3EHI

Crystal Structure of Human Thymidyalte Synthase M190K with Loop 181-197 stabilized in the inactive conformation


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.238 
  • R-Value Work: 0.198 
  • R-Value Observed: 0.200 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Variants of human thymidylate synthase with loop 181-197 stabilized in the inactive conformation.

Lovelace, L.L.Johnson, S.R.Gibson, L.M.Bell, B.J.Berger, S.H.Lebioda, L.

(2009) Protein Sci 18: 1628-1636

  • DOI: https://doi.org/10.1002/pro.171
  • Primary Citation of Related Structures:  
    3EGY, 3EHI

  • PubMed Abstract: 

    Loop 181-197 of human thymidylate synthase (hTS) populates two major conformations, essentially corresponding to the loop flipped by 180 degrees . In one of the conformations, the catalytic Cys195 residue lies distant from the active site making the enzyme inactive. Ligands stabilizing this inactive conformation may function as allosteric inhibitors. To facilitate the search for such inhibitors, we have expressed and characterized several mutants designed to shift the equilibrium toward the inactive conformer. In most cases, the catalytic efficiency of the mutants was only somewhat impaired with values of k(cat)/K(m) reduced by factors in a 2-12 range. One of the mutants, M190K, is however unique in having the value of k(cat)/K(m) smaller by a factor of approximately 7500 than the wild type. The crystal structure of this mutant is similar to that of the wt hTS with loop 181-197 in the inactive conformation. However, the direct vicinity of the mutation, residues 188-194 of this loop, assumes a different conformation with the positions of C(alpha) shifted up to 7.2 A. This affects region 116-128, which became ordered in M190K while it is disordered in wt. The conformation of 116-128 is however different than that observed in hTS in the active conformation. The side chain of Lys190 does not form contacts and is in solvent region. The very low activity of M190K as compared to another mutant with a charged residue in this position, M190E, suggests that the protein is trapped in an inactive state that does not equilibrate easily with the active conformer.


  • Organizational Affiliation

    Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, USA.


Macromolecules
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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Thymidylate synthaseA [auth X]313Homo sapiensMutation(s): 1 
Gene Names: TYMSTSOK/SW-cl.29
EC: 2.1.1.45
UniProt & NIH Common Fund Data Resources
Find proteins for P04818 (Homo sapiens)
Explore P04818 
Go to UniProtKB:  P04818
PHAROS:  P04818
GTEx:  ENSG00000176890 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP04818
Sequence Annotations
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  • Reference Sequence
Small Molecules
Modified Residues  1 Unique
IDChains TypeFormula2D DiagramParent
CME
Query on CME
A [auth X]L-PEPTIDE LINKINGC5 H11 N O3 S2CYS
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.238 
  • R-Value Work: 0.198 
  • R-Value Observed: 0.200 
  • Space Group: P 31 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 96.053α = 90
b = 96.053β = 90
c = 80.064γ = 120
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
REFMACrefinement
PDB_EXTRACTdata extraction
SERGUIdata collection
CNSphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2009-08-18
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Advisory, Version format compliance
  • Version 1.2: 2017-10-25
    Changes: Refinement description
  • Version 1.3: 2021-10-20
    Changes: Database references, Derived calculations
  • Version 1.4: 2023-08-30
    Changes: Data collection, Refinement description