1TMM

Crystal structure of ternary complex of E.coli HPPK(W89A) with MGAMPCPP and 6-Hydroxymethylpterin


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.25 Å
  • R-Value Free: 0.164 
  • R-Value Observed: 0.132 

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Ligand Structure Quality Assessment 


This is version 1.6 of the entry. See complete history


Literature

Is the Critical Role of Loop 3 of Escherichia coli 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase in Catalysis Due to Loop-3 Residues Arginine-84 and Tryptophan-89? Site-Directed Mutagenesis, Biochemical, and Crystallographic Studies.

Li, Y.Blaszczyk, J.Wu, Y.Shi, G.Ji, X.Yan, H.

(2005) Biochemistry 44: 8590-8599

  • DOI: https://doi.org/10.1021/bi0503495
  • Primary Citation of Related Structures:  
    1TMJ, 1TMM

  • PubMed Abstract: 

    Deletion mutagenesis, biochemical, and X-ray crystallographic studies have shown that loop 3 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is required for the assembly of the active center, plays an important role in the stabilization of the ternary complex of HPPK with MgATP and 6-hydroxymethyl-7,8-dihydropterin (HP), and is essential for catalysis. Whether the critical functional importance of loop 3 is due to the interactions between residues R84 and W89 and the two substrates has been addressed by site-directed mutagenesis, biochemical, and X-ray crystallographic studies. Substitution of R84 with alanine causes little changes in the dissociation constants and kinetic constants of the HPPK-catalyzed reaction, indicating that R84 is not important for either substrate binding or catalysis. Substitution of W89 with alanine increases the K(d) for the binding of MgATP by a factor of 3, whereas the K(d) for HP increases by a factor of 6, which is due to the increase in the dissociation rate constant. The W89A mutation decreases the rate constant for the chemical step of the forward reaction by a factor of 15 and the rate constant for the chemical step of the reverse reaction by a factor of 25. The biochemical results of the W89A mutation indicate that W89 contributes somewhat to the binding of HP and more significantly to the chemical step. The crystal structures of W89A show that W89A has different conformations in loops 2 and 3, but the critical catalytic residues are positioned for catalysis. When these results are taken together, they suggest that the critical functional importance of loop 3 is not due to the interactions of the R84 guanidinium group or the W89 indole ring with the substrates.


  • Organizational Affiliation

    Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase
A, B
158Escherichia coliMutation(s): 1 
Gene Names: FOLKB0142
EC: 2.7.6.3
UniProt
Find proteins for P26281 (Escherichia coli (strain K12))
Explore P26281 
Go to UniProtKB:  P26281
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP26281
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 6 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
APC
Query on APC

Download Ideal Coordinates CCD File 
K [auth A],
R [auth B]
DIPHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER
C11 H18 N5 O12 P3
CAWZRIXWFRFUQB-IOSLPCCCSA-N
HHR
Query on HHR

Download Ideal Coordinates CCD File 
L [auth A],
S [auth B]
6-HYDROXYMETHYLPTERIN
C7 H7 N5 O2
XGWIBNWDLMIPNF-UHFFFAOYSA-N
GOL
Query on GOL

Download Ideal Coordinates CCD File 
M [auth A],
T [auth B]
GLYCEROL
C3 H8 O3
PEDCQBHIVMGVHV-UHFFFAOYSA-N
ACT
Query on ACT

Download Ideal Coordinates CCD File 
G [auth A],
H [auth A],
I [auth A],
Q [auth B]
ACETATE ION
C2 H3 O2
QTBSBXVTEAMEQO-UHFFFAOYSA-M
CL
Query on CL

Download Ideal Coordinates CCD File 
F [auth A],
J [auth A],
P [auth B]
CHLORIDE ION
Cl
VEXZGXHMUGYJMC-UHFFFAOYSA-M
MG
Query on MG

Download Ideal Coordinates CCD File 
C [auth A],
D [auth A],
E [auth A],
N [auth B],
O [auth B]
MAGNESIUM ION
Mg
JLVVSXFLKOJNIY-UHFFFAOYSA-N
Binding Affinity Annotations 
IDSourceBinding Affinity
HHR Binding MOAD:  1TMM Kd: 1100 (nM) from 1 assay(s)
APC Binding MOAD:  1TMM Kd: 230 (nM) from 1 assay(s)
PDBBind:  1TMM Kd: 230 (nM) from 1 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.25 Å
  • R-Value Free: 0.164 
  • R-Value Observed: 0.132 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 47.992α = 90
b = 47.392β = 104.13
c = 71.438γ = 90
Software Package:
Software NamePurpose
SHELXmodel building
SHELXL-97refinement
DENZOdata reduction
SCALEPACKdata scaling
AMoREphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2005-06-21
    Type: Initial release
  • Version 1.1: 2008-04-30
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Non-polymer description, Version format compliance
  • Version 1.3: 2021-10-27
    Changes: Database references, Derived calculations
  • Version 1.4: 2023-08-23
    Changes: Data collection, Refinement description
  • Version 1.5: 2023-08-30
    Changes: Database references, Structure summary
  • Version 1.6: 2024-03-13
    Changes: Source and taxonomy, Structure summary