2IBT

Crystal structure of the adenine-specific DNA methyltransferase M.TaqI complexed with the cofactor analog AETA and a 10 bp DNA containing 2-aminopurine at the target position and an abasic site analog at the target base partner position


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.70 Å
  • R-Value Free: 0.203 
  • R-Value Work: 0.167 
  • R-Value Observed: 0.169 

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This is version 1.4 of the entry. See complete history


Literature

2-Aminopurine Flipped into the Active Site of the Adenine-Specific DNA Methyltransferase M.TaqI: Crystal Structures and Time-Resolved Fluorescence

Lenz, T.Bonnist, E.Y.M.Pljevaljcic, G.Neely, R.K.Dryden, D.T.F.Scheidig, A.J.Jones, A.C.Weinhold, E.

(2007) J Am Chem Soc 129: 6240-6248

  • DOI: https://doi.org/10.1021/ja069366n
  • Primary Citation of Related Structures:  
    2IBS, 2IBT

  • PubMed Abstract: 

    We report the crystal structure of the DNA adenine-N6 methyltransferase, M.TaqI, complexed with DNA, showing the fluorescent adenine analog, 2-aminopurine, flipped out of the DNA helix and occupying virtually the same position in the active site as the natural target adenine. Time-resolved fluorescence spectroscopy of the crystalline complex faithfully reports this state: base flipping is accompanied by the loss of the very short ( approximately 50 ps) lifetime component associated with fully base-stacked 2-aminopurine in DNA, and 2-aminopurine is subject to considerable quenching by pi-stacking interactions with Tyr108 in the catalytic motif IV (NPPY). This proves 2-aminopurine to be an excellent probe for studying base flipping by M.TaqI and suggests similar quenching in the active sites of DNA and RNA adenine-N6 as well as DNA cytosine-N4 methyltransferases sharing the conserved motif IV. In solution, the same distinctive fluorescence response confirms complete destacking from DNA and is also observed when the proposed key residue for base flipping by M.TaqI, the target base partner thymine, is substituted by an abasic site analog. The corresponding cocrystal structure shows 2-aminopurine in the active site of M.TaqI, demonstrating that the partner thymine is not essential for base flipping. However, in this structure, a shift of the 3' neighbor of the target base into the vacancy left after base flipping is observed, apparently replicating a stabilizing role of the missing partner thymine. Time-resolved fluorescence and acrylamide quenching measurements of M.TaqI complexes in solution provide evidence for an alternative binding site for the extra-helical target base within M.TaqI and suggest that the partner thymine assists in delivering the target base into the active site.


  • Organizational Affiliation

    Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, D-52056 Aachen, Germany.


Macromolecules

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Entity ID: 3
MoleculeChains Sequence LengthOrganismDetailsImage
Modification methylase TaqIE [auth A],
F [auth D]
421Thermus aquaticusMutation(s): 0 
Gene Names: taqIM
EC: 2.1.1.72
UniProt
Find proteins for P14385 (Thermus aquaticus)
Explore P14385 
Go to UniProtKB:  P14385
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP14385
Sequence Annotations
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  • Reference Sequence

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Entity ID: 1
MoleculeChains LengthOrganismImage
5'-D(*GP*TP*TP*CP*GP*(2PR)P*TP*GP*TP*C)-3'A [auth B],
C [auth E]
10N/A
Sequence Annotations
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  • Reference Sequence

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Entity ID: 2
MoleculeChains LengthOrganismImage
5'-D(*GP*AP*CP*AP*(3DR)P*CP*GP*(6MA)P*AP*C)-3'B [auth C],
D [auth F]
10N/A
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 2 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
NEA
Query on NEA

Download Ideal Coordinates CCD File 
G [auth A],
K [auth D]
5'-DEOXY-5'-[2-(AMINO)ETHYLTHIO]ADENOSINE
C12 H18 N6 O3 S
APAPOJUCRZTCHD-WOUKDFQISA-N
GOL
Query on GOL

Download Ideal Coordinates CCD File 
H [auth A]
I [auth A]
J [auth A]
L [auth D]
M [auth D]
H [auth A],
I [auth A],
J [auth A],
L [auth D],
M [auth D],
N [auth D],
O [auth D]
GLYCEROL
C3 H8 O3
PEDCQBHIVMGVHV-UHFFFAOYSA-N
Binding Affinity Annotations 
IDSourceBinding Affinity
NEA PDBBind:  2IBT Kd: 500 (nM) from 1 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.70 Å
  • R-Value Free: 0.203 
  • R-Value Work: 0.167 
  • R-Value Observed: 0.169 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 59.339α = 90
b = 68.699β = 92.09
c = 114.022γ = 90
Software Package:
Software NamePurpose
REFMACrefinement
ProDCdata collection
MAR345data collection
XDSdata scaling
MOLREPphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2007-05-29
    Type: Initial release
  • Version 1.1: 2008-05-01
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Non-polymer description, Version format compliance
  • Version 1.3: 2017-10-18
    Changes: Refinement description
  • Version 1.4: 2023-10-25
    Changes: Data collection, Database references, Derived calculations, Refinement description