1QQQ

CRYSTAL STRUCTURE ANALYSIS OF SER254 MUTANT OF ESCHERICHIA COLI THYMIDYLATE SYNTHASE


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.50 Å
  • R-Value Free: 0.238 
  • R-Value Work: 0.224 
  • R-Value Observed: 0.224 

wwPDB Validation   3D Report Full Report


This is version 2.1 of the entry. See complete history


Literature

Drug-resistant variants of Escherichia coli thymidylate synthase: effects of substitutions at Pro-254.

Fantz, C.Shaw, D.Jennings, W.Forsthoefel, A.Kitchens, M.Phan, J.Minor, W.Lebioda, L.Berger, F.G.Spencer, H.T.

(2000) Mol Pharmacol 57: 359-366

  • Primary Citation of Related Structures:  
    1QQQ

  • PubMed Abstract: 

    Drug-resistant variants of thymidylate synthase (TS) can potentially be used in gene therapy applications to decrease the myelosuppressive side effects of TS-directed anticancer agents or to select genetically modified cells in vivo. Mutations of proline 303 of human TS confer resistance to TS-directed fluoropyrimidines and antifolates (). We generated the corresponding variants in Escherichia coli TS (ecTS), position 254, to better understand the mechanism by which mutations at this residue confer resistance. In addition, because ecTS is intrinsically resistant to several antifolates when compared with human TS, we suspected that greater resistance could be achieved with the bacterial enzyme. The P254L enzyme conferred >100-fold resistance to both raltitrexed and 5-fluoro-2'-deoxyuridine (FdUrd) compared with wild-type ecTS. Four additional mutants (P254F, P254S, P254G, and P254D), each of which complemented growth of a TS-deficient cell line, were generated, isolated, and characterized. Steady-state values of K(m) for dUMP and k(cat) were not substantially different among the variants and were comparable with the wild-type values, but K(m) for methylenetetrahydrofolate (CH(2)H(4)PteGlu) was >10-fold higher for P254D. Values of k(on) and k(off) for nucleotide binding, which were obtained by stopped-flow spectroscopy, were virtually unchanged among the mutants. Drastic differences were observed for CH(2)H(4)PteGlu binding, with K(d) values >15-fold higher than observed with the wild-type enzyme; surprisingly, the proposed isomerization reaction that is very evident for the wild-type enzyme is not observed with P254S. The decrease in affinity for CH(2)H(4)PteGlu correlates well with K(i) values obtained for three TS-directed inhibitors. These results show that mutations at Pro-254 specifically affect the initial binding interactions between enzyme and cofactor and also alter the ability of the mutant enzymes to undergo conformational changes that occur on ternary complex formation. The crystal structure of P254S was determined at 1.5 A resolution and is the most precise structure of TS available. When compared with wild-type TS, the structure shows local conformational changes affecting mostly Asp-253; its carbonyl is rotated approximately 40 degrees, and the side chain forms an ion pair with Arg-225.


  • Organizational Affiliation

    Department of Chemistry, Columbia, South Carolina, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
THYMIDYLATE SYNTHASE264Escherichia coliMutation(s): 1 
EC: 2.1.1.45
UniProt
Find proteins for P0A884 (Escherichia coli (strain K12))
Explore P0A884 
Go to UniProtKB:  P0A884
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP0A884
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Modified Residues  2 Unique
IDChains TypeFormula2D DiagramParent
CME
Query on CME
A
L-PEPTIDE LINKINGC5 H11 N O3 S2CYS
CXM
Query on CXM
A
L-PEPTIDE LINKINGC6 H11 N O4 SMET
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.50 Å
  • R-Value Free: 0.238 
  • R-Value Work: 0.224 
  • R-Value Observed: 0.224 
  • Space Group: I 21 3
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 132.437α = 90
b = 132.437β = 90
c = 132.437γ = 90
Software Package:
Software NamePurpose
AMoREphasing
CNSrefinement
DENZOdata reduction
SCALEPACKdata scaling

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 1999-06-14
    Type: Initial release
  • Version 1.1: 2008-04-27
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Derived calculations, Version format compliance
  • Version 2.0: 2021-11-03
    Changes: Atomic model, Database references, Derived calculations
  • Version 2.1: 2022-04-13
    Changes: Database references, Structure summary