Assessment by Molecular Dynamics Simulations of the Structural Determinants of DNA-binding Specificity for Transcription Factor Sp1

Abstract

The DNA-binding domain (DBD) of the ubiquituous transcription factor Sp1 consists of three consecutive zinc fingers that recognize a number of nucleotide sequences different from, but related to and sometimes overlapping, those recognized by the structurally better characterized early growth response protein 1 (EGR1, also known as Zif268, Krox-24, and NGFI-A). The accepted consensus binding sequence for Sp1 is usually defined by the asymmetric hexanucleotide core GGGCGG but this sequence does not include, among others, the GAG ( ¼ CTC) repeat that constitutes a high-affinity site for Sp1 binding to the wt1 promoter. Since no 3D structure of the whole DBD of Sp1 is available, either alone or in complex with DNA, a homology-based model was built and its interaction with two DNA 14-mers was studied using nanosecond molecular dynamics simulations in the presence of explicit water molecules. These oligonucleotides represent Sp1 target sites that are present in the promoters of the mdr1 and wt1 genes. For comparative purposes and validation of the protocol, the complex between the DBD of EGR1 and its DNA target site within the proximal mdr1 promoter was simulated under the same conditions. Some water molecules were seen to play an important role in recognition and stabilization of the protein-DNA complexes. Our results, which are supported by the available experimental evidence, suggest that the accuracy in the prediction of putative Sp1-binding sites can be improved by interpreting a set of rules, which are a blend of both stringency and tolerance, for the juxtaposed triplet subsites to which each zinc finger binds. Our approach can be extrapolated to WT1 and other related natural or artificial zinc-finger-containing DNA-binding proteins and may aid in the assignment of particular DNA stretches as allowed or disallowed-binding sites.

Essential Interaction of Egr-1 at an Islet-specific Response Element for Basal and Gastrin-dependent Glucagon Gene Transactivation in Pancreatic α-Cells

“…First, the fact that Egr-1 controls the basal activity of the glucagon gene although this is not the case for basal transactivation of the chromogranin A gene demonstrates that the functional relevance of the transcription factor is different in the two situations. Moreover, an interplay of Egr-1 and Sp1, resulting in overlapping consensus motifs (26), is crucial for gastrin regulation of the chromogranin A promoter. In the context of the glucagon gene promoter, Sp1 is absent from the DNA-protein complex induced by gastrin, indicating no functional interaction between these two factors.…”

Structural Basis for the Binding of Didemnins to Human Elongation Factor eEF1A and Rationale for the Potent Antitumor Activity of These Marine Natural Products

“…The cutoff distance for the nonbonded interactions was 9 Å. The simulation protocol for relaxing this system was essentially as described, 71 involving a series of progressive energy minimizations followed by 200 ps of unrestrained molecular dynamics at 300 K and 1 atm using the SANDER module in AMBER 6.0 (http:// amber.scripps.edu/). 72 The ensuing steered MD (sMD) procedure consisted of pairwise forcing the distances between CR atoms of residues 77-90, 76-91, 75-92, 74-93, and 73-94 in the original eEF1B-bound form of eEF1A to adopt the same values that are measured between CR atoms of equivalent residues (67-80, 66-81, 65-82, 64-83, and 63-84) in the crystallographic GTP form of EF1A.…”

The p38 MAPK Pathway Mediates Transcriptional Activation of the Plasma Platelet-activating Factor Acetylhydrolase Gene in Macrophages Stimulated with Lipopolysaccharide

“…These investigators utilized consensus Sp1 probes whose sequences differ from those present in the CD14 and IL-10 promoters they were studying, respectively. Marco et al (55) "recognition code" exists that correlates specific residues in the recognition helix of Sp1 with particular bases in the DNA site. Although the code is known to tolerate some level of degeneracy, small alterations in the sequences within both the core motif and the flanking regions result in altered affinities.…”