Until now, it has been reasonably assumed that specific base-pair recognition is the only mechanism controlling the specificity of transcription factor (TF)−DNA binding. Contrary to this assumption, here we show that nonspecific DNA sequences possessing certain repeat symmetries, when present outside of specific TF binding sites (TFBSs), statistically control TF−DNA binding preferences. We used highthroughput protein−DNA binding assays to measure the binding levels and free energies of binding for several human TFs to tens of thousands of short DNA sequences with varying repeat symmetries. Based on statistical mechanics modeling, we identify a new protein−DNA binding mechanism induced by DNA sequence symmetry in the absence of specific base-pair recognition, and experimentally demonstrate that this mechanism indeed governs protein−DNA binding preferences. protein−DNA binding is an important biophysical mechanism operating in a living cell (1). This seminal work makes it possible to interpret experiments that measured how transcription factors (TFs) search for their specific target sites flanked by nonconsensus sequence elements (1-10). A specific consensus motif is a short DNA sequence, typically 6-20 base pairs (bp), that possesses an enhanced binding affinity for a particular TF. For example, the sequence CACGTG represents the specific consensus motif for the human protein Max used in this study (Fig. 1). The process of establishing specific, consensus protein−DNA binding requires the formation of precise geometrical fit between the protein and its consensus DNA motif, accompanied by the formation of specific hydrogen and electrostatic contacts at the protein−DNA binding interface (6, 7) ( Fig. 1). In addition to binding to their consensus DNA motifs, transcription factors can also bind, albeit with lower affinity, to DNA regions lacking any consensus motifs. The term "nonspecific protein−DNA binding" (6) is typically used to describe these weaker interactions. Von Hippel and Berg suggested classifying nonspecific protein−DNA binding into two related mechanisms (6). The first mechanism includes protein binding to its mutated specific motifs that retain some residual, reduced specificity. The second mechanism is largely DNA sequence independent, and it involves electrostatic binding modulated by the overall DNA geometry (6). Despite significant experimental progress, molecular mechanisms responsible for these two types of nonspecific binding remain poorly understood, and the free energy of nonspecific protein−DNA binding has not been systematically characterized (11)(12)(13)(14). The interplay between consensus and nonconsensus DNA sequence elements emerges as a dominant factor that governs protein−DNA binding preferences. However, this interplay is also poorly understood (15, 16). Until now, it has been reasonably assumed that specific (consensus) base-pair recognition must control the genome-wide specificity of TF−DNA binding.Contrary to this assumption, here we identify a general mechanism for protein−DNA bi...

A mutation in the allosteric site of the caspase 3 dimer interface of Val266 to histidine abolishes activity of the enzyme, and models predict that the mutation mimics the action of small molecule allosteric inhibitors by preventing formation of the active site. Mutations were coupled to His266 at two sites in the interface, E124A and Y197C. We present results from X-ray crystallography, enzymatic activity and molecular dynamics simulations for seven proteins, consisting of single, double and triple mutants. The results demonstrate that considering allosteric inhibition of caspase 3 as a shift between discrete ‘off-state’ or ‘on-state’ conformations is insufficient. Although His266 is accommodated in the interface, the structural defects are propagated to the active site through a helix on the protein surface. A more comprehensive view of allosteric regulation of caspase 3 requires the representation of an ensemble of inactive states and shows that subtle structural changes lead to the population of the inactive ensemble.

SUMMARY Paralogous transcription factors (TFs) are oftentimes reported to have identical DNA-binding motifs, despite the fact that they perform distinct regulatory functions. Differential genomic targeting by paralogous TFs is generally assumed to be due to interactions with protein co-factors or the chromatin environment. Using a computational-experimental framework called iMADS (integrative modeling and analysis of differential specificity), we show that, contrary to previous assumptions, paralogous TFs bind differently to genomic target sites even in vitro. We used iMADS to quantify, model, and analyze specificity differences between 11 TFs from 4 protein families. We found that paralogous TFs have diverged mainly at mediumand low-affinity sites, which are poorly captured by current motif models. We identify sequence and shape features differentially preferred by paralogous TFs, and we show that the intrinsic differences in specificity among paralogous TFs contribute to their differential in vivo binding. Thus, our study represents a step forward in deciphering the molecular mechanisms of differential specificity in TF families.

BackgroundThe Myc-Max heterodimer is a transcription factor that regulates expression of a large number of genes. Genome occupancy of Myc-Max is thought to be driven by Enhancer box (E-box) DNA elements, CACGTG or variants, to which the heterodimer binds in vitro.ResultsBy analyzing ChIP-Seq datasets, we demonstrate that the positions occupied by Myc-Max across the human genome correlate with the RNA polymerase II, Pol II, transcription machinery significantly better than with E-boxes. Metagene analyses show that in promoter regions, Myc is uniformly positioned about 100 bp upstream of essentially all promoter proximal paused polymerases with Max about 15 bp upstream of Myc. We re-evaluate the DNA binding properties of full length Myc-Max proteins. Electrophoretic mobility shift assay results demonstrate Myc-Max heterodimers display significant sequence preference, but have high affinity for any DNA. Quantification of the relative affinities of Myc-Max for all possible 8-mers using universal protein-binding microarray assays shows that sequences surrounding core 6-mers significantly affect binding. Compared to the in vitro sequence preferences, Myc-Max genomic occupancy measured by ChIP-Seq is largely, although not completely, independent of sequence specificity.ConclusionsWe quantified the affinity of Myc-Max to all possible 8-mers and compared this with the sites of Myc binding across the human genome. Our results indicate that the genomic occupancy of Myc cannot be explained by its intrinsic DNA specificity and suggest that the transcription machinery and associated promoter accessibility play a predominant role in Myc recruitment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0482-3) contains supplementary material, which is available to authorized users.

Background: The Myc-Max heterodimer is a transcription factor that regulates expression of a large number of genes. Genome occupancy of Myc-Max is thought to be driven by Enhancer box (E-box) DNA elements, CACGTG or variants, to which the heterodimer binds in vitro. Results: By analyzing ChIP-Seq datasets, we demonstrate that the positions occupied by Myc-Max across the human genome correlate with the RNA polymerase II, Pol II, transcription machinery significantly better than with E-boxes. Metagene analyses show that in promoter regions, Myc is uniformly positioned about 100 bp upstream of essentially all promoter proximal paused polymerases with Max about 15 bp upstream of Myc. We re-evaluate the DNA binding properties of full length Myc-Max proteins. Electrophoretic mobility shift assay results demonstrate Myc-Max heterodimers display significant sequence preference, but have high affinity for any DNA. Quantification of the relative affinities of Myc-Max for all possible 8-mers using universal protein-binding microarray assays shows that sequences surrounding core 6-mers significantly affect binding. Compared to the in vitro sequence preferences, Myc-Max genomic occupancy measured by ChIP-Seq is largely, although not completely, independent of sequence specificity.

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 highresolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection.C aspase function in cell development and cell death results from a continuum of enzyme activity, in which an as-yetundefined activity threshold is required for cell death. At subthreshold levels, caspase activity is important for a variety of physiological reactions (referred to as adaptive responses), including remodeling the cytoplasm (1), cell differentiation (2), neuron pruning (3), receptor endocytosis (4), macrophage function (5), and development of the eye lens (6) and inner ear (7). The roles of caspases in apoptosis are well known, but their roles in adaptive responses are less clear, particularly in regard to how cells set the threshold of caspase activity to limit apoptosis while ensuring sufficient activity for signaling and differentiation.Cells use two general mechanisms to modify caspase activity, through modulating levels of active caspase or through allosteric mechanisms that change the distribution of conformations in the native ensemble, although the two are not mutually exclusive. Levels of caspase-3 are controlled by cleavage of the inactive zymogen to yield a dimer of protomers (Fig. 1A) (8,9), and this process is responsive to several signaling pathways, such as transient expression of the Bad-Bax cascade (10) or phosphorylation of the zymogen (Fig. 1B) (11). Alternatively, inhibitor of apoptosis proteins (IAPs) affect levels of active caspase-3 by dire...

The dimer interface of caspase-3 contains a bifunctional allosteric site in which the enzyme can be activated or inactivated, depending on the context of the protein. In the mature caspase-3, the binding of allosteric inhibitors to the interface results in an order-to-disorder transition in the active site loops. In procaspase-3, by contrast, the binding of allosteric activators to the interface results in a disorder-to-order transition in the active site. We have utilized the allosteric site to identify a small molecule activator of procaspase and to characterize it's binding to the protease. The data suggest that an efficient activator must stabilize the active conformer of the zymogen by expelling the intersubunit linker from the interface, and it must interact with active site residues found in the allosteric site. Small molecule activators that fulfill the two requirements should provide scaffolds for drug candidates as a therapeutic strategy for directly promoting procaspase-3 activation in cancer cells.

The conformational ensemble of procaspase 3, the primary executioner in apoptosis, contains two major forms, inactive and active, with the inactive state favored in the native ensemble. A region of the protein known as the intersubunit linker (IL) is cleaved during maturation, resulting in movement of the IL out of the dimer interface and subsequent active site formation (activation-by-cleavage mechanism). We examined two models for the role of the IL in maintaining the inactive conformer, an IL-extension model versus a hydrophobic cluster model, and we show that increasing the length of the IL by introducing 3-5 alanines results in constitutively active procaspases. Active site labeling and subsequent analyses by mass spectrometry show that the full-length zymogen is enzymatically active. We also show that minor populations of alternately cleaved procaspase result from processing at D169 when the normal cleavage site, D175, is unavailable. Importantly, the alternately cleaved proteins have little to no activity, but increased flexibility of the linker increases the exposure of D169. The data show that releasing the strain of the short IL, in and of itself, is not sufficient to populate the active conformer of the native ensemble. The IL must also allow for interactions that stabilize the active site, possibly from a combination of optimal length, flexibility in the IL, and specific contacts between the IL and interface. The results provide further evidence that substantial energy is required to shift the protein to the active conformer. As a result, the activation-by-cleavage mechanism dominates in the cell.