Lysyl-tRNA Synthetase-generated Lysyl-Adenylate Is a Substrate for Histidine Triad Nucleotide Binding Proteins*

Histidine triad nucleotide binding proteins (Hints) are the most ancient members of the histidine triad protein superfamily of nucleotidyltransferases and hydrolyases. Protein-protein interaction studies have found that complexes of the transcription factors MITF or USF2 and lysyl-tRNA synthetase (LysRS) are associated with human Hint1. Therefore, we hypothesized that lysyl-AMP or the LysRS·lysyl-AMP may be a native substrate for Hints. To explore the biochemical relationship between Hint1 and LysRS, a series of catalytic radiolabeling, mutagenesis, and kinetic experiments was conducted with purified LysRSs and Hints from human and Escherichia coli. After incubation of the E. coli or human LysRS with Hints and [α-32P]ATP, but not [α-32P]GTP, 32P-labeled Hints were observed. By varying time and the concentrations of lysine, Mg2+, or LysRS, the adenylation of Hint was found to be dependent on the formation of lysyl-AMP. Site-directed mutagenesis studies of the active site histidine triad revealed that Hint labeling could be abolished by substitution of either His-101 of E. coli hinT or His-112 of human Hint1 by either alanine or glycine. Ap4A, believed to be synthesized by LysRS in vivo, and Zn2+ were shown to inhibit the formation of Hint-AMP with an IC50 value in the low micromolar range. Consistent with pyrophosphate being an inhibitor for aminoacyl-tRNA synthetase, incubations in the presence of pyrophosphatase resulted in enhanced formation of Hint-AMP. These results demonstrate that the lysyl-AMP intermediate formed by LysRS is a natural substrate for Hints and suggests a potential highly conserved regulatory role for Hints on LysRS and possibly other aminoacyl-tRNA synthetases.

Histidine triad nucleotide binding protein (Hint) 2 belongs to a histidine triad (HIT) superfamily that has a characteristic C-terminal active site motif, HXHXHXX, where X is a hydrophobic residue (1). HIT enzymes are a ubiquitous superfamily consisting primarily of nucleoside phosphoramidases, dinucle-otide hydrolyases, and nucleotidyltransferases (1). Five distinct branches of HIT superfamily have been recently classified by a phylogenetic study of HIT proteins (2,3). Recently, Hint1 homologs isolated from rabbit (4), human (5), chicken (6), yeast (4), and Escherichia coli (5) have been shown to be purine nucleoside phosphoramidases. Human Hint1 has been shown to associate with, and possibly regulate several transcription factors such as TFIIH (7), MITF (8,9), and USF2 (10). Additionally, Hint1 knock out mice have been shown to have an increased susceptibility to the induction of ovarian and mammary tumors by the carcinogen dimethylbenzanthracene and to spontaneous tumors (11). Up-regulation of Hint1 and the significantly reduced in vivo tumorigenicity of 5-aza-dC-treated non-smallcell lung cancer cell line NCI-H522 suggested that hHint1 might be a tumor suppressor (12). Recently, Weiske and Huber (13) reported that Hint1 triggers apoptosis independent of its phosphoramidase activity (13). Human Hint2, which is 61% identical to Hint1, has recently been shown to be a mitochondrial apoptotic sensitizer that is down-regulated in hepatocellular carcinomas (14). Although Hints are efficient hydrolases of purine nucleoside phosphoramidates, cellular function and biochemical relevance of the enzymatic phosphoramidase activity has not been determined.
The Fhit (fragile histidine triad) branch of the HIT superfamily is only found in eukaryotes. Like Hint, Fhit is also a homodimer with tumor suppressor activity (15). Human Fhit is a diadenosine P 1 ,P 3 -triphosphate (Ap 3 A) hydrolyase as well as phosphoramidase (16,17). The precise mechanism of action by which it affects tumor development is not well understood, although site-directed mutagenesis studies have indicated that the Fhit tumor suppressor function is likely not dependent on its Ap 3 A hydrolase activity (18). In contrast to the first two branches, the galactose-1-phosphate uridylyltransferase branch has been shown to be the second enzyme in the Leloir pathway necessary for galactose utilization (19,20). Although, galactose-1-phosphate uridylyltransferase is a homodimer with little overall sequence homology with Hint1 or Fhit, it does share some tertiary structure similarity (21). The fourth family is aprataxin, which is mutated in ataxia-oculomotor apraxia1 (22,23) and possesses phosphoramidase and Ap 4 A hydrolase activity as well as DNA/RNA binding properties (2); recently, the physiological substrate for aprataxin has been shown to be abortive adenylated DNA ligation intermediates (3). The last family is the scavenger mRNA decapping enzyme, DcpS/ DCS-1, a 7-methyl-GpppG hydrolase (24,25).
Recently, hHint1 has been isolated from complexes with lysyl-tRNA synthetase (LysRS) and MITF or USF2 transcription factors (9, 10). To elucidate the biochemical connection * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTAL PROCEDURES
Protein Purification-The E. coli BS68 strain harboring the pBAS39 (derived from pET3a) plasmid for expressing ecLysU as a C-terminal His 6 -tag fusion protein was a gift from Dr. Paul Schimmel (The Scripps Research Institute) (26). The plasmid pM368 encoding hLysRS expresses a fusion protein containing the N-terminal MRGSHHHHHHSSGWVD sequence appended to full-length hLysRS was a gift from Dr. Karin Musier-Forsyth (University of Minnesota) (27). Both LysRS enzymes were purified by using Ni 2ϩ -agarose binding according to a previously published procedure (27). Wild type and mutants of hHint1 and echinT were purified by an AMP-agarose column from echinT knock out strain (BB2) as described previously (5).
Lysyl-AMP-dependent Adenylation of Hints by E. coli LysU and Human LysRS-The concentration of ecLysU, hLysRS, lysine, [␣-32 P]ATP, Mg 2ϩ , Zn 2ϩ , nucleotides, yeast inorganic pyrophosphatase (Sigma), Hint proteins, and incubation time period for each experiment is described in the legends to Figs. 3-9. The reactions were terminated by the addition of SDS sample buffer (4ϫ), and samples were analyzed as described above. Quantitation of the intensity of the 32 P signal was carried out with ImageQuant software (GE Healthcare).

Measurement of AMP Production by TLC Assay-
The TLC assay was employed to measure the formation of AMP by aminoacyl-tRNA synthetase (28,29). E. coli LysU (1 M) was added to the reaction mixture containing ATP (250 M), lysine (200 FIGURE 1. X-ray crystallographic structure of hHint1. A, overall dimer structure with bound AMPCP (Protein Data Bank code 1AV5). Shown is the active site region in one monomer with bound AMPCP (Protein Data Bank code 1AV5) (B) and with bound AMP (C); histidine 112 of hHint1 is the nucleophilic residue that forms a covalent P-N bond with its substrate (Protein Data Bank code 1KPF) (48). M), [␣-32 P]ATP (7.5 M) in buffer B (100 mM Tris HCl, pH 7.0, 10 mM NaCl, 10 mM MgCl 2 , 5 mM dithiothreitol, 0.03 unit/l inorganic pyrophosphatase) at 23°C. Aliquots (2 l) were withdrawn and quenched in ice-cold sodium acetate solution (200 mM, pH 5.0). After 10 or 30 min, echinT (2.5 M) was added to the remaining reaction mixture, and AMP formation was monitored for an additional 10 min. Aliquots of quenched reactions (1 l) were spotted in duplicate on polyethyleneimine-cellulose plates (20 ϫ 10 cm, Selecto Scientific) pre-developed with water. The air-dried TLC plate was developed in ammonium acetate (100 mM), 5% acetic acid solution. The 32 P signal was detected and quantitated as described above.

RESULTS
The Chimeric Mutant of hHint1 and echinT-Human Hint1 and echinT shared 50% sequence identity over 107 alignable amino acids, but their C termini are distinct. The C terminus may play an important role on determining substrate specificity. Homology modeling studies revealed that the C termini of echinT are likely to reside in a similar location to that of the hHint1. 3 To address this possibility, a human/E. coli chimera was created by replacing the C terminus of hHint1 (Gln-120 to Gly-126) with the C terminus of echinT (Pro-109 to Leu-119).
Nucleotidylation of Hints by ecLysU-Adenylation but not guanylation of Hints was observed after incubation with ecLysU ( Fig. 2A), whereas the control experiments without ecLysU ( Fig Enhanced Adenylation of Hint by ecLysU by the Addition of Lysine-To first investigate whether the lysyl-tRNA synthetase activity associated with ecLysU was responsible for the adenylation of Hints, the effect of lysine on Hint-AMP formation was evaluated. Incubation of ecLysU (0.22 M) was carried out at 23°C for 10 min with [␣-32 P]ATP (0.38 M) in buffer A followed by the addition of echinT (0.28 or 2.8 M) for 10 min (Fig. 3, lanes 2 and 4) and in buffer A containing lysine (2.    FEBRUARY 16, 2007 • VOLUME 282 • NUMBER 7

JOURNAL OF BIOLOGICAL CHEMISTRY 4721
observed. Because the small amount of Hint-AMP was observed in the absence of added lysine (Fig. 3, lanes 2 and 4), it is highly likely that a small amount of contaminating lysine or lysyl-AMP bound with ecLysU is present with the purified enzyme. In addition, at the higher echinT concentration, we do observe small amounts of labeled ecLysU (Fig. 3, lanes 4  revealed that the intensity of the Hint-AMP was proportional to the added amount of echinT, reaching a plateau between 1.4 and 2.8 M. Moreover, labeling of ecLysU was noticeable when incubations were carried out with at least a 56-fold excess of echinT. The prevalence of an additional higher M r band, consistent in size with covalently linked ecLysU and echinT, was observed at the highest echinT concentration (14 M) (Fig. 5). The possibility that this band may result from the labeling of LysRS at multiple sites cannot be ruled out. Labeling of the high M r band and ecLysU is likely to suppress the amount of observed echinT-AMP.

Adenylation of Hint Is Dependent on the Concentration of
Lysine and Mg 2ϩ -Because lysine is one of the substrates and Mg 2ϩ is absolutely required for lysyl-AMP formation (30), it is of interest to determine the effect of both molecules on adenylation of echinT in more detail. Hence, various concentrations of lysine (0.025-2500 M) were examined to reveal a saturation-type behavior with respect to the amount of lysine; under these conditions maximal intensity was observed at 25 M lysine (Fig. 6A). In addition, labeling was observed only when Mg 2ϩ was present in the reaction mixture, with maximal intensity reached at a concentration of 1500 M (Fig. 6B). Taken together, the observed reliance of echinT adenylation on lysine and Mg 2ϩ is consistent with a labeling reaction dependent on the formation of lysyl-AMP by ecLysU.

Inhibition of Hint Adenylation by Zn 2ϩ , Ap 4 A, and Ap 3 A-Divalent Zn 2ϩ has been shown to stimulate Ap 4 A formation by
ecLysU. The mechanism has been shown to rely on the ability of Zn 2ϩ to stimulate nucleophilic attack by the ␥-phosphate of ATP on lysyl-AMP (31). In addition, Zn 2ϩ , but not Ap 4 A, has been shown to inhibit Hint activity with an IC 50 of (9.5 Ϯ 2.1) M. 4 Therefore, we chose to test the effect of Zn 2ϩ , Ap 4 A, and other nucleotides on the formation of Hint-AMP. As shown in    (Fig. 8A). When the ecLysU concentration was varied from 0.025 to 14 M with a fixed echinT concentration (6.25 M), the intensity of Hint-AMP was found to increase with increasing amounts of ecLysU (Fig. 8B).
Effect of Inorganic Pyrophosphate (PPi) on Hint-AMP Formation-Yeast inorganic pyrophosphatase, which hydrolyzes PPi, has been used to enhance the catalytic activity of LysRS by reducing product inhibition by PPi (32). The addition of inorganic pyrophosphatase to aminoacyl-tRNA synthetase reaction mixtures has been suggested to more closely mimic the in vivo conditions for aminoacylation (33,34). Therefore, adenylation of echinT was also carried out with and without inorganic pyrophosphatase to evaluate the effect of PPi on Hint-AMP formation. As shown in supplemental Fig. 2, in the presence of inorganic pyrophosphatase-enhanced Hint-AMP formation was observed (3-fold).
Effect of Hint on AMP Formation by ecLysU-To further establish the effect of Hint on LysRS-mediated reaction, a direct measurement of AMP production by ecLysU with or without echinT was compared using the described TLC assay (28,29). The observed rate constant for AMP formation by ecLysU is 0.08 Ϯ 0.01 min Ϫ1 and 0.16 Ϯ 0.02 min Ϫ1 in the presence of echinT. Variants are represented by S.D.
Adenylation of Hint Mutants by ecLysU and hLysRS-Mutation of His-101 of echinT and His-112 of hHint1 to Ala or Gly completely abolished formation of Hint-AMP (Fig. 9), indicating that the adenylation site was the nucleophilic active site histidine. As shown in Fig. 9A, incubation of ecLysU with echinT, the chimera, and hHint1 revealed similar labeling   FEBRUARY 16, 2007 • VOLUME 282 • NUMBER 7 intensities for echinT and the chimera but about 100-fold lower intensity for hHint1. Labeling by hLysRS demonstrated a 10-fold higher intensity for echinT than that for hHint1 and a 47-fold higher intensity for the chimera (Fig. 9B), suggesting that the Hint C terminus is responsible for mediating the differential labeling observed by the ecLysU and hLysRS.

DISCUSSION
Aminoacyl-tRNA synthetases are essential enzymes that charge amino acids with their corresponding cognate tRNAs to form aminoacyl-tRNAs, which are a prerequisite for protein biosynthesis. Aminoacyl-tRNA synthetases synthesize aminoacyl-tRNA in a two-step reaction (35). In the first step, a carboxylate of an amino acid reacts with ATP to form an activated aminoacyl-AMP intermediate. In the second step, a tRNA reacts with the intermediate to form the aminoacyl-tRNA. In addition to the aminoacylation reaction, lysyl-tRNA synthetase and several other aminoacyl-tRNA synthetases are known to catalyze the formation of Ap 4 A (36 -38).
In addition to their well recognized aminoacylation activity, several aminoacyl-tRNA synthetases appear to have alternative cellular and possibly physiological roles (39). For example, the association of human LysRS with the human immunodeficiency virus proteins Vrp (40) and Gag (41,42) and the involvement of histidyl-tRNA synthetase and aspartyl-tRNA synthetase with pro-inflammatory cytokines (43) suggest that aminoacyl-tRNA synthetases have multiple physiological functions. Recently, hLysRS has been shown to suppress gene transcription in complexes with hHint1 and MITF (9) or USF2 (10). Dissociation of hHint1 from the complex by Ap 4 A leads to an observable increase of the transcription of specific genes. Nevertheless, the ubiquitous presence of Hints suggests that an underlining and highly conserved physiological function is likely to exist. Although protein phosphoramidate adenylates  such as adenylated DNA ligase are well known, thus far they have been shown not to be substrates for Hints (4). Because no small molecule nucleoside phosphoramidates are known to exist, we hypothesized that, based on the association of hHint1 with hLysRS, aminoacyl-adenylates may be natural substrates for Hints.
To investigate the relationship between LysRS and Hint-AMP formation, a series of experiments was carried out under various conditions to verify the necessity for lysyl-AMP formation. Consistent with the preference of aminoacyl-tRNA synthetase for ATP over GTP, labeling of Hints was only observed for incubations with [␣-32 P]ATP (Fig. 2). In addition, GTP was found to be a significantly poorer inhibitor of Hint-AMP formation than ATP. In support of our findings, recent modeling studies attempting to develop a molecular rationale for the substrate specificity of ecLysU have concluded that the enzyme has a greater propensity to bind ATP in the necessary productive conformation than GTP (44).
Given the catalytic processes involving LysRS (Fig. 11), we proposed that formation of Hint-AMP is dependent on the formation of lysyl-AMP. As can be seen in Figs. 2 and 5, increasing the amount of lysine in the reaction solution resulted in enhanced and saturable Hint labeling, as expected from an enzymatic process. Hint labeling was found to be dependent on Mg 2ϩ , which is required for aminoacyl-adenylate formation, whereas the addition of Zn 2ϩ , which favors Ap 4 A formation, was found to be inhibitory. In addition, our observation that Ap 4 A and Ap 3 A, which are neither substrates nor inhibitors of Hints, are potent inhibitors of Hint-AMP formation is consistent with the inhibitory effect of Zn 2ϩ and the necessity of lysyl-AMP for labeling. That we can observe a plateau in the amount of labeling as we increase the amount of echinT is indicative of a substrate-saturable enzymatic reaction, with an apparent K m of (0.85 Ϯ 0.34) M, which is about 25-fold lower than that observed for the cognate tRNA Lys (45).
The removal of PPi, a byproduct of aminoacyl-tRNA synthetase aminoacyl-adenylate formation, by pyrophosphatase has been shown to significantly enhance tRNA aminoacylation (34). Because the binding site for both PPi and the incoming tRNA are overlapping, inorganic pyrophosphatase has been shown to aid the reaction by reducing product inhibition and the rate of the reverse reaction. Similarly, a greater amount of Hint adenylation was observed with reaction mixtures containing inorganic pyrophosphatase.
Previously, it has been proposed that Hints, like other members of the HIT family, carry out substrate hydrolysis by employing nucleophilic catalysis (1,46). Of the four conserved active site histidines, one has been shown to be catalytically essential and presumably required for intermediate formation.
Adenylated enzymes were not observed when the putative nucleophilic histidine in either echinT (His-101) or hHint1 (His-112, Fig. 1 (47). In addition, the rate of AMP production by ecLysU increases by ϳ2-fold in the presence of echinT. Taken together, these results are consistent with formation of a covalent P-N bond between the active site histidine and AMP. As observed for other HIT proteins, such as Fhit and galactose-1-phosphate uridylyltransferase (19,48,49), which also proceed through active site histidine-AMP intermediates, the catalytic mechanism used by Hints proceeds through formation of an adenylated enzyme intermediate (Fig. 10). The extent to which Hint hydrolysis of the aminoacyl-adenylate is dependent on proteinprotein transfer or the release of lysyl-AMP from the active site of LysRS remains to be determined (Fig. 11).
Although E. coli and human Hints are nearly 50% sequence similar and their active sites consist of a conserved set of side chains, the C termini of each enzyme is quite unique. X-ray crystal structure analysis of the human and rabbit structures has clearly demonstrated that the C termini of one monomer is in close proximity to the active site of the opposite monomer (21,48,50). Consequently, we hypothesized that a chimeric Hint in which the C terminus of the human enzyme was replaced by that the C terminus of the E. coli enzyme may be responsible for substrate specificity differences. Indeed, regard-  FEBRUARY 16, 2007 • VOLUME 282 • NUMBER 7 less of the LysRS, the chimera was more effectively labeled than either of the two parental Hints. In particular, the bacterial C terminus improved adenylation of hHint1 by ecLysU by nearly 100-fold, with the chimeric protein appearing to be as effectively labeled as the echinT by ecLysU. Surprisingly, hLysRS more effectively labeled echinT than hHint1. As can be seen in Fig. 9B, the E. coli C terminus appears to be at least partially responsible for enhanced adenylate formation, since chimera labeling is enhanced 47-fold. The additional 4-fold increase in chimera labeling relative to echinT may reflect additional hHint1 specific determinants of adenylation or difference in the half-life of the Hint-AMP intermediate.

Hint Is a Lysyl-AMP Hydrolase
Taken together, these results demonstrate that the lysyl-AMP is a physiological substrate for Hints. In retrospect, the ability of Hints to hydrolyze aminoacyl-adenylates should not be surprising given the similar structure and the greater reactivity when compared with nucleoside phosphoramidates. Given the broad conservation of Hints in all three kingdoms of life and their wide spread tissue distribution, a common evolutionarily conserved function appears likely. Although it is possible that Hints are cellular scavengers of inadvertently released aminoacyl-adenylates, the results of recent E. coli protein-protein interaction studies demonstrating that elongation factor (EF-Tu) binds not only to lysyl, alanyl-, and isoleucyl-tRNA synthetase but also echinT suggests that the ability of Hints to hydrolyze aminoacyl-adenylates may possibly have a role to play as regulators of protein translation (51). Whether this role may be to assist in pre-transfer editing or the regulation of adenylate formation remains to be determined. In addition, in mammalian cells, the adenylation of Hint by LysRS might be responsible for the regulation of complex formation with the transcription factors, USF2 and MITF. Ongoing enzyme-and cell-based studies should clarify their apparently necessary cellular role.