NF-kappa B binding mechanism: A nuclear magnetic resonance and modeling study of a GGG -> CTC mutation

We present the solution structure of the nonpalindromic 16 bp DNA (5')d(CTG CTCACTTTCCAGG)(3'). (5')d(CCTGGAAAGTGAGCAG) (3') containing a mutated kappa B Site for which the mutation of a highly conserved GGG tract of the nativ e kappa B HIV-1 site to CTC abolishes NF-kappa B binding. H-1 and P-31 NMR spectroscopies have been used together with molecular modeling to determine the fine structure of the duplex. NMR data show evidence for a BI-BII equi librium of the CpA.TpG steps at the 3'-end of the oligomer. Models for the extreme conformations reached by the mutated duplex (denoted 16M) are propo sed in agreement with the NMR data. Since the distribution of BII sites is changed in the mutated duplex compared to that of the native duplex (denote d 16N), large differences are induced in the intrinsic structural propertie s of both duplexes. in particular, in BII structures, 16M shows a kink loca ted at the 3'-end of the duplex, and in contrast, 16N exhibits an intrinsic global curvature toward the major groove. Whereas 16N can reach a conforma tion very favorable for the interaction with NF-kappa B, 16M cannot mimic s uch a conformation and, moreover, its deeper and narrower major groove coul d hinder the DNA-protein interactions.