NF-κB Binding Mechanism:  A Nuclear Magnetic Resonance and Modeling Study of a GGG → CTC Mutation

Abstract

We present the solution structure of the nonpalindromic 16 bp DNA 5'd(CTGCTCACTTTCCAGG)3'. 5'd(CCTGGAAAGTGAGCAG)3' containing a mutated kappaB site for which the mutation of a highly conserved GGG tract of the native kappaB HIV-1 site to CTC abolishes NF-kappaB binding. 1H and 31P 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 equilibrium 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 proposed 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 (denoted 16N), large differences are induced in the intrinsic structural properties of both duplexes. In particular, in BII structures, 16M shows a kink located 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 conformation very favorable for the interaction with NF-kappaB, 16M cannot mimic such a conformation and, moreover, its deeper and narrower major groove could hinder the DNA-protein interactions.

Programmable oligomers targeting 5′-GGGG-3′ in the minor groove of DNA and NF-κB binding inhibition

“…1). 13,15,24,25 Polyamides targeted to this region have previously been shown to inhibit NF-κB binding. 15 The possibility of steric or allosteric inhibition exists due to numerous contacts between the p50 protein and the phosphate backbone or direct protein-DNA contacts in the major groove, opposite the minor groove polyamide binding site.…”

Regulation of DNA binding by Rel/NF-κB transcription factors: structural views

“…NMR studies have been done on the HIV-1 LTR 5'-GGGACTTTC-3' and a mutated kB DNA sequence (Tisne et al, 1998(Tisne et al, , 1999. The wildtype kB site exists in an equilibrium between being inherently straight or bent 208 towards the major groove while the mutations, biochemically observed to abrogate protein binding (Nabel and Baltimore, 1987), e ect a pronounced kink that precludes protein binding (Tisne et al, 1999). Indeed, in vitro DNA bending experiments show that the various kB sites do have inherently di erent bend angles (Schreck et al, 1990).…”

DNA conformational transitions inferred from re-evaluation ofm|Fo| −D|Fc| electron-density maps

“…NMR data revealed evidence for a BI-BII equilibrium in steps flanking the NF-B binding site and a curvature of the DNA duplex induced by the equilibrium (Tisné et al, 1998;Wecker et al, 2002). A mutation study showed that the distribution of BI-BII conformational equilibria had a critical effect on the binding affinity (Tisné et al, 1999). Molecular-dynamics (MD) simulations of the binding sites of papillomavirus type 1 E2 protein (Djuranovic & Hartmann, 2005;Robertson & Cheatham, 2015) demonstrated that a DNA sequence with stronger affinity adopts the BII conformer more frequently, and the BII conformer is also preserved in protein-DNA complexes.…”