The Zinc-binding Site of Escherichia coli Glutamyl-tRNA Synthetase Is Located in the Acceptor-binding Domain

Jinhua Liu,Yves Gagnon,Jolle Gauthier,Lars Furenlid,Pierre-Jean L'Heureux,Michèle Auger,Osamu Nureki,Shigeyuki Yokoyama,Jacques Lapointe

doi:10.1074/jbc.270.25.15162

Abstract

The zinc contents of fragments of Escherichia coli glutamyl-tRNA synthetase, as well as the conservation of the CYC sequence only in zinc-containing glutamyl-tRNA synthetases, suggested that the 98CYCX24-CRHSHEHHADDEPC138 includes some or all residues involved in binding its zinc atom (Liu, J., Lin, S.-X., Blochet, J.-E., Pézolet, M., and Lapointe, J. (1993) Biochemistry 32, 11390-11396). Extended x-ray absorption fine structure (EXAFS) shows that this zinc atom has a four-coordinate non-planar coordination environment with 3 sulfur and 1 nitrogen atoms with bond lengths, respectively, 2.37 +/- 0.02 A and 2.01 +/- 0.02 A, presumably belonging to 3 cysteine residues and 1 histidine residue. Conservative replacement of each histidine and cysteine residue of the 98C-138C segment, respectively, with glutamine (Q) and serine (S), yields variants H129Q, H131Q, H132Q, and C138S (which sustain the growth at 42 degrees C of E. coli JP1449, whose glutamyl-tRNA synthetase is thermosensitive) and C98S, C100S, C125S, and H127Q (which do not). The amount of this enzyme in these mutants is at least 1 order of magnitude larger than that in a wild type strain; however, no glutamyl-tRNA synthetase activity is detectable in extracts of the variants C100S and C125S, whereas its specific activity in those of C98S and H127Q is about 10-fold lower than in cells overproducing the wild type enzyme or the variants H129Q, H131Q, H132Q, and C138S. These results indicate that the zinc atom present in E. coli glutamyl-tRNA synthetase is bound by the 2 evolutionarily conserved cysteines at positions 98 and 100, and by Cys125 and His127. Molecular modeling of the N-terminal half of this enzyme, using the known structure of E. coli glutaminyl-tRNA synthetase, supports this conclusion and suggests that the 98C-127H segment does not have the characteristics of the classical zinc fingers.

Highlights

The zinc contents offragments ofEscherichia coli glutamyl-tRNA synthetase, as well as the conservation of the CYC sequence only in zinc-containing glutamyltRNA synthetases, suggested that the 98CYCX24" CRHSllEHHADDEPC138 includes some or all residues involved in binding its zinc atom (Liu, J., Lin, S.-x., Blochet, J.-E., Pezolet, M., and Lapointe, J. (1993) Biochemistry 32, 11390-11396)
Using extended x-ray absorption fine structure (EXAFS) analysis to identify the coordination environment of zinc, site-directed mutagenesis to change each cysteine and histidine of the 98C_138C region, respectively, to serine and glutamine, and molecular modeling of E. coli GluRS based on the known three-dimensional structure of E. coli GlnRS, we report here that 3 sulfur and 1 nitrogen atoms belonging to Cys98, CyslOO, Cys125, and His127 are the zinc ligands in E. coli GluRS
The Coordination Environment of Zinc in E. coli GluRS, Identified by Extended x-ray absorption fine structure (EXAFS)-Analysis of spectra grouped in subsets for error estimation revealed that the enzyme sample was slowly changing with time over the course of the 24 h it was illuminated with x-rays

Summary

Introduction

The zinc contents offragments ofEscherichia coli glutamyl-tRNA synthetase, as well as the conservation of the CYC sequence only in zinc-containing glutamyltRNA synthetases, suggested that the 98CYCX24" CRHSllEHHADDEPC138 includes some or all residues involved in binding its zinc atom (Liu, J., Lin, S.-x., Blochet, J.-E., Pezolet, M., and Lapointe, J. (1993) Biochemistry 32, 11390-11396). Conservative replacement of each histidine and cysteine residue of the 98C·138C segment, respectively, with glutamine (Q) and serine (S), yields variants H129Q, H131Q, H132Q, and Cl38S (which sustain the growth at 42°C of E. coli JP1449, whose glutamyl.tRNA synthetase is thermosensitive) and C98S, CIOOS, C125S, and Hl27Q (which do not) The amount of this enzyme in these mutants is at least I order ofmagnitude larger than that in a wild type strain; no glutamyl·tRNA synthetase activity is detectable in extracts of the variants CIOOS and C125S, whereas its specific activity in those of C98S and Hl27Q is about 10-fold lower than in cells overproducing the wild type enzyme or the variants H129Q, HI3IQ, H132Q, and C138S. This property is not always conserved among the aaRSs specific for a given amino acid, as illustrated by the presence of zinc in E. coli GluRS (Liu et al, 1993) and its absence in T. thermophilus GluRS (Nureki et al, 1993)

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Conclusion