Solution structure of a specific DNA complex of the Myb DNA-binding domain with cooperative recognition helices (original) (raw)
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European Journal of Biochemistry, 1996
A range of double and triple resonance heteronuclear NMK experiments has been used lo obtain nearly complete sequence-specific 15N, 13C and 'H resonance assignments for a 1 10-rcsidue protein corresponding to the B-Myb DNA-binding domain (B-MybR2R3) and to determine its secondary structure in solution. The protein was found to contain two stablc heliccs in rcpeat-2 (R2) and lhree in rcpeat-3 (R3L involving residues K12-K24 (R2-1), W30-H36 (K2-2), E64-V76 (K3-1), W81-LX7 (K3-2) and D93-K1OS (R3-3). In addition, the chemical shift and nuclear Overhauser effect data suggest that amino acids Q44-W49 near the C-terminus of R2 form an unstable or nascent helix, which could be stabilised on binding to a specific DNA target site. The two N-terminal heliccs in K2 and R3 occupy essentially identical positions in the two domains, consistent with the high lcvel of sequcnce similarity between thcse regions. In contrast, the C-terminal region forming the third helix in R3 shows low scquencc similarity with R2, accounting for the differences in secondary structure. In the ciise of B-MybR2R3, there is clear chemical shift and line-broadening evidence for the existence of multiple conformations in the C-terminal region ofR2, which is believed to form one half of the DNA-binding site. We propose that confortriational instability of pait of the DNA-binding motif is a way of incrcasing the specificity of Myb proteins for a relatively short (6-bp) DNA target site by reducing their affinity for lion-specific 13NA sequences compared to specific sites.
Nucleic Acids Research, 2001
In the Myb family, as in other families of transcription factors sharing similar DNA-binding domains (DBDs), diversity of function is believed to rely mainly on the less conserved parts of the proteins and on their distinct patterns of expression. However, small conserved differences between DBDs of individual members could play a role in finetuning their function. We have compared the highly conserved DBDs of the three vertebrate Myb proteins (A-, Band c-Myb) and found distinct functional differences. While A-and c-Myb behaved virtually identically in a variety of DNA-binding assays, B-Myb formed complexes of comparatively lower stability, rapidly dissociating under competitive conditions and showing less tolerance to binding site variations. The three protein domains also differed as substrates for protein kinases. Whereas PKA in theory should target the DBDs of A-and c-Myb, but not B-Myb, only c-Myb was phosphorylated by PKA. CK2 phosphorylated all three proteins, although on different sites in the N-terminal region. Finally, B-Myb was remarkably sensitive to cysteine-directed oxidation compared to the other Myb proteins. Our data suggest that the small differences that have evolved between individual Myb family members lead to clear differences in DBD properties even if their sequence recognition remains the same.
European Journal of Biochemistry, 1993
The DNA-binding domain of the c-Myb oncoprotein contains two repeats, R2 and R3, both of which have been proposed to be related to the helix-turn-helix(HTH) motif. As a first step towards determination of the three-dimensional structure of this domain and of the mode of interaction with the DNA, we have undertaken multidimensional heteronuclear NMR studies using uniformly "N-labeled and 13C, 15N double-labeled R2R3 and, a selectively I5N-enriched sample on all lysine, histidine and leucine residues of R2R3. We present almost complete assignments of the backbone 'H, "N and 'TC" atoms and determine the secondary structure of R2R3 in solution.
Nucleic Acids Research, 2015
The recent upgrade of nucleic acid-protein interaction database (NPIDB, http://npidb.belozersky.msu. ru/) includes a newly elaborated classification of complexes of protein domains with double-stranded DNA and a classification of families of related complexes. Our classifications are based on contacting structural elements of both DNA: the major groove, the minor groove and the backbone; and protein: helices, beta-strands and unstructured segments. We took into account both hydrogen bonds and hydrophobic interaction. The analyzed material contains 1942 structures of protein domains from 748 PDB entries. We have identified 97 interaction modes of individual protein domain-DNA complexes and 17 DNA-protein interaction classes of protein domain families. We analyzed the sources of diversity of DNA-protein interaction modes in different complexes of one protein domain family. The observed interaction mode is sometimes influenced by artifacts of crystallization or diversity in secondary structure assignment. The interaction classes of domain families are more stable and thus possess more biological sense than a classification of single complexes. Integration of the classification into NPIDB allows the user to browse the database according to the interacting structural elements of DNA and protein molecules. For each family, we present average DNA shape parameters in contact zones with domains of the family.
Nucleic Acids Research, 2001
Bas1p, a divergent yeast member of the Myb family of transcription factors, shares with the proteins of this family a highly conserved cysteine residue proposed to play a role in redox regulation. Substitutions of this residue in Bas1p (C153) allowed us to establish that, despite its very high conservation, it is not strictly required for Bas1p function: its substitution with a small hydrophobic residue led to a fully functional protein in vitro and in vivo. C153 was accessible to an alkylating agent in the free protein but was protected by prior exposure to DNA. The reactivity of cysteines in the first and third repeats was much lower than in the second repeat, suggesting a more accessible conformation of repeat 2. Proteolysis protection, fluorescence quenching and circular dichroism experiments further indicated that DNA binding induces structural changes making Bas1p less accessible to modifying agents. Altogether, our results strongly suggest that the second repeat of the DNA-binding domain of Bas1p behaves similarly to its Myb counterpart, i.e. a DNA-induced conformational change in the second repeat leads to formation of a full helix-turn-helix-related motif with the cysteine packed in the hydrophobic core of the repeat.
Proceedings of the National Academy of Sciences, 1993
The DNA-binding domain of c-Myb consists of three homologous tandem repeats of 52 amino acids. The structure of the third (C-terminal) repeat obtained by NMR analysis has a conformation related to the helix-turn-helix motif. To identify the role of each repeat in the sequence recognition of DNA, we analyzed specific interactions between c-Myb and DNA by measuring binding affinities for systematic mutants of Myb-binding DNA sites and various truncated c-Myb mutants. We found that specific interactions are localized unevenly in the AACTGAC region in the consensus binding site of c-Myb: The first adenine, third cytosine, and fifth guanine are involved in very specific interactions, in which any base substitutions reduce the binding affinity by > 500-fold. On the other hand, the interaction at the second adenine is less specific, with the affinity reduction in the range of 6- to 15-fold. The seventh cytosine involves a rather peculiar interaction, in which only guanine substitution a...
Structure of the DNA binding domain of E. coli SSB bound to ssDNA
Nature structural biology, 2000
The structure of the homotetrameric DNA binding domain of the single stranded DNA binding protein from Escherichia coli (Eco SSB) bound to two 35-mer single stranded DNAs was determined to a resolution of 2.8 A. This structure describes the vast network of interactions that results in the extensive wrapping of single stranded DNA around the SSB tetramer and suggests a structural basis for its various binding modes.
Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries
Acta Crystallographica Section D Biological Crystallography, 2014
Protein-DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein-DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite the fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein-DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein-DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain -helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.