Abstract
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 [alpha-(32)P]ATP, but not [alpha-(32)P]GTP, (32)P-labeled Hints were observed. By varying time and the concentrations of lysine, Mg(2+), 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. Ap(4)A, believed to be synthesized by LysRS in vivo, and Zn(2+) were shown to inhibit the formation of Hint-AMP with an IC(50) 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.
Highlights
FEBRUARY 16, 2007 VOLUME 282 NUMBER 7 otide hydrolyases, and nucleotidyltransferases [1]
These results demonstrate that the lysyl-AMP intermediate formed by lysyl-tRNA synthetase (LysRS) is a natural substrate for Histidine triad nucleotide binding proteins (Hints) and suggests a potential highly conserved regulatory role for Hints on LysRS and possibly other aminoacyl-tRNA synthetases
HHint1 has been isolated from complexes with lysyl-tRNA synthetase (LysRS) and MITF or USF2 transcription factors [9, 10]
Summary
FEBRUARY 16, 2007 VOLUME 282 NUMBER 7 otide hydrolyases, and nucleotidyltransferases [1]. Hint Is a Lysyl-AMP Hydrolase between LysRS and Hint1, both E. coli LysRS (ecLysU) and human LysRS (hLysRS) were purified and shown to label echinT and hHint1 with [␣-32P]ATP but not with [␥-32P]ATP, [␣-32P]GTP, or [␥-32P]GTP.
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