5ON3

Quaternary complex of mutant T252A of E. coli leucyl-tRNA synthetase with tRNA(leu), leucyl-adenylate analogue, and post-transfer editing analogue of leucine in the aminoacylation conformation


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 3.10 Å
  • R-Value Free: 0.278 
  • R-Value Work: 0.241 
  • R-Value Observed: 0.243 

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Ligand Structure Quality Assessment 


This is version 1.5 of the entry. See complete history


Literature

Kinetic Origin of Substrate Specificity in Post-Transfer Editing by Leucyl-tRNA Synthetase.

Dulic, M.Cvetesic, N.Zivkovic, I.Palencia, A.Cusack, S.Bertosa, B.Gruic-Sovulj, I.

(2018) J Mol Biol 430: 1-16

  • DOI: https://doi.org/10.1016/j.jmb.2017.10.024
  • Primary Citation of Related Structures:  
    5OMW, 5ON2, 5ON3, 5ONH

  • PubMed Abstract: 

    The intrinsic editing capacities of aminoacyl-tRNA synthetases ensure a high-fidelity translation of the amino acids that possess effective non-cognate aminoacylation surrogates. The dominant error-correction pathway comprises deacylation of misaminoacylated tRNA within the aminoacyl-tRNA synthetase editing site. To assess the origin of specificity of Escherichia coli leucyl-tRNA synthetase (LeuRS) against the cognate aminoacylation product in editing, we followed binding and catalysis independently using cognate leucyl- and non-cognate norvalyl-tRNA Leu and their non-hydrolyzable analogues. We found that the amino acid part (leucine versus norvaline) of (mis)aminoacyl-tRNAs can contribute approximately 10-fold to ground-state discrimination at the editing site. In sharp contrast, the rate of deacylation of leucyl- and norvalyl-tRNA Leu differed by about 10 4 -fold. We further established the critical role for the A76 3'-OH group of the tRNA Leu in post-transfer editing, which supports the substrate-assisted deacylation mechanism. Interestingly, the abrogation of the LeuRS specificity determinant threonine 252 did not improve the affinity of the editing site for the cognate leucine as expected, but instead substantially enhanced the rate of leucyl-tRNA Leu hydrolysis. In line with that, molecular dynamics simulations revealed that the wild-type enzyme, but not the T252A mutant, enforced leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water. Our data demonstrated that the LeuRS editing site exhibits amino acid specificity of kinetic origin, arguing against the anticipated prominent role of steric exclusion in the rejection of leucine. This feature distinguishes editing from the synthetic site, which relies on ground-state discrimination in amino acid selection.


  • Organizational Affiliation

    Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia.


Macromolecules

Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Leucine--tRNA ligaseA,
C [auth D]
880Escherichia coli K-12Mutation(s): 1 
Gene Names: leuSb0642JW0637
EC: 6.1.1.4
UniProt
Find proteins for P07813 (Escherichia coli (strain K12))
Explore P07813 
Go to UniProtKB:  P07813
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP07813
Sequence Annotations
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  • Reference Sequence
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Entity ID: 2
MoleculeChains LengthOrganismImage
tRNA(leu)B,
D [auth E]
87Escherichia coli
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Binding Affinity Annotations 
IDSourceBinding Affinity
LSS BindingDB:  5ON3 Ki: 0.14 (nM) from 1 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 3.10 Å
  • R-Value Free: 0.278 
  • R-Value Work: 0.241 
  • R-Value Observed: 0.243 
  • Space Group: C 1 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 159α = 90
b = 68.7β = 102.8
c = 227.2γ = 90
Software Package:
Software NamePurpose
REFMACrefinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2017-11-15
    Type: Initial release
  • Version 1.1: 2017-11-22
    Changes: Derived calculations
  • Version 1.2: 2017-12-27
    Changes: Database references
  • Version 1.3: 2018-10-24
    Changes: Data collection, Source and taxonomy
  • Version 1.4: 2019-10-16
    Changes: Data collection
  • Version 1.5: 2024-01-17
    Changes: Data collection, Database references, Refinement description