Structural determinants of specific DNA-recognition by the THAP zinc finger

Human THAP1 is the prototype of a large family of cellular factors sharing an original THAP zinc-finger motif responsible for DNA binding. Human THAP1 regulates endothelial cell proliferation and G1/S cell-cycle progression, through modulation of pRb/E2F cell-cycle target genes including rrm1. Recently, mutations in THAP1 have been found to cause DYT6 primary torsion dystonia, a human neurological disease. We report here the first 3D structure of the complex formed by the DNA-binding domain of THAP1 and its specific DNA target (THABS) found within the rrm1 target gene. The THAP zinc finger uses its double-stranded β-sheet to fill the DNA major groove and provides a unique combination of contacts from the β-sheet, the N-terminal tail and surrounding loops toward the five invariant base pairs of the THABS sequence. Our studies reveal unprecedented insights into the specific DNA recognition mechanisms within this large family of proteins controlling cell proliferation, cell cycle and pluripotency.     

Ensemble-based computational approach discriminates functional activity of p53 cancer and rescue mutants

The tumor suppressor protein p53 can lose its function upon single-point missense mutations in the core DNA-binding domain (“cancer mutants”). Activity can be restored by second-site suppressor mutations (“rescue mutants”). This paper relates the functional activity of p53 cancer and rescue mutants to their overall molecular dynamics (MD), without focusing on local structural details. A novel global measure of protein flexibility for the p53 core DNA-binding domain, the number of clusters at a certain RMSD cutoff, was computed by clustering over 0.7 µs of explicitly solvated all-atom MD simulations. For wild-type p53 and a sample of p53 cancer or rescue mutants, the number of clusters was a good predictor of in vivo p53 functional activity in cell-based assays. This number-of-clusters (NOC) metric was strongly correlated (r² = 0.77) with reported values of experimentally measured ΔΔG protein thermodynamic stability. Interpreting the number of clusters as a measure of protein flexibility: (i) p53 cancer mutants were more flexible than wild-type protein, (ii) second-site rescue mutations decreased the flexibility of cancer mutants, and (iii) negative controls of non-rescue second-site mutants did not. This new method reflects the overall stability of the p53 core domain and can discriminate which second-site mutations restore activity to p53 cancer mutants.     

Structure-function analysis of the THAP zinc finger of THAP1, a large C2CH DNA-binding module linked to Rb/E2F pathways

THAP1, the founding member of a previously uncharacterized large family of cellular proteins (THAP proteins), is a sequence-specific DNA-binding factor that has recently been shown to regulate cell proliferation through modulation of pRb/E2F cell cycle target genes. THAP1 shares its DNA-binding THAP zinc finger domain with Drosophila P element transposase, zebrafish E2F6, and several nematode proteins interacting genetically with the retinoblastoma protein pRb. In this study, we report the three-dimensional structure and structure-function relationships of the THAP zinc finger of human THAP1. Deletion mutagenesis and multidimensional NMR spectroscopy revealed that the THAP domain of THAP1 is an atypical zinc finger of approximately 80 residues, distinguished by the presence between the C2CH zinc coordinating residues of a short antiparallel beta-sheet interspersed by a long loop-helix-loop insertion. Alanine scanning mutagenesis of this loop-helix-loop motif resulted in the identification of a number of critical residues for DNA recognition. NMR chemical shift perturbation analysis was used to further characterize the residues involved in DNA binding. The combination of the mutagenesis and NMR data allowed the mapping of the DNA binding interface of the THAP zinc finger to a highly positively charged area harboring multiple lysine and arginine residues. Together, these data represent the first structure-function analysis of a functional THAP domain, with demonstrated sequence-specific DNA binding activity. They also provide a structural framework for understanding DNA recognition by this atypical zinc finger, which defines a novel family of cellular factors linked to cell proliferation and pRb/E2F cell cycle pathways in humans, fish, and nematodes.     

Fluorescence studies of the binding of bacteriophage M13 gene V mutant proteins to polynucleotides.

This investigation describes how the binding characteristics of the single-stranded DNA-binding protein encoded by gene V of bacteriophage M13, are affected by single-site amino acid substitutions. The series of mutant proteins tested includes mutations in the purported monomer-monomer interaction region as well as mutations in the DNA-binding domain at positions which are thought to be functionally involved in monomer-monomer interaction or single-stranded DNA binding. The characteristics of the binding of the mutant proteins to the homopolynucleotides poly(dA), poly(dU) and poly(dT), were studied by means of fluorescence-titration experiments. The binding stoichiometry and fluorescence quenching of the mutant proteins are equal to, or lower than, the wild-type gene V protein values. In addition, all proteins measured bind a more-or-less co-operative manner to single-stranded DNA. The binding affinities for poly(dA) decrease in the following order: Y61H greater than wild-type greater than F68L and R16H greater than Y41F and Y41H greater than F73L greater than R21C greater than Y34H greater than G18D/Y56H. Possible explanations for the observed differences are discussed. The conservation of binding affinity, also for mutations in the single-stranded DNA-binding domain, suggests that the binding to homopolynucleotides is largely non-specific.     

The Yeast a1 and α2 Homeodomain Proteins Do Not Contribute Equally to Heterodimeric DNA Binding

In diploid cells of the yeast Saccharomyces cerevisiae, the α2 and a1 homeodomain proteins bind cooperatively to sites in the promoters of haploid cell-type-specific genes (hsg) to repress their expression. Although both proteins bind to the DNA, in the α2 homeodomain substitutions of residues that are involved in contacting the DNA have little or no effect on repression in vivo or cooperative DNA binding with a1 protein in vitro. This result brings up the question of the contribution of each protein in the heterodimer complex to the DNA-binding affinity and specificity. To determine the requirements for the a1-α2 homeodomain DNA recognition, we systematically introduced single base-pair substitutions in an a1-α2 DNA-binding site and examined their effects on repression in vivo and DNA binding in vitro. Our results show that nearly all substitutions that significantly decrease repression and DNA-binding affinity are at positions which are specifically contacted by either the α2 or a1 protein. Interestingly, an α2 mutant lacking side chains that make base-specific contacts in the major groove is able to discriminate between the wild-type and mutant DNA sites with the same sequence specificity as the wild-type protein. These results suggest that the specificity of α2 DNA binding in complex with a1 does not rely solely on the residues that make base-specific contacts. We have also examined the contribution of the a1 homeodomain to the binding affinity and specificity of the complex. In contrast to the lack of a defective phenotype produced by mutations in the α2 homeodomain, many of the alanine substitutions of residues in the a1 homeodomain have large effects on a1-α2-mediated repression and DNA binding. This result shows that the two proteins do not make equal contributions to the DNA-binding affinity of the complex.     

Simian virus 40 origin DNA-binding domain on large T antigen.

Fifty variant forms of simian virus 40 (SV40) large T antigen bearing point, multiple point, deletion, or termination mutations within a region of the protein thought to be involved in DNA binding were tested for their ability to bind to SV40 origin DNA. A number of the mutant large T species including some with point mutations were unable to bind, whereas many were wild type in this activity. The clustering of the mutations that are defective in origin DNA binding both reported here and by others suggests a DNA-binding domain on large T maps between residues 139 and approximately 220, with a particularly sensitive sequence between amino acids 147 and 166. The results indicate that the domain is involved in binding to both site I and site II on SV40 DNA, but it remains unclear whether it is responsible for binding to cellular DNA. Since all the mutants retain the ability to transform Rat-1 cells, we conclude that the ability of large T to bind to SV40 origin DNA is not a prerequisite for its transforming activity.     

Analysis of the herpes simplex virus type 1 OriS sequence: mapping of functional domains.

The herpes simplex virus type 1 (HSV-1) OriS region resides within a 90-bp sequence that contains two binding sites for the origin-binding protein (OBP), designated sites I and II. A third presumptive OBP-binding site (III) within OriS has strong sequence similarity to sites I and II, but no sequence-specific OBP binding has yet been demonstrated at this site. We have generated mutations in sites I, II, and III and determined their replication efficiencies in a transient in vivo assay in the presence of a helper virus. Mutations in any one of the sites reduced DNA replication significantly. To study the role of OriS sequence elements in site I and the presumptive site III in DNA replication, we have also generated a series of mutations that span from site I across the presumptive binding site III. These mutants were tested for their ability to replicate and for the ability to bind OBP by using gel shift analyses. The results indicate that mutations across site I drastically reduce DNA replication. Triple-base-pair substitution mutations that fall within the crucial OBP-binding domain, 5'-YGYTCGCACT-3' (where Y represents C or T), show a reduced level of OBP binding and DNA replication. Substitution mutations in site I that are outside this crucial binding sequence show a more detrimental effect on DNA replication than on OBP binding. This suggests that these sequences are required for initiation of DNA replication but are not critical for OBP binding. Mutations across the presumptive OBP-binding site III also resulted in a loss in efficiency of DNA replication. These mutations influenced OBP binding to OriS in gel shift assays, even though the mutated sequences are not contained within known OBP-binding sites. Replacement of the wild-type site III with a perfect OBP-binding site I results in a drastic reduction of DNA replication. Thus, our DNA replication assays and in vitro DNA-binding studies suggest that the binding of the origin sequence by OBP is not the only determining factor for initiation of DNA replication in vivo.     

Mapping DNA-binding domains of the autoimmune regulator protein

The human autoimmune regulator (AIRE) gene encodes a putative DNA-binding protein, which is mutated in patients affected by the autoimmune polyglandular syndrome type 1 or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. We have recently reported that AIRE can bind to two different DNA sequence motifs, suggesting the existence of at least two DNA-binding domains in the AIRE protein. By expressing a series of recombinant AIRE protein fragments, we demonstrate here that the two well-known plant homeodomains (PHD) domains in AIRE can bind to the ATTGGTTA sequence motif. The first ATTGGTTA-binding domain is mapped to amino acids 299-355 and the second ATTGGTTA-binding domain to amino acids 434-475. Furthermore, the SAND domain of AIRE is shown to bind to TTATTA motif. Results presented herein show that the residues at position 189-196 of AIRE (QRAVAMSS) are required for its binding to the TTATTA motif. The required sequence for DNA binding in the SAND domain of AIRE is remarkably different from other SAND-containing proteins such as Sp-100b and NUDR. Data presented in this paper indicate that the two PHD domains contained in AIRE, in addition to the SAND domain, can bind to specific DNA sequence motifs.     

Effects of temperature on the p53-DNA binding interactions and their dynamical behavior: comparing the wild type to the R248Q mutant

Background

The protein p53 plays an active role in the regulation of cell cycle. In about half of human cancers, the protein is inactivated by mutations located primarily in its DNA-binding domain. Interestingly, a number of these mutations possess temperature-induced DNA-binding characteristics. A striking example is the mutation of Arg248 into glutamine or tryptophan. These mutants are defective for binding to DNA at 310 K although they have been shown to bind specifically to several p53 response elements at sub-physiological temperatures (298–306 K).

Methodology/Principal Findings

This important experimental finding motivated us to examine the effects of temperature on the structure and configuration of R248Q mutant and compare it to the wild type protein. Our aim is to determine how and where structural changes of mutant variants take place due to temperature changes. To answer these questions, we compared the mutant to the wild-type proteins from two different aspects. First, we investigated the systems at the atomistic level through their DNA-binding affinity, hydrogen bond networks and spatial distribution of water molecules. Next, we assessed changes in their long-lived conformational motions at the coarse-grained level through the collective dynamics of their side-chain and backbone atoms separately.

Conclusions

The experimentally observed effect of temperature on the DNA-binding properties of p53 is reproduced. Analysis of atomistic and coarse-grained data reveal that changes in binding are determined by a few key residues and provide a rationale for the mutant-loss of binding at physiological temperatures. The findings can potentially enable a rescue strategy for the mutant structure.     

Assessing the plasticity of DNA target site recognition of the PI-SceI homing endonuclease using a bacterial two-hybrid selection system

The PI-SceI protein from Saccharomyces cerevisiae is a member of the LAGLIDADG family of homing endonucleases that have been used in genomic engineering. To assess the flexibility of the PI-SceI-binding interaction and to make progress towards the directed evolution of homing endonucleases that cleave specified DNA targets, we applied a two-hybrid method to select PI-SceI variants from a randomized expression library that bind to different DNA substrates. In particular, the codon for Arg94, which is located in the protein splicing domain and makes essential contacts to two adjacent base-pairs, and the codons for four proximal residues were randomized. There is little conservation of the wild-type amino acid residues at the five randomized positions in the variants that were selected to bind to the wild-type site, yet one of the purified derivatives displays DNA-binding specificity and DNA endonuclease activity that is similar to that of the wild-type enzyme. A spectrum of DNA-binding behaviors ranging from partial relaxation of specificity to marked shifts in target site recognition are present in variants selected to bind to sites containing mutations at the two base-pairs. Our results illustrate the inherent plasticity of the PI-SceI/DNA interface and demonstrate that selection based on DNA binding is an effective means of altering the DNA cleavage specificity of homing endonucleases. Furthermore, it is apparent that homing endonuclease target specificity derives, in part, from constraints on the flexibility of DNA contacts imposed by hydrogen bonds to proximal residues.     

Analysis of the DNA-binding domain of Escherichia coli DnaA protein

The DNA-binding domain of the Escherichia coli DnaA protein is represented by the 94 C-terminal amino acids (domain 4, aa 374-467). The isolated DNA-binding domain acts as a functional repressor in vivo, as monitored with a mioC:lacZ translational fusion integrated into the chromosome of the indicator strain. In order to identify residues required for specific DNA binding, site-directed and random PCR mutagenesis were performed, using the mioC:lacZ construct for selection. Mutations defective in DNA binding were found all over the DNA-binding domain with some clustering in the basic loop region, within presumptive helix B and in a highly conserved region at the N-terminus of presumptive helix C. Surface plasmon resonance (SPR) analysis revealed different binding classes of mutant proteins. No or severely reduced binding activity was demonstrated for amino acid substitutions at positions R399, R407, Q408, H434, T435, T436 and A440. Altered binding specificity was found for mutations in a 12 residue region close to the N-terminus of helix C. The defects of the classical temperature sensitive mutants dnaA204, dnaA205 and dnaA211 result from instability of the proteins at higher temperatures. dnaX suppressors dnaA71 and dnaA721 map to the region close to helix C and bind DNA non-specifically.     

Rett syndrome-causing mutations in human MeCP2 result in diverse structural changes that impact folding and DNA interactions

Most cases of Rett syndrome (RTT) are caused by mutations in the methylated DNA-binding protein, MeCP2. Here, we have shown that frequent RTT-causing missense mutations (R106W, R133C, F155S, T158M) located in the methylated DNA-binding domain (MBD) of MeCP2 have profound and diverse effects on its structure, stability, and DNA-binding properties. Fluorescence spectroscopy, which reports on the single tryptophan in the MBD, indicated that this residue is strongly protected from the aqueous environment in the wild type but is more exposed in the R133C and F155S mutations. In the mutant proteins R133C, F155S, and T158M, the thermal stability of the domain was strongly reduced. Thermal stability of the wild-type protein was increased in the presence of unmethylated DNA and was further enhanced by DNA methylation. DNA-induced thermal stability was also seen, but to a lesser extent, in each of the mutant proteins. Circular dichroism (CD) of the MBD revealed differences in the secondary structure of the four mutants. Upon binding to methylated DNA, the wild type showed a subtle but reproducible increase in alpha-helical structure, whereas the F155S and R106W did not acquire secondary structure with DNA. Each of the mutant proteins studied is unique in terms of the properties of the MBD and the structural changes induced by DNA binding. For each mutation, we examined the extent to which the magnitude of these differences correlated with the severity of RTT patient symptoms.