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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Biochemistry. Author manuscript; available in PMC 2010 December 29.
Published in final edited form as:
PMCID: PMC2807364
NIHMSID: NIHMS167092

DNA Plasticity Is a Key Determinant of the Energetics of Binding of Jun-Fos Heterodimeric Transcription Factor to Genetic Variants of TGACGTCA Motif

Abstract

The Jun-Fos heterodimeric transcription factor is a target of a diverse array of signaling cascades that initiate at the cell surface and converge in the nucleus and ultimately result in the expression of genes involved in a multitude of cellular processes central to health and disease. Here, using isothermal titration calorimetry in conjunction with circular dichroism, we report the effect of introducing single nucleotide variations within the TGACGTCA canonical motif on the binding of bZIP domains of Jun-Fos heterodimer to DNA. Our data reveal that the Jun-Fos heterodimer exhibits differential energetics in binding to such genetic variants in the physiologically relevant micromolar-submicromolar range with the TGACGTCA canonical motif affording the highest affinity. Although binding energetics are largely favored by enthalpic forces and accompanied by entropic penalty, neither the favorable enthalpy nor the unfavorable entropy correlate with overall free energy of binding in agreement with enthalpy-entropy compensation phenomenon widely observed in biological systems. However, a number of variants including the TGACGTCA canonical motif bind to the Jun-Fos heterodimer with high affinity through having overcome such enthalpy-entropy compensation barrier, arguing strongly that better understanding of the underlying invisible forces driving macromolecular interactions may be the key to future drug design. Our data also suggest that the Jun-Fos heterodimer has a preference for binding to TGACGTCA variants with higher AT content, implying that the DNA plasticity may be an important determinant of protein-DNA interactions. This notion is further corroborated by the observation that the introduction of genetic variations within the TGACGTCA motif allows it to sample a much greater conformational space. Taken together, these new findings further our understanding of the role of DNA sequence and conformation on protein-DNA interactions in thermodynamic terms.

Keywords: AP1-DNA thermodynamics, Jun-Fos heterodimer, bZIP domain, Single nucleotide variants, DNA plasticity

Transcription factors present the terminal link between the transfer of extracellular information in the form of growth factors and cytokines to the site of DNA transcription within the nucleus in a wide variety of cellular processes central to health and disease. This feat is in part executed by virtue of transcription factors to bind to specific recognition sites, termed response elements, within the promoters of target genes. Although such response elements are envisioned to comprise of a canonical sequence, typically spanning between 6–10 consecutive nucleotides, the promoters of many target genes in essence contain genetic variations of these response elements and, in particular, single nucleotide variants are extremely common within the eukaryotic genomes. Given that the nucleotide sequence is a key determinant of the ability of DNA to behave as a flexible polymer and undergo physical phenomena such as bending, stretching, deformation and distortion coupled with its ability to exist in various structural conformations (such as the B-DNA, A-DNA and Z-DNA) (13), our knowledge of how genetic variations within the response elements influence the ability of transcription factors to bind and subsequently affect gene transcription remains largely elusive. In an attempt to embark on this challenge, our earlier work indicated that the single nucleotide variants (SNVs) of the TGACTCA response element tightly modulate energetics and orientation of binding of the Jun-Fos heterodimeric transcription factor with important consequences on the recruitment of other cellular factors necessary for transcriptional machinery (4).

Jun-Fos heterodimer belongs to the AP1 (activator protein 1) family of transcription factors involved in executing the terminal stage of many critical signaling cascades that initiate at the cell surface and reach their climax in the nucleus (57). Upon activation by MAP kinases, AP1 binds to the promoters of a multitude of genes as Jun-Jun homodimer or Jun-Fos heterodimer. In so doing, Jun and Fos recruit the transcriptional machinery to the site of DNA and switch on expression of genes involved in a diverse array of cellular processes such as cell growth and proliferation, cell cycle regulation, embryonic development and cancer (811). Jun and Fos recognize the two closely related canonical TGACTCA and TGACGTCA response elements, respectively referred to as the TPA (12-O-tetradecanoylphorbol-13-acetate) response element (TRE) and the cAMP response element (CRE), within the promoters of target genes through their so-called basic zipper (bZIP) domains (Figure 1a). The bZIP domain can be further dissected into two well-defined functional subdomains termed the basic region (BR) at the N-terminus followed by the leucine zipper (LZ) at the C-terminus. The leucine zipper is a highly conserved protein module found in a wide variety of cellular proteins and usually contains a signature leucine at every seventh position within the five successive heptads of amino acid residues. The leucine zippers adapt continuous α-helices in the context of Jun-Jun homodimer or Jun-Fos heterodimer by virtue of their ability to wrap around each other in a coiled coil dimer (7, 12, 13). Such intermolecular arrangement brings the basic regions at the N-termini of bZIP domains into close proximity and thereby enables them to insert into the major grooves of DNA at the promoter regions in an optimal fashion in a manner akin to a pair of forceps (13). While the α-helices are held together by numerous inter-helical hydrophobic contacts and salt bridges, hydrogen bonding between the sidechains of basic residues in the basic regions and the DNA bases accounts for high affinity binding of bZIP domains to DNA.

Figure 1
Protein and DNA analysis. (a) 3D structural representation of Jun-Fos heterodimer in complex with dsDNA oligo containing the TGACGTCA motif. The leucine zipper (LZ) and basic region (BR) domains are shown in brown and blue, respectively. Within the dsDNA ...

In an effort to further expand on our earlier efforts (4), we employ here isothermal titration calorimetry (ITC) in conjunction with circular dichroism (CD) to shed light on the effect of introducing single nucleotide variations within the TGACGTCA response element on the binding of Jun-Fos heterodimer to DNA. Such genetic variations are widely encountered in the promoters of a diverse array of genes under the control of AP1 transcription factor (1436). Our new findings suggest that the genetic variations within the TGACGTCA canonical motif are an important determinant of DNA plasticity and that such conformational flexibility may in turn intimately dictate the affinity of protein-DNA interactions central to gene transcription and regulation. Taken together, these new findings further our understanding of the role of DNA sequence and conformation on protein-DNA interactions in thermodynamic terms.

MATERIALS and METHODS

Protein preparation

bZIP domains of human Jun and Fos were cloned and expressed as described previously (4, 37). Briefly, the proteins were cloned into pET102 bacterial expression vector, with an N-terminal thioredoxin (Trx)-tag and a C-terminal polyhistidine (His)-tag, using Invitrogen TOPO technology. Additionally, thrombin protease sites were introduced at both the N- and C-termini of the proteins to aid in the removal of tags after purification. Proteins were subsequently expressed in Escherichia coli Rosetta2(DE3) bacterial strain (Novagen) and purified on a Ni-NTA affinity column using standard procedures. Further treatment of bZIP domains of Jun and Fos on a Hiload Superdex 200 size-exclusion chromatography (SEC) column coupled to GE Akta FPLC system led to purification of recombinant domains to apparent homogeneity as judged by SDS-PAGE analysis. The identity of recombinant proteins was confirmed by MALDI-TOF mass spectrometry analysis. Final yields were typically between 10–20mg protein of apparent homogeneity per liter of bacterial culture. As noted previously (37), the treatment of recombinant proteins with thrombin protease significantly destabilized the bZIP domains of both Jun and Fos and both domains appeared to be proteolytically unstable. For this reason, all experiments reported herein were carried out on recombinant fusion bZIP domains of Jun and Fos containing a Trx-tag at the N-terminus and a His-tag at the C-terminus. The tags were found to have no effect on the binding of these domains to DNA under all conditions used here. Protein concentrations were determined as described earlier (37). Jun-Fos bZIP heterodimers were generated by mixing equimolar amounts of the purified bZIP domains of Jun and Fos. The efficiency of bZIP heterodimerization was close to 100% as judged by Native-PAGE and SEC analysis.

DNA synthesis

16-mer DNA oligos containing the TGACGTCA consensus motif and all possible single nucleotide variants thereof were commercially obtained from Sigma Genosys. The flanking nucleotides were appropriately chosen to prevent self-annealing and to favor the formation of heteroduplexes from sense and antisense strands. The design of such oligos and the numbering of various nucleotides relative to the central CG bases are depicted in Figures 1b–d. Oligo concentrations were determined spectrophotometrically on the basis of their extinction co-efficients derived from their nucleotide sequences using the online software OligoAnalyzer 3.0 (Integrated DNA Technologies) based on the nearest-neighbor model (38). Double-stranded DNA (dsDNA) oligos were generated as described earlier (37). Annealed dsDNA oligos were verified using Native-PAGE and circular dichroism (CD) analysis.

ITC measurements

Isothermal titration calorimetry (ITC) experiments were performed on a Microcal VP-ITC instrument and data were acquired and processed using Microcal ORIGIN software. All measurements were repeated 2–3 times. Briefly, the bZIP domains of Jun-Fos heterodimer and dsDNA oligos were prepared in 50mM Tris, 200mM NaCl, 1mM EDTA and 5mM β-mercaptoethanol at pH 8.0. The experiments were initiated by injecting 25 × 10μl aliquots of 50–100μM of dsDNA oligo from the syringe into the calorimetric cell containing 1.8ml of 5–10μM of the bZIP domains of Jun-Fos heterodimer at 25°C. The data were fit to a one-site model derived from the binding of a ligand to a macromolecule using the law of mass action to extract the various thermodynamic parameters as described previously (37). To ensure that the Trx- and His-tags did not interfere with the binding of DNA to Jun-Fos heterodimer, control experiments were conducted. Titration of a protein construct containing thioredoxin with a C-terminal His-tag (Trx-His) in the calorimetric cell with dsDNA oligos in the syringe produced no observable signal, implying that the tags do not interact with DNA. In a similar manner, titration of bZIP domains of Jun-Fos heterodimer in the calorimetric cell with Trx-His construct in the syringe produced no observable signal, implying that the tags do not interact with bZIP domains. Additionally, the fact that the affinities obtained for the binding of bZIP domains of Jun-Fos heterodimer to dsDNA oligos containing the CRE site are in an excellent agreement with those reported previously for non-tagged bZIP domains suggests that the Trx- and His-tags do not interfere with protein-DNA interactions reported here (12, 3942).

CD analysis

Circular dichroism (CD) measurements were conducted on a Bio-Logic MOS450 spectrometer equipped with a CD accessory and thermostatically controlled with a water bath. All data were acquired and processed using the Biokine software. For each experiment, 10μM of dsDNA oligo containing the TGACGTCA motif or a single nucleotide variant thereof was pre-equilibrated with an equimolar amount of bZIP domains of Jun-Fos heterodimer in 50mM Tris, 200mM NaCl, 1mM EDTA and 5mM β-mercaptoethanol at pH 8.0. CD spectra were collected at 25°C using a quartz cuvette with a 2-mm pathlength in the wavelength range 200–300nm and normalized against a reference spectrum to remove the contribution of protein to DNA ellipticity. The reference spectrum was obtained in a similar manner on a 10μM solution of bZIP domains of Jun-Fos heterodimer in the same buffer. Data were recorded with a slit bandwidth of 2nm at a scan rate of 3nm/min. Each data set represents an average of 2–3 scans acquired at 1nm intervals.

RESULTS and DISCUSSION

Single nucleotide variants of the TGACGTCA motif dictate the energetics of binding of Jun-Fos heterodimer

Given that they are widely encountered in the promoters of a diverse array of genes (1436), addressing the issue of how single nucleotide variants of the TGACGTCA motif influence the binding of Jun-Fos heterodimer is of paramount interest. Figure 2 shows a set of representative ITC isotherms obtained for the TGACGTCA canonical motif and two variants. The complete thermodynamic profiles for the entire set of variants are provided in Table 1. It is clearly evident from our data that the Jun-Fos heterodimer binds to all TGACGTCA variants with physiologically-relevant affinity in the micromolar-submicromolar range. Remarkably, the TGACGTCA canonical motif affords the most optimal affinity that is close to an order of magnitude stronger than the TGACTTCA variant, the sequence that is energetically least favorable for binding to the Jun-Fos heterodimer. Given its occurrence in the gene promoters with higher frequency combined with its higher affinity for binding to Jun-Fos heterodimer relative to other variants, the TGACGTCA canonical motif will be hereinafter referred to as the wildtype (WT) motif for the purpose of distinguishing it from other variants which have been assigned nomenclature according to the position of the nucleotide substitution (Table 1). It should also be noted here that the binding of all TGACGTCA variants to Jun-Fos heterodimer is largely driven by favorable entahlpic forces accompanied by unfavorable entropic penalty — an observation in agreement with the knowledge that protein-DNA interactions are predominantly driven by electrostatic interactions and hydrogen bonding. Comparison of these data with the binding of Jun-Fos heterodimer to nucleotide variants of the TGACTCA pseudo-palindrome suggests that the nucleotide variations within the TGACGTCA palindrome display a much tighter binding window stretching just under 10-fold while it spanned over nearly 60-fold in the case of the pseudo-palindrome (4). One likely scenario for the rather higher tolerance of the TGACGTCA palindrome to genetic variations in terms of its ability to bind to Jun-Fos heterodimer relative to the TGACTCA pseudo-palindrome may be due to the larger separation of the TGA and TCA half-sites within the palindrome and thereby allowing the protein greater freedom to slide between these half-sites in response to specific genetic changes. Although the role of sliding is not strange to protein-DNA interactions (4346), further studies are clearly warranted to assess the extent to which the ability of Jun-Fos heterodimer to slide along its response elements within the promoters of target genes may factor into its energetics of binding.

Figure 2
Representative ITC isotherms for the binding of bZIP domains of Jun-Fos heterodimer to dsDNA oligos containing the promoter sites TGACGTCA (a), TGAAGTCA (b) and TGACTTCA (c). In each case, 8μM of dimer-equivalent of bZIP domains in the calorimetric ...
Table 1
Experimentally determined thermodynamic parameters for the binding of bZIP domains of Jun-Fos heterodimer to dsDNA oligos containing the wild-type (WT) TGACGTCA canonical motif and all possible single nucleotide variants thereof obtained from ITC measurements ...

Jun-Fos heterodimer binds to all possible single nucleotide variants of the TGACGTCA motif without a preferred orientation

In a previous study, we reported that the certain variants of the TGACTCA motif result in the binding of of Jun-Fos heterodimer with a preferred orientation due to the ability of each monomer to engage in asymmetric protein-DNA contacts (4). In an attempt to assess the extent to which the Jun-Fos heterodimer might be able to display such orientational preference in complex with TGACGTCA variants, we constructed a plot showing the relative binding of symmetrically-related pairs of TGACGTCA variants to Jun-Fos heterodimer (Figure 3). Note that the symmetrically-related pairs of TGACGTCA variants are related by a two-fold symmetry, whereby the upper strand of one dsDNA oligo is identical to the lower strand of the other within the TGACGTCA sequence. Given such symmetrical relationship, one would expect the binding of Jun-Fos heterodimer to symmetrically-related pairs of TGACGTCA variants to be energetically-equivalent and thus result in indistinguishable affinities. The only exception to this rule would arise if the two monomers were not able to exchange freely due to non-equivalent contacts with flanking nucleotides in one strand over the other. Such asymmetric binding would be expected to result in a preferred orientation of Jun-Fos heterdoimer to the specific TGACGTCA variants. As shown in Figure 3, our data reveal that the Jun-Fos heterodimer binds to all TGACGTCA variants with relative Kd values of close to unity, implying that the binding largely occurs via non-preferred orientation due to little or negligible differences in the energetics of binding. The small deviations from unity in the values of relative Kd could be accounted for by the differences in the flanking nucleotides for the symmetrically-related pairs of TGACGTCA variants. Whether such small differences in the binding energetics could be sufficient to orient the Jun-Fos heterodimer clearly warrants further studies. It should be noted however that the orientation of transcription factors in complex with DNA plays a critical role in defining the nature of other cellular factors that can be recruited to the site of DNA transcription in a temporal and spatial manner. In this context, the possibility that the Jun-Fos heterodimer may bind in an oriented manner to some TGACGTCA variants cannot be ruled out on the basis of data presented herein.

Figure 3
Analysis of relative binding of symmetrically-related pairs of dsDNA oligos containing TGACGTCA variants to bZIP domains of Jun-Fos heterodimer. Relative binding is defined as the ratio of the Kd of the binding of one dsDNA oligo to Jun-Fos heterodimer ...

High-affinity binding of TGACGTCA motif is in part owed to overcoming the enthalpy-entropy compensation barrier

Enthalpy-entropy compensation is a phenomenon that is widely observed in biological systems (4751). In a nutshell, it postulates that enthalpic contributions to macromolecular interactions are compensated by equal but opposing entropic changes such that there is no net gain in the overall free energy. Figure 4 shows the enthalpy-entropy compensation plot for the binding of TGACGTCA canonical motif and all single nucleotide variants thereof to Jun-Fos heterodimer. It should be noted however that the enthalpy-entropy compensation is not an absolute thermodynamic law nor it ought to be obeyed universally. Indeed, if it were to be obeyed, macromolecular interactions would be expected to be of similar affinity across the board in lieu of ranging from weak interactions in the micromolar range to ultra-tight interactions in the picomolar range observed in diverse cellular processes. Supporting these arguments, enthalpy-entropy compensation plot for the binding of Jun-Fos heterodimer to TGACGTCA variants suggests that a number of motifs, including the TGACGTCA canonical motif, indeed overcome the enthalpy-entropy barrier to bind with affinities much higher than would have been possible otherwise (Figure 4). Close scrutiny of the thermodynamics of TGACGTCA canonical motif suggests that it somehow manages to release about −1 kcal/mol of favorable enthalpy without paying anything for it in entropy penalty upon binding to the Jun-Fos heterodimer. Had it not been for such escape from enthalpy-entropy compensation, the binding of TGACGTCA canonical motif to Jun-Fos heterodimer would have been reduced to an affinity in the micromolar range in lieu of the experimentally observed sub-micromolar range (Figure 4 and Table 1) and thereby rendering it one of the weakest variants to recognize the Jun-Fos heterodimer. The TGACGTCA (WT) canonical motif is immediately flanked between TGACGGCA (G+1) and TCACGTCA (C-2) variants on the enthalpy-entropy compensation plot (Figure 4, lower panel). Note that, unlike the WT motif, G+1 and C-2 motifs faithfully obey the enthalpy-entropy compensation. In comparison with G+1 and C-2 motifs, the ability of the WT motif to defeat the enthalpy-entropy compensation barrier must reside in its distinguishing chemistry at positions +1 and −2 that allows it to engage in intermolecular contacts that favor the release of enthalpy but cause minimal changes in unfavorable entropy. Close scrutinization of 3D structure of Jun-Fos heterodimer in complex with DNA reveals that the nucleotides (or their complementary pairs) at +1 and −2 positions reside in the major groove and engage in hydrogen bonding with the sidechain of N262 within the Jun monomer and the sidechain of N147 within the Fos monomer, respectively (Figure 1a). It is thus conceivable that the distinguishing chemical nature of nucleotides at +1 and −2 positions within the WT motif versus the G+1 and C-2 variants could account for their distinguishing thermodynamic features. Although complete analysis of the underlying forces that allow the WT motif to escape the enthalpy-entropy barrier but not the G+1 and C-2 motifs is beyond the scope of this study, our data presented herein nonetheless provide a glimpse into the interacting forces that determine the overall binding affinity of protein-DNA interactions. Our data also argue strongly that the design of future drugs sporting greater efficacy coupled with low toxicity could benefit from the consideration of underlying thermodynamic forces.

Figure 4
Enthalpy (ΔH)-entropy (TΔS) plot for the binding of TGACGTCA variants to Jun-Fos heterodimer. The top panel shows the ΔH-TΔS plot for the binding of Jun-Fos heterodimer to all possible single nucleotide variants of the ...

Enthalpic and entropic contributions do not correlate with the energetics of binding of Jun-Fos heterodimer to TGACGTCA variants

Given that protein-DNA interactions are usually driven by enthalpy accompanied by entropic penalty (5257), we next asked the question whether there was any correlation between enthalpy and entropy versus the overall free energy for the binding of TGACGTCA variants to the Jun-Fos heterodimer. As illustrated in Figure 5, our data show that an increase in free energy neither correlates with favorable enthalpy nor favorable entropy in agreement with the enthalpy-entropy compensation phenomenon discussed above. In other words, a small favorable enthalpy may be sufficient to generate a high binding affinity by virtue of a small corresponding entropic penalty. Conversely, a large favorable enthalpy does not necessarily result in tighter binding due to a compensating increase in entropic penalty. Despite the lack of such an overall correlation in the entire cohort of TGACGTCA variants (Figures 5a and 5b, upper panels), we have identified a subset of variants that exquisitely display a correlation between enthalpy and entropy versus the free energy. These include the A-0, T-0 and WT motifs, whose favorable enthalpy and unfavorable entropy positively correlate with free energy enhancement in what would be referred to as “enthalpically-optimized” variants (Figures 5a and 5b, lower panels). Note that all of these motifs differ from each other in the nature of nucleotide at −0 position — WT motif is identical to A-0 and T-0 except for the presence of a cytosine at −0 position. What can we learn from such findings? It is clear that higher binding affinity is achievable through optimizing both the enthalpic and entropic contributions and thus overcoming the enthalpy-entropy compensation barrier that too often acts as a bottle-neck to rationale drug design. In the case of A-0, T-0 and WT variants, optimization of enthalpic contribution results in an enhancement of binding affinity by nearly an order of magnitude from a value of 2.41μM for the A-0 motif to a value of 0.27μM for the WT motif (Table 1). Close analysis of 3D structure of the Jun-Fos heterodimer in complex with DNA suggests that the nucleotides at −0 position and their complementary pairs on the opposite strand engage in hydrogen bonding with R270 within the Jun monomer and R155 within the Fos monomer (Figure 1a). In light of these observations, the most likely scenario for the increase in binding affinity as a direct result of an increase in favorable enthalpy for the A-0, T-0 and WT motifs is that the smaller thymine and cytosine bases instead of the larger adenine at −0 position are enthalpically preferred. Taken together, our data provide precedence for the design of higher affinity ligands on thermodynamic grounds through optimization of enthalpic and entropic contributions. Although our data suggest that enthalpically-optimized ligands may be more amenable to design, there is a growing school of thought that the future design of drugs could also benefit from favorable entropic contributions to the free energy in what has come to be termed as “entropically-optimized drugs”.

Figure 5
Dependence of enthalpic (ΔH) and entropic (TΔS) contributions to the free energy (ΔG) of binding of TGACGTCA variants to Jun-Fos heterodimer. (a) The upper panel shows ΔH-ΔG plot for the binding of Jun-Fos heterodimer ...

A/T substitutions are preferred over G/C substitutions within the TGACGTCA motif for binding to Jun-Fos heterodimer

It is widely believed that A/T sequences within DNA account for its intrinsic conformational flexibility such as bending and curvature (1, 2, 5863). Such intrinsic propensity of A/T sequences to undergo bending is believed to be largely due to increased propeller twist of these sequences by virtue of the fact that A-T base pairs are held together by only two hydrogen bonds in lieu of three formed between G-C base pairs. In an attempt to analyze how single nucleotide substitutions of A/T versus G/C affect the binding of TGACGTCA variants to Jun-Fos heterodimer, we generated thermodynamic plots shown in Figure 6. It is immediately evident that G/C substitutions are enthalpically more favorable at all positions within the TGACGTCA motif but −1 and +2, presumably by virtue of their ability to engage in greater intermolecular contacts via hydrogen bonding and hydrophobic forces (Figure 6a, top panel). In contrast, A/T substitutions are entropically more favorable at all positions within the TGACGTCA motif but −1 and +1, indicative of their intrinsic flexibility allowing them to engage in close intermolecular contacts (Figure 6a, middle panel). Given the antagonistic actions of A/T and G/C substitutions on underlying thermodynamic forces, it might be inferred that the overall free energy of binding may not correlate with A/T or G/C substitutions at any one given position within the TGACGTCA motif. On the contrary, A/T substitutions are preferred at all positions but −3 for the optimal binding of Jun-Fos heterodimer (Figure 6a, bottom panel). Taken together, these salient observations imply that the conformational plasticity such as the ability of TGACGTCA variants to bend and wrap around Jun and Fos to attain a close molecular fit is critical for high-affinity binding. Although DNA bending is not observed in the crystal structure of Jun-Fos heterodimer bound to the related TGACTCA motif (13), several solution studies argue in support of the role of intrinsic bendability of DNA as being a key factor in its ability to bind to Jun-Fos heterodimer with high-affinity (6466).

Figure 6
Dependence of enthalpy (ΔH), entropy (TΔS) and free energy (ΔG) on the position of nucleotide substitution within the TGACGTCA motif. The changes in various thermodynamic parameters upon the introduction of A, T, G and C substitutions ...

DNA conformational flexibility appears to play a key role in the differential binding of TGACGTCA variants to Jun-Fos heterodimer

In light of the above observations coupled with the notion that the nucleotide sequence is a key determinant of the ability of DNA to behave as a flexible polymer and undergo physical phenomena such as bending and curvature (13), we further analyzed the conformational flexibility of TGACGTCA motif and all the variants thereof in complex with Jun-Fos heterodimer by CD spectroscopy. As shown in Figure 7, the CD spectra of bound DNA (with the protein contributions largely removed) exhibit features characteristic of a right-handed double-stranded B-DNA with bands centered around 220nm and 280nm. It should be noted here that while the 220-nm band arises from secondary structural DNA features, the 280-nm band probes the 3D conformation of DNA and therefore it is highly sensitive to physical changes in DNA such as bending and curvature. It is clearly evident that the CD spectra of the variant oligos do not superimpose upon the CD spectrum of the TGACGTCA motif but rather fluctuate around it and fan out forming a cluster of closely related optical spectra. This salient observation implies that the introduction of single nucleotide substitutions within the TGACGTCA motif tightly governs its conformational flexibility and that the varying flexibility is likely to be an integral feature of their ability to bind to Jun-Fos heterodimer with distinct underlying energetics. While the intensity of the 280-nm band is related to overall 3D conformation of DNA, it is not easily interpretable in structural terms such as bending and curvature. Nonetheless, our CD data indicate that the introduction of genetic variations within the TGACGTCA motif allows it to sample a much greater conformational space that might be a key feature of the ability of its variants to bind to the Jun-Fos heterodimer at distinct promoters in a selective manner.

Figure 7
CD spectra of dsDNA oligos containing the TGACGTCA motif and all possible single nucleotide variants thereof bound to Jun-Fos heterodimer (with the protein contributions removed). The CD spectrum of TGACGTCA motif (red) is superimposed upon CD spectra ...

CONCLUSIONS

Transcription factors play a central role in the transduction of cellular signals into gene expression via their ability to recognize DNA in a sequence-specific manner. The precise sequence of DNA determines its physical properties, such as bending and curvature, allowing it to adapt into a complementary shape to provide the best fit for the incoming protein partner (13). Our data presented herein indeed add to this school of thought. Our study shows that genetic variations within the TGACGTCA motif modulate energetics of binding of the Jun-Fos heterodimer by virtue of their ability to bend and curve accordingly to obtain the optimal molecular fit between the two interacting partners. The genetic variations within the response elements may thus play an important role in regulating the biological activity of Jun-Fos heterodimer at specific promoters. Our findings also bear important consequences for the occurrence of single nucleotide polymorphisms (SNPs) within promoter regions containing the AP1-responsive site. It has indeed been shown that a SNP that changes the TGACGTCA canonical motif to TGACGTTA variant alters transcription factor binding (67). While many proteins bind to unperturbed DNA, the plasticity of the TGACGTCA variants appears to be a key feature of their ability to bind to Jun-Fos heterodimer on the basis of data reported herein and elsewhere (6466). In particular, the content and position of A/T nucleotides within the TGACGTCA variants may be an important determinant of their ability to bend and deform upon binding. It is widely believed that A/T sequences within DNA account for its intrinsic conformational flexibility such as bending and curvature (1, 2, 5863). These observations are further supported by our findings that A/T substitutions are preferred at all positions within the TGACGTCA motif but −3 for the optimal binding of Jun-Fos heterodimer, implying that the structural plasticity of DNA is necessary for high-affinity protein-DNA interactions. Our data also suggest that the TGACGTCA canonical motif defies the enthalpy-entropy compensation barrier in order to bind with a high affinity. Although the enthalpy-entropy compensation barrier is widely observed in biological systems and presents a bottle-neck for the design of high-affinity drugs to combat disease, our findings clearly lay the groundwork for furthering our understanding of the invisible forces driving macromolecular interactions and may lead to unlocking the secrets to overcoming this thermodynamic barrier for the design of next-generation drugs sporting greater efficacy coupled with low toxicity. The design of high-affinity ligands on thermodynamic grounds through optimization of enthalpic and entropic contributions may indeed be one of the major frontiers that we have to conquer to improve the clinical design of future therapies. In conclusion, the present study provides a comprehensive analysis of the effect of genetic variations within the TGACGTCA motif on the energetics of binding of Jun-Fos heterodimer. Our data strongly argue that such genetic variations control the plasticity and conformational deformability of the target DNA and thereby bear important consequences for the physiological role of this key transcription factor central to health and disease.

ABBREVIATIONS

AP1
Activator protein 1
BR
Basic region
His
Polyhistidine tag
ITC
Isothermal titration calorimetry
LZ
Leucine zipper
MAP
Mitogen-activated protein
PAGE
Polyacrylamide gel electrophoresis
SDS
Sodium dodecyl sulfate
SNP
Single nucleotide polymorphism
SNV
Single nucleotide variant
Trx
Thioredoxin
bZIP
Basic zipper

Footnotes

This work was supported by funds from the National Institutes of Health (Grant# R01-GM083897), the American Heart Association (Grant# 0655087B) and the USylvester Braman Family Breast Cancer Institute to AF. CBM is a recipient of a postdoctoral fellowship from the National Institutes of Health (Award# T32-CA119929). BJD and AF are members of the Sheila and David Fuente Graduate Program in Cancer Biology at the Sylvester Comprehensive Cancer Center of the University of Miami.

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