PDBsum entry 1o0c

Ligase/RNA

PDB id: 1o0c

Contents

Protein chain

529 a.a. *

DNA/RNA

Ligands

SO4 ×2

GLU

AMP

Waters ×144

* Residue conservation analysis

PDB id:

1o0c

Name:

Ligase/RNA

Title:

Crystal structure of l-glutamate and ampcpp bound to glutamine aminoacyl tRNA synthetase

Structure:

Glutaminyl tRNA. Chain: b. Synonym: tRNA(gln). Transfer RNA-gln ii. Engineered: yes. Mutation: yes. Glutaminyl-tRNA synthetase. Chain: a. Synonym: glutamine--tRNA ligase. Glutamine tRNA synthetase. Glnrs. Engineered: yes.

Source:

Synthetic: yes. Other_details: product of runoff t7 polymerase transcription from a double helical DNA template. Escherichia coli. Organism_taxid: 562. Gene: glns. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

2.70Å R-factor: 0.213 R-free: 0.265

Authors:

T.L.Bullock,J.J.Perona

Key ref:

T.L.Bullock et al. (2003). Amino acid discrimination by a class I aminoacyl-tRNA synthetase specified by negative determinants. J Mol Biol, 328, 395-408. PubMed id: 12691748 DOI: 10.1016/S0022-2836(03)00305-X

Date:

20-Feb-03 Release date: 15-Apr-03

Protein chain

P00962  (SYQ_ECOLI) -  Glutamine--tRNA ligase from Escherichia coli (strain K12)

Seq: 554 a.a.

Struc: 529 a.a.

DNA/RNA chain

G-G-G-G-U-A-U-C-G-C-C-A-A-G-C-G-G-U-A-A-G-G-C-A-C-C-G-G-A-U-U-C-U-G-A-U-U-C-C- 74 bases

Enzyme reactions

• Enzyme class: E.C.6.1.1.18 - glutamine--tRNA ligase.
• Reaction: tRNA(Gln) + L-glutamine + ATP = L-glutaminyl-tRNA(Gln) + AMP + diphosphate

tRNA(Gln) + L-glutamine (Bound ligand (Het Group name = GLU) matches with 81.82% similarity) + ATP = L-glutaminyl-tRNA(Gln) (Bound ligand (Het Group name = AMP) corresponds exactly) + AMP + diphosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/S0022-2836(03)00305-X

J Mol Biol 328:395-408 (2003)

PubMed id: 12691748

Amino acid discrimination by a class I aminoacyl-tRNA synthetase specified by negative determinants.

T.L.Bullock, N.Uter, T.A.Nissan, J.J.Perona.

ABSTRACT

The 2.5 A crystal structure of Escherichia coli glutaminyl-tRNA synthetase in a quaternary complex with tRNA(Gln), an ATP analog and glutamate reveals that the non-cognate amino acid adopts a distinct binding mode within the active site cleft. In contrast to the binding of cognate glutamine, one oxygen of the charged glutamate carboxylate group makes a direct ion-pair interaction with the strictly conserved Arg30 residue located in the first half of the dinucleotide fold domain. The nucleophilic alpha-carboxylate moiety of glutamate is mispositioned with respect to both the ATP alpha-phosphate and terminal tRNA ribose groups, suggesting that a component of amino acid discrimination resides at the catalytic step of the reaction. Further, the other side-chain carboxylate oxygen of glutamate is found in a position identical to that previously proposed to be occupied by the NH(2) group of the cognate glutamine substrate. At this position, the glutamate oxygen accepts hydrogen bonds from the hydroxyl moiety of Tyr211 and a water molecule. These findings demonstrate that amino acid specificity by GlnRS cannot arise from hydrogen bonds donated by the cognate glutamine amide to these same moieties, as previously suggested. Instead, Arg30 functions as a negative determinant to drive binding of non-cognate glutamate into a non-productive orientation. The poorly differentiated cognate amino acid-binding site in GlnRS may be a consequence of the late emergence of this enzyme from the eukaryotic lineage of glutamyl-tRNA synthetases.

Selected figure(s)

Figure 3.
Figure 3. (A) Schematic drawing of a proposed network of hydrogen bonds in the amino acid-binding pocket of GlnRS when substrate glutamine is bound. Arrowheads point toward the hydrogen bond acceptor of each pair, and numerals indicate the distance in Å units between the two electronegative atoms of the pair, as estimated from this 2.3 Å crystal structure. The closest approach of Arg30 to the glutamine substrate is 4 Å. MC indicates main-chain. The donor-acceptor pairings and directions of the hydrogen bonds in this model are identical to those proposed by Rath et al.10 based on the structure of GlnRS bound to the QSI analog (except that WAT4 was not considered in that analysis). The correspondence between the nomenclature of the water molecules is: WAT1 corresponds to WAT1050 of Rath et al.10 WAT2 corresponds to WAT1052, WAT3 corresponds to WAT1136, and WAT4 corresponds to WAT1081. (B) Schematic drawing of a proposed network of hydrogen bonds in the amino acid-binding pocket of GlnRS when non-cognate glutamate is bound. Glu makes two additional hydrogen bonds with Arg30 and WAT1. Differences in proposed hydrogen-bonding structure to accommodate the acceptor oxygen atoms of Glu are (i) bifurcation of the Og hydrogen of Ser227. This hydrogen lies 2.3 Å from WAT1 and 2.6 Å from the Asp219 carboxylate and is thus well-positioned to bifurcate; in the structure bound to Glu, the hydrogen in the refined coordinates rotates toward WAT1, which is itself re-oriented to donate a proton to the substrate as depicted. (ii) One proton of WAT3 is now bifurcated between the Asp212 carboxylate and Asn236 main-chain acceptors (bottom). Rotation of this water by graphics modeling shows that one proton can be oriented midway between the two acceptors at 2.6 Å distance from each, while the second proton then points in-line toward WAT2. This orients the two acceptor positions generally toward WAT4.

Figure 4.
Figure 4. Time-course for glutamylation of E. coli tRNA[2]^Gln by GlnRS. The inset shows a thin-layer chromatography plate in which misacylated Glu-AMP is formed to approximately 50% aminoacylation levels (see Materials and Methods for details). The % aminoacylation on the ordinate is derived from the ratio of intensities of the spots corresponding to Glu-AMP and AMP (right).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 328, 395-408) copyright 2003.

Figures were selected by an automated process.