4R64

Binary complex crystal structure of E295K mutant of DNA polymerase Beta


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.243 
  • R-Value Work: 0.190 
  • R-Value Observed: 0.195 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Substrate-induced DNA Polymerase beta Activation.

Beard, W.A.Shock, D.D.Batra, V.K.Prasad, R.Wilson, S.H.

(2014) J Biol Chem 289: 31411-31422

  • DOI: https://doi.org/10.1074/jbc.M114.607432
  • Primary Citation of Related Structures:  
    4R63, 4R64, 4R65, 4R66

  • PubMed Abstract: 

    DNA polymerases and substrates undergo conformational changes upon forming protein-ligand complexes. These conformational adjustments can hasten or deter DNA synthesis and influence substrate discrimination. From structural comparison of binary DNA and ternary DNA-dNTP complexes of DNA polymerase β, several side chains have been implicated in facilitating formation of an active ternary complex poised for chemistry. Site-directed mutagenesis of these highly conserved residues (Asp-192, Arg-258, Phe-272, Glu-295, and Tyr-296) and kinetic characterization provides insight into the role these residues play during correct and incorrect insertion as well as their role in conformational activation. The catalytic efficiencies for correct nucleotide insertion for alanine mutants were wild type ∼ R258A > F272A ∼ Y296A > E295A > D192A. Because the efficiencies for incorrect insertion were affected to about the same extent for each mutant, the effects on fidelity were modest (<5-fold). The R258A mutant exhibited an increase in the single-turnover rate of correct nucleotide insertion. This suggests that the wild-type Arg-258 side chain generates a population of non-productive ternary complexes. Structures of binary and ternary substrate complexes of the R258A mutant and a mutant associated with gastric carcinomas, E295K, provide molecular insight into intermediate structural conformations not appreciated previously. Although the R258A mutant crystal structures were similar to wild-type enzyme, the open ternary complex structure of E295K indicates that Arg-258 stabilizes a non-productive conformation of the primer terminus that would decrease catalysis. Significantly, the open E295K ternary complex binds two metal ions indicating that metal binding cannot overcome the modified interactions that have interrupted the closure of the N-subdomain.


  • Organizational Affiliation

    From the Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709.


Macromolecules

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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
DNA polymerase beta335Homo sapiensMutation(s): 1 
Gene Names: POLB
EC: 2.7.7.7 (PDB Primary Data), 4.2.99 (PDB Primary Data)
UniProt & NIH Common Fund Data Resources
Find proteins for P06746 (Homo sapiens)
Explore P06746 
Go to UniProtKB:  P06746
PHAROS:  P06746
GTEx:  ENSG00000070501 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP06746
Sequence Annotations
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  • Reference Sequence

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Entity ID: 2
MoleculeChains LengthOrganismImage
DNA (5'-D(*CP*CP*GP*AP*CP*AP*GP*CP*GP*CP*AP*TP*CP*AP*GP*C)-3')B [auth T]16synthetic construct
Sequence Annotations
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  • Reference Sequence

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Entity ID: 3
MoleculeChains LengthOrganismImage
DNA (5'-D(*GP*CP*TP*GP*AP*TP*GP*CP*GP*C)-3')C [auth P]10synthetic construct
Sequence Annotations
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  • Reference Sequence

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Entity ID: 4
MoleculeChains LengthOrganismImage
DNA (5'-D(P*GP*TP*CP*GP*G)-3')5synthetic construct
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.243 
  • R-Value Work: 0.190 
  • R-Value Observed: 0.195 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 53.525α = 90
b = 79.009β = 106.12
c = 54.505γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
PHENIXrefinement
PDB_EXTRACTdata extraction
HKL-2000data collection
PHENIXphasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2014-10-08
    Type: Initial release
  • Version 1.1: 2014-10-15
    Changes: Database references
  • Version 1.2: 2014-11-26
    Changes: Database references
  • Version 1.3: 2017-11-22
    Changes: Refinement description
  • Version 1.4: 2023-09-20
    Changes: Data collection, Database references, Derived calculations, Refinement description