5W3Q

L28F E.coli DHFR in complex with NADPH


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.40 Å
  • R-Value Free: 0.168 
  • R-Value Work: 0.139 
  • R-Value Observed: 0.140 

Starting Model: experimental
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This is version 1.3 of the entry. See complete history


Literature

Defining the Structural Basis for Allosteric Product Release from E. coli Dihydrofolate Reductase Using NMR Relaxation Dispersion.

Oyen, D.Fenwick, R.B.Aoto, P.C.Stanfield, R.L.Wilson, I.A.Dyson, H.J.Wright, P.E.

(2017) J Am Chem Soc 139: 11233-11240

  • DOI: https://doi.org/10.1021/jacs.7b05958
  • Primary Citation of Related Structures:  
    5W3Q

  • PubMed Abstract: 

    The rate-determining step in the catalytic cycle of E. coli dihydrofolate reductase is tetrahydrofolate (THF) product release, which can occur via an allosteric or an intrinsic pathway. The allosteric pathway, which becomes accessible when the reduced cofactor NADPH is bound, involves transient sampling of a higher energy conformational state, greatly increasing the product dissociation rate as compared to the intrinsic pathway that obtains when NADPH is absent. Although the kinetics of this process are known, the enzyme structure and the THF product conformation in the transiently formed excited state remain elusive. Here, we use side-chain proton NMR relaxation dispersion measurements, X-ray crystallography, and structure-based chemical shift predictions to explore the structural basis of allosteric product release. In the excited state of the E:THF:NADPH product release complex, the reduced nicotinamide ring of the cofactor transiently enters the active site where it displaces the pterin ring of the THF product. The p-aminobenzoyl-l-glutamate tail of THF remains weakly bound in a widened binding cleft. Thus, through transient entry of the nicotinamide ring into the active site, the NADPH cofactor remodels the enzyme structure and the conformation of the THF to form a weakly populated excited state that is poised for rapid product release.


  • Organizational Affiliation

    Department of Integrative Structural and Computational Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Dihydrofolate reductase159Escherichia coli K-12Mutation(s): 1 
Gene Names: folAtmrAb0048JW0047
EC: 1.5.1.3
UniProt
Find proteins for P0ABQ4 (Escherichia coli (strain K12))
Explore P0ABQ4 
Go to UniProtKB:  P0ABQ4
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP0ABQ4
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.40 Å
  • R-Value Free: 0.168 
  • R-Value Work: 0.139 
  • R-Value Observed: 0.140 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 34.337α = 90
b = 45.166β = 90
c = 97.77γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
HKL-2000data reduction
SCALEPACKdata scaling
PHASERphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)United StatesGM75995

Revision History  (Full details and data files)

  • Version 1.0: 2017-08-09
    Type: Initial release
  • Version 1.1: 2017-08-23
    Changes: Database references
  • Version 1.2: 2022-03-23
    Changes: Author supporting evidence, Database references, Derived calculations
  • Version 1.3: 2023-10-04
    Changes: Data collection, Refinement description