Use of super(13)C, super(15)N-Labeled DNA in a Non-Sequence-Specific Protein-DNA Complex Resolves Ambiguous Assignments of Intermolecular NOEs

• Published in: Journal of the American Chemical Society 1999-04, Vol.121 (14), p.3547-3548
• Main Authors: Masse, JE, Allain, FH-T, Yen, Y-M, Johnson, R C, Feigon, J
• Format: Article
• Language: English
• Subjects: NHP6A protein; Non-histone protein 6A; Saccharomyces cerevisiae
• ISSN: 0002-7863
• DOI: 10.1021/ja9839926

To date, NMR structures of protein-DNA complexes have been determined by using solely homonuclear data or with only the protein component isotopically labeled. All of these complexes have involved sequence-specific DNA binding proteins which make specific contacts with the DNA bases, with one recent exception. The recent development of protocols for the production of uniformly super(15)N, super(13)C-labeled DNA in sufficient quantity for multidimensional NMR now makes the acquisition of the complementary data possible. We have applied heteronuclear multidimensional NMR spectroscopy to study the interaction of the 93 amino acid yeast Non-Histone Protein 6A (NHP6A), a nonsequence specific HMG box protein, with the 15 base-pair DNA duplex d(GGGGTGATTGTTCAG) times d(CTGAACAATCACCCC). The DNA sequence contains the seven nucleotide recognition sequence of the sequence-specific HMG box protein SRY (a sex-determining factor in mammals), and was chosen to compare the mode of binding of a sequence-specific and non-sequence-specific HMG box protein on the same DNA sequence. Assignment of intermolecular NOEs in protein-DNA complexes is often particularly difficult to obtain due in part to line broadening at the protein DNA interface, in addition to spectral overlap especially for the deoxyribose resonances. Here we show that the use of isotopically labeled DNA in this protein-DNA complex was essential to obtain unambiguous assignment of intermolecular NOEs which were unresolved in the spectra of the complex using uniformly super(15)N, super(13)C-labeled protein alone. These NOEs, which could not have been assigned without the use of labeled DNA, allowed a precise positioning of the protein on the DNA. This is the first time that intermolecular NOEs have been reported and a precise side-chain localization achieved in a protein-DNA complex with a non-sequence-specific HMG box protein, despite several attempts. Surprisingly, the protein binds at a different site and in an opposite orientation on the DNA to what is observed for the sequence-specific proteins.