1NXE

A Novel NADH Allosteric Regulator Site is Found on the Surface of the Hexameric Type II Phe383Ala Variant of Citrate Synthase


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.30 Å
  • R-Value Work: 0.170 
  • R-Value Observed: 0.170 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Insights into the evolution of allosteric properties. The NADH binding site of hexameric type II citrate synthases.

Maurus, R.Nguyen, N.T.Stokell, D.J.Ayed, A.Hultin, P.G.Duckworth, H.W.Brayer, G.D.

(2003) Biochemistry 42: 5555-5565

  • DOI: https://doi.org/10.1021/bi020622s
  • Primary Citation of Related Structures:  
    1NXE, 1NXG

  • PubMed Abstract: 

    Study of the hexameric and allosterically regulated citrate synthases (type II CS) provides a rare opportunity to gain not only an understanding of a novel allosteric mechanism but also insight into how such properties can evolve from an unregulated structural platform (the dimeric type I CS). To address both of these issues, we have determined the structure of the complex of NADH (a negative allosteric effector) with the F383A variant of type II Escherichia coli CS. This variant was chosen because its kinetics indicate it is primarily in the T or inactive allosteric conformation, the state that strongly binds to NADH. Our structural analyses show that the six NADH binding sites in the hexameric CS complex are located at the interfaces between dimer units such that most of each site is formed by one subunit, but a number of key residues are drawn from the adjacent dimer. This arrangement of interactions serves to explain why NADH allosteric regulation is a feature only of hexameric type II CS. Surprisingly, in both the wild-type enzyme and the NADH complex, the two subunits of each dimer within the hexameric conformation are similar but not identical in structure, and therefore, while the general characteristics of NADH binding interactions are similar in each subunit, the details of these are somewhat different between subunits. Detailed examination of the observed NADH binding sites indicates that both direct charged interactions and the overall cationic nature of the sites are likely responsible for the ability of these sites to discriminate between NADH and NAD(+). A particularly novel characteristic of the complex is the horseshoe conformation assumed by NADH, which is strikingly different from the extended conformation found in its complexes with most proteins. Sequence homology studies suggest that this approach to binding NADH may arise out of the evolutionary need to add an allosteric regulatory function to the base CS structure. Comparisons of the amino acid sequences of known type II CS enzymes, from different Gram-negative bacteria taxonomic groups, show that the NADH-binding residues identified in our structure are strongly conserved, while hexameric CS molecules that are insensitive to NADH have undergone key changes in the sequence of this part of the protein.


  • Organizational Affiliation

    Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Citrate synthase
A, B
427Escherichia coliMutation(s): 1 
Gene Names: GLTA OR GLUT OR ICDB OR B0720
EC: 2.3.3.1
UniProt
Find proteins for P0ABH7 (Escherichia coli (strain K12))
Explore P0ABH7 
Go to UniProtKB:  P0ABH7
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP0ABH7
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.30 Å
  • R-Value Work: 0.170 
  • R-Value Observed: 0.170 
  • Space Group: H 3
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 165.243α = 90
b = 165.243β = 90
c = 157.557γ = 120
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
CNSrefinement
CNSphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2003-04-08
    Type: Initial release
  • Version 1.1: 2008-04-29
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2021-10-27
    Changes: Database references, Derived calculations
  • Version 1.4: 2023-08-16
    Changes: Data collection, Refinement description