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Updated: Sep 20, 2026
X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
Published on: May 13, 2020
Solution structure and NH exchange studies of the MutT pyrophosphohydrolase complexed with Mg(2+) and 8-oxo-dGMP, a
Michael A Massiah1, Vibhor Saraswat, Hugo F Azurmendi
1Department of Biological Chemistry, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA.
Abstract:
To learn the structural basis for the unusually tight binding of 8-oxo-nucleotides to the MutT pyrophosphohydrolase of Escherichia coli (129 residues), the solution structure of the MutT-Mg(2+)-8-oxo-dGMP product complex (K(D) = 52 nM) was determined by standard 3-D heteronuclear NMR methods. Using 1746 NOEs (13.5 NOEs/residue) and 186 phi and psi values derived from backbone (15)N, Calpha, Halpha, and Cbeta chemical shifts, 20 converged structures were computed with NOE violations
Insights
The Escherichia coli MutT enzyme binds 8-oxo-dGMP tightly due to ligand-induced conformational changes that narrow the nucleotide-binding site. This structural adaptation enhances enzyme-product complex stability and affinity.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Biology
Background:
- MutT pyrophosphohydrolase from Escherichia coli is crucial for preventing mutations.
- 8-oxo-nucleotides are mutagenic DNA damage products.
- Understanding MutT's tight binding mechanism is key to its biological function.
Purpose of the Study:
- To elucidate the structural basis for the high affinity of MutT for 8-oxo-dGMP.
- To determine the solution structure of the MutT-Mg(2+)-8-oxo-dGMP complex.
Main Methods:
- 3D heteronuclear NMR spectroscopy was employed.
- Nuclear Overhauser Effect (NOE) data and chemical shifts were used to calculate structures.
- Residual dipolar couplings refined the structural model.
Main Results:
- A well-defined structure of the MutT-Mg(2+)-8-oxo-dGMP complex was determined.
- Binding of 8-oxo-dGMP induced significant conformational changes, narrowing the nucleotide-binding cleft.
- The enzyme buried a large surface area (71-78%) of the bound 8-oxo-dGMP.
- Ligand binding led to slower backbone amide proton exchange rates, indicating a more compact structure.
Conclusions:
- The unusually tight binding of 8-oxo-dGMP to MutT is attributed to extensive ligand-induced conformational changes.
- These changes narrow the nucleotide-binding site, stabilizing the enzyme-product complex.
- Specific hydrogen bonds involving Asn-119 and Arg-78 likely contribute to the high affinity.
