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Atomic resolution structure of Escherichia coli dUTPase determined ab initio
A González1, G Larsson, R Persson
1European Molecular Biology Laboratory (EMBL), Hamburg Outstation, Notkestrasse 85, 22603 Hamburg, Germany. ana@slac.stanford.edu
Summary
High-resolution structures of Escherichia coli dUTPase reveal significant protein mobility even when frozen. Mercury derivatization aids crystal lattice stabilization for detailed structural analysis.
Area of Science:
- Biochemistry
- Structural Biology
- Crystallography
Background:
- Escherichia coli dUTPase is crucial for DNA repair.
- Understanding its structure is key to developing therapeutic strategies.
- Previous structural studies were limited in resolution.
Purpose of the Study:
- To determine the high-resolution structure of Escherichia coli dUTPase.
- To investigate the effects of cryocooling and mercury derivatization on enzyme structure.
- To assess the utility of mercury derivatization for future structural studies.
Main Methods:
- X-ray crystallography of cryocooled mercury-derivatized and native Escherichia coli dUTPase crystals.
- Data collection to atomic resolution (1.05 Å and 1.45 Å).
- Structure determination using molecular replacement and refinement with ARP/wARP.
Main Results:
- A highly accurate atomic model of Escherichia coli dUTPase was obtained.
- Significant protein mobility was observed in the frozen state, with disordered main and side chains.
- Glycerol molecules were found in the deoxyribose-binding site.
- Mercury binding did not adversely affect the active site.
- Mercury derivatization unexpectedly stabilized the crystal lattice.
Conclusions:
- Cryocooled Escherichia coli dUTPase exhibits considerable intrinsic disorder.
- Mercury derivatization is a valuable technique for stabilizing crystals and achieving high-resolution structures.
- This approach facilitates detailed studies of enzyme-inhibitor interactions.