Structural analysis of the DNA target site and its interaction with Mbp1

Abstract

The solution structure of a 14 base-pair non-self complementary DNA duplex containing the consensus-binding site of the yeast transcription factor Mbp1 has been determined by NMR using a combination of scalar coupling analysis, time-dependent NOEs, residual dipolar couplings and 13C-edited NMR spectroscopy of a duplex prepared with one strand uniformly labeled with 13C-nucleotides. As expected, the free DNA duplex is within the B-family of structures, and within experimental limits is straight. However, there are clear local structural variations associated with the consensus CGCG element in the binding sequence that are important for sequence recognition. In the complex, the DNA bends around the protein, which also undergoes some conformational rearrangement in the C-terminal region. Structural constraints derived from paramagnetic perturbation experiments with spin-labeled DNA, chemical shift perturbation experiments of the DNA, previous cross-saturation, chemical shift perturbation experiments on the protein, information from mutational analysis, and electrostatics calculations have been used to produce a detailed docked structure using the known solution conformation of the free protein and other spectroscopic information about the Mbp1:DNA complex. A Monte Carlo-based docking procedure with restrained MD in a fully solvated system subjected to available experimental constraints produced models that account for the available structural data, and can rationalize the extensive thermodynamic data about the Mbp1:DNA complex. The protein:DNA interface is closely packed and is associated with a small number of specific contacts. The structure shows an extensive positively charged surface that accounts for the high polyelectrolyte contribution to binding.

Revisiting the Association of Cationic Groove-Binding Drugs to DNA Using a Poisson-Boltzmann Approach

“…4, extracting DG el directly from thermodynamic binding data is not possible, even though Eq. 4 is used widely (51,(69)(70)(71) to do this. The common practice of inferring that electrostatic interactions are more important when the magnitude of SK obs is large (72), as implied by Eq.…”

Coordinated Genome-Wide Modifications within Proximal Promoter Cis-regulatory Elements during Vertebrate Evolution

“…It is possible that other mechanisms may also play a role in motif frequency changes, including posttranslational modifications of proteins, mutational biases, and changes in effective population size (Berg et al 2004; Chernatynskaya et al 2009; Ezkurdia et al 2009). For instance, methylation of the cytosine ring in CG dinucleotides causes increased rates of biased mutation (Baele et al 2008, 2010; Illingworth and Bird 2009) and may well influence evolutionary changes in GC-rich motifs.…”

Structural basis of DNA recognition by PCG2 reveals a novel DNA binding mode for winged helix-turn-helix domains

“…In the past decade, a number of studies have characterized the interaction between MBP1 and MCB–DNA sequences ( 12 – 20 ). The structure of the DBD of MBP1 has been determined by both X-ray crystallography and nuclear magnetic resonance (NMR) methods and revealed that MBP1–DBD has the same topology as the winged helix-turn-helix (wHTH) domain ( 12 , 13 , and 17 ), although they share very low amino acid sequence identity ( 15 ).…”

“…RFX1 has an atypical binding module in the DNA complex, in which the wing instead of helix B recognizes the DNA. The wing region and helix B in the DBD of MBP1 were also predicted to interact with DNA ( 15 , 20 ). However, the molecular details for the interactions between the DNA binding elements of MBP1 family proteins and DNA remain unknown.…”