title: Cosolute paramagnetic relaxation enhancements detect transient conformations of human uracil DNA glycosylase (hUNG).
abstract: The human DNA repair enzyme uracil DNA glycosylase (hUNG) locates and excises rare uracil bases that arise in DNA from cytosine deamination or through dUTP incorporation by DNA polymerases. Previous NMR studies of hUNG have revealed millisecond time scale dynamic transitions in the enzyme-nonspecific DNA complex, but not the free enzyme, that were ascribed to a reversible clamping motion of the enzyme as it scans along short regions of duplex DNA in its search for uracil. Here we further probe the properties of the nonspecific DNA binding surface of {(2)H(12)C}{(15)N}-labeled hUNG using a neutral chelate of a paramagnetic Gd(3+) cosolute (Gd(HP-DO3A)). Overall, the measured paramagnetic relaxation enhancements (PREs) on R(2) of the backbone amide protons for free hUNG and its DNA complex were in good agreement with those calculated based on their relative exposure observed in the crystal structures of both enzyme forms. However, the calculated PREs systematically underestimated the experimental PREs by large amounts in discrete regions implicated in DNA recognition and catalysis: active site loops involved in DNA recognition (268-274, 246-250), the uracil binding pocket (143-148, 169-170), a transient extrahelical base binding site (214-216), and a remote hinge region (129-132) implicated in dynamic clamping. These reactive hot spots were not correlated with structural, hydrophobic, or solvent exchange properties that might be common to these regions, leaving the possibility that the effects arise from dynamic sampling of exposed conformations that are distinct from the static structures. Consistent with this suggestion, the above regions have been previously shown to be flexible based on relaxation dispersion measurements and course-grained normal-mode analysis. A model is suggested where the intrinsic dynamic properties of these regions allows sampling of transient conformations where the backbone amide groups have greater average exposure to the cosolute as compared to the static structures. We conclude that PREs derived from the paramagnetic cosolute reveal dynamic hot spots in hUNG and that these regions are highly correlated with substrate binding and recognition.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, United States.
authors.forename: Yan
authors.initials: Y
authors.lastname: Sun
authors.forename: Joshua I
authors.initials: JI
authors.lastname: Friedman
authors.forename: James T
authors.initials: JT
authors.lastname: Stivers
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D002614:Chelating Agents / Q000737:chemistry; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D051981:Uracil-DNA Glycosidase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C544520:10-(2-hydroxypropyl)-1,4,7-tetraazacyclododecane-1,4,7-triacetic acid; D002614:Chelating Agents; D006573:Heterocyclic Compounds, 1-Ring; D051981:Uracil-DNA Glycosidase
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