluorescence of dATP† was monitored (excitation ⫽ 295 nm; emission ⫽ 440 nm), and tRNAVal (312 nM) was added to stimulate editing (arrow). The fluorescence traces were normalized to correct for the quenching of the energy-transfer-dependent fluorescence of dATP† caused by the innerfilter effect from tRNAVal. (Inset) The dATP† fluorescence increase during the first 3 s after tRNAVal addition, depicting the rapid phase of dATP† fluorescence increase during editing.

Fig. 4. Comparison of the rates of translocation of threonine, ␣-aminobutyrate, and cysteine at pH 7.5, 20°C. ValRS (900 nM) was incubated with dATP† (10 ␮M), pyrophosphatase (4 nM), and threonine (1.2 mM), ␣-aminobutyrate (1.2 mM), or cysteine (4 mM) for 4 min to ensure that all of the ValRS was complexed to aminoacyl adenylate. The energy-transfer-dependent fluorescence of dATP† was monitored (excitation ⫽ 295 nm; emission ⫽ 440 nm), and tRNAVal (105 nM) was added to stimulate editing (arrow). The fluorescence traces were normalized to correct for the quenching of the energy-transferdependent fluorescence of dATP† caused by the inner-filter effect from tRNAVal.

three amino acids, the translocation rates are similar to their respective overall editing rates (2.7–3.5 s⫺1). The rate of translocation is considerably slower than the maximum rate of hydrolysis of exogenously added misacylated tRNAVal (20–40 s⫺1) (18, 19). Thus, once the misactivated amino acid is translocated to the site for editing, the chemical step for hydrolysis is relatively instantaneous. Consequently, under normal circumstances where a noncognate amino acid is mixed with tRNAVal, translocation is rate-limiting for editing. The close similarities in the translocation rate constants measured for threonine and cysteine establish that the editing 1. 2. 3. 4. 5. 6. 7. 8. 9.

Giege´, R., Sissler, M. & Florentz, C. (1998) Nucleic Acids Res. 26, 5017–5035. Baldwin, A. N. & Berg, P. (1966) J. Biol. Chem. 241, 839–845. Eldred, E. W. & Schimmel, P. R. (1972) J. Biol. Chem. 247, 2961–2964. Ebel, J. P., Giege´, R., Bonnet, J., Kern, D., Befort, N., Bollack, C., Fasiolo, F., Gangloff, J. & Dirheimer, G. (1973) Biochimie 55, 547–557. Jakubowski, H. & Goldman, E. (1992) Microbiol. Rev. 56, 412–429. Fersht, A. (1999) Structure and Mechanism in Protein Science (Freeman, New York). Schmidt, E. & Schimmel, P. (1994) Science 264, 265–267. Nureki, O., Vassylyev, D. G., Tateno, M., Shimada, A., Nakama, T., Fukai, S., Konno, M., Hendrickson, T. L., Schimmel, P. & Yokoyama, S. (1998) Science 280, 578–582. Silvian, L. F., Wang, J. & Steitz, T. A. (1999) Science 285, 1074–1077.

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domain is not designed to select a specific misactivated amino acid. This possibility is supported by our observation that ␣-aminobutyrate also is efficiently translocated during editing, even though it is an unnatural amino acid in E. coli (hence, the existence of evolutionary pressure on the E. coli editing domain to select ␣-aminobutyrate as a substrate is not obvious). Instead, to carry out its role as the ‘‘fine sieve’’ in the double-sieve-editing model, the editing domain probably is structured to accept all misactivated amino acids and to strictly prevent the entrance of the activated cognate amino acid. The translocation of misactivated amino acid during ValRS editing is thought to occur primarily by the posttransfer pathway, that is, by the deacylation of mischarged tRNAVal (20). A possible molecular mechanism for posttransfer editing in IleRS has been inferred from the structure of IleRS complexed to tRNAIle (9). Specifically, the last three nucleotides of the tRNA (74–76) are proposed to adopt two conformations in the IleRS䡠tRNAIle complex. The hairpinned conformation projects the A76 nucleotide into the active site of the enzyme, where it can be aminoacylated, whereas the stacked conformation places the A76 nucleotide in the editing domain, where an incorrect amino acid (attached to A76) can be hydrolyzed. The switch from the hairpinned to the stacked conformation could therefore provide the mechanism for translocation during editing. A similar mechanism might be responsible for the translocation of misactivated amino acids during editing by ValRS. In such a mechanism, the rate of translocation will depend primarily on the rate of the tRNA switching from the hairpinned to the stacked conformation, and not on the side chain of the amino acid attached to the tRNA. This prediction is consistent with our observation that threonine, ␣-aminobutyrate, and cysteine are translocated with similar efficiencies. It also implies that there is no physical contact between the amino acid side chain and the surface of the enzyme that lies between the synthetic active site and the editing site. If the side chain of an aminoacyl group made contact with the enzyme during translocation, then the difference between the more polar threonine and cysteine, on the one hand, and the hydrophobic ␣-aminobutyrate, on the other, would probably be reflected in different rates of translocation. Thus, we infer that the amino acid side chain points out, away from the surface of the protein during translocation. We thank Dr. Chien Chia Wang for the cloning of the E. coli ValRS gene into a plasmid that allowed for protein expression. This work was supported by National Institutes of Health Grant GM 15539. 10. Heck, J. D. & Hatfield, G. W. (1988) J. Biol. Chem. 263, 868–877. 11. Burbaum, J. J. & Schimmel, P. (1991) J. Biol. Chem. 266, 16965–16968. 12. Hou, Y. M., Shiba, K., Mottes, C. & Schimmel, P. (1991) Proc. Natl. Acad. Sci. USA 88, 976–980. 13. Starzyk, R. M., Webster, T. A. & Schimmel, P. (1987) Science 237, 1614–1618. 14. Lin, L., Hale, S. P. & Schimmel, P. (1996) Nature (London) 384, 33–34. 15. Nomanbhoy, T. K., Hendrickson, T. L. & Schimmel, P. (1999) Mol. Cell 4, 519–528. 16. Hale, S. P. & Schimmel, P. (1996) Proc. Natl. Acad. Sci. USA 93, 2755–2758. 17. Hiratsuka, T. (1983) Biochim. Biophys. Acta 742, 496–508. 18. Jakubowski, H. & Fersht, A. R. (1981) Nucleic Acids Res. 9, 3105–3117. 19. Fersht, A. R. & Dingwall, C. (1979) Biochemistry 18, 1238–1245. 20. Fersht, A. R. & Kaethner, M. M. (1976) Biochemistry 15, 3342–3346.

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