title: Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase.
abstract: X-ray crystallography provides excellent structural data on protein-DNA interfaces, but crystallographic complexes typically contain only small fragments of large DNA molecules. We present a new approach that can use longer DNA substrates and reveal new protein-DNA interactions even in extensively studied systems. Our approach combines rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). DXMS identifies solvent-exposed protein surfaces; docking is used to create a 3-dimensional model of the protein-DNA interaction. We investigated the enzyme uracil-DNA glycosylase (UNG), which detects and cleaves uracil from DNA. UNG was incubated with a 30 bp DNA fragment containing a single uracil, giving the complex with the abasic DNA product. Compared with free UNG, the UNG-DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210-220 and 251-264. Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG-DNA crystallographic structures. Our results can be explained by separation of the two DNA strands on one side of the active site. These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway.
authors.affiliation: San Diego Supercomputer Center, University of California, San Diego, 9500 Gilman Drive, MC 0505, La Jolla, CA 92093, USA. vickie@sdsc.edu
authors.forename: Victoria A
authors.initials: VA
authors.lastname: Roberts
authors.forename: Michael E
authors.initials: ME
authors.lastname: Pique
authors.forename: Simon
authors.initials: S
authors.lastname: Hsu
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.forename: Geir
authors.initials: G
authors.lastname: Slupphaug
authors.forename: Robert P
authors.initials: RP
authors.lastname: Rambo
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Jamison
authors.forename: Tong
authors.initials: T
authors.lastname: Liu
authors.forename: Jun H
authors.initials: JH
authors.lastname: Lee
authors.forename: John A
authors.initials: JA
authors.lastname: Tainer
authors.forename: Lynn F
authors.initials: LF
authors.lastname: Ten Eyck
authors.forename: Virgil L
authors.initials: VL
authors.lastname: Woods
mesh_terms: D000465:Algorithms; D020134:Catalytic Domain; D019295:Computational Biology / Q000379:methods; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D051981:Uracil-DNA Glycosidase / Q000737:chemistry*
chemical_list: D004268:DNA-Binding Proteins; D004247:DNA; D051981:Uracil-DNA Glycosidase