Abstract
Escherichia coli endonuclease VIII (Nei) excises oxidized pyrimidines from DNA. It shares significant sequence homology and similar mechanism with Fpg, a bacterial 8-oxoguanine glycosylase. The structure of a covalent Nei-DNA complex has been recently determined, revealing critical amino acid residues which are important for DNA binding and catalysis. Several Fpg structures have also been reported; however, analysis of structural dynamics of Fpg/Nei family proteins has been hindered by the lack of structures of uncomplexed and DNA-bound enzymes from the same source. We report a 2.8 Å resolution structure of free wild-type Nei and two structures of its inactive mutants, Nei-E2A (2.3 Å) and Nei-R252A (2.05 Å). All three structures are virtually identical, demonstrating that the mutations did not affect the overall conformation of the protein in its free state. The structures show a significant conformational change compared with the Nei structure in its complex with DNA, reflecting a ∼50° rotation of the two main domains of the enzyme. Such interdomain flexibility has not been reported previously for any DNA glycosylase and may present the first evidence for a global DNA-induced conformational change in this class of enzymes. Several local but functionally relevant structural changes are also evident in other parts of the enzyme.
| Original language | English |
|---|---|
| Pages (from-to) | 5006-5016 |
| Number of pages | 11 |
| Journal | Nucleic Acids Research |
| Volume | 33 |
| Issue number | 15 |
| DOIs | |
| State | Published - 2005 |
Bibliographical note
Funding Information:We thank the staff at the National Synchrotron Light Source facility (X25 and X26C beamlines) of the Brookhaven National Laboratory and the staff at the Advanced Photon Source synchrotron facility (5-ID-B beamline) of the Argonne National Laboratory for their help in the X-ray synchrotron data collection and analysis. This research was supported in part by grant (CA17395) (to A.P.G) from the National Institutes of Health. D.O.Z. acknowledges support from the Presidium of the Russian Academy of Sciences (program 10.5), Russian Foundation for Basic Research (04-04-48254, 05-04-48619), Russian Science Support Foundation, US Civil Research and Development Foundation, and the Wellcome Trust UK (070244/Z/03/Z). G.G. was supported by fellowships from the Wolf Foundation (Israel), the Chorafas Foundation (Switzerland) and the Clore Foundation (Israel). Funding to pay the Open Access publication charges for this article was provided by HU internal funds.