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Electrostatic protein/DNA interactions arise from the neutralization of the DNA phosphodiester backbone as well as coupled exchanges by charged protein residues as salt bridges or with mobile ions. Much focus has been and continues to be paid to interfacial ion pairs with DNA. The role of extra-interfacial ionic interactions, particularly as dynamic drivers of DNA sequence selectivity, remain poorly known. The ETS family of transcription factors represents an attractive model for addressing this knowledge gap given their diverse ionic composition in primary structures that fold to a tightly conserved DNA-binding motif. To probe the importance of extra-interfacial salt bridges in DNA recognition, we compared the salt-dependent binding by Elk1 with ETV6, two ETS homologs differing markedly in ionic composition. While both proteins exhibit salt-dependent binding with cognate DNA that corresponds to interfacial phosphate contacts, their nonspecific binding diverges from cognate binding as well as each other. Molecular dynamics simulations in explicit solvent, which generated ionic interactions in agreement with the experimental binding data, revealed distinct salt-bridge dynamics in the nonspecific complexes formed by the two proteins. Impaired DNA contact by ETV6 resulted in fewer backbone contacts in the nonspecific complex, while Elk1 exhibited a redistribution of extra-interfacial salt bridges via residues that are non-conserved between the two ETS relatives. Thus, primary structure variation in ionic residues can encode highly differentiated specificity mechanisms in a highly conserved DNA-binding motif.

Sequence selectivity is a critical attribute of DNA-binding ligands and underlines the need for detailed molecular descriptions of binding in representative sequence contexts. We investigated the binding and volumetric properties of DB1976, a model bis(benzimidazole)-selenophene diamidine compound with emerging therapeutic potential in acute myeloid leukemia, debilitating fibroses, and obesity-related liver dysfunction. To sample the scope of cognate DB1976 target sites, we evaluated three dodecameric duplexes spanning >10(3)-fold in binding affinity. The attendant changes in partial molar volumes varied substantially, but not in step with binding affinity, suggesting distinct modes of interactions in these complexes. Specifically, whereas optimal binding was associated with loss of hydration water, low-affinity binding released more hydration water. Explicit-atom molecular dynamics simulations showed that minor groove binding perturbed the conformational dynamics and hydration at the termini and interior of the DNA in a sequence-dependent manner. The impact of these distinct local dynamics on hydration was experimentally validated by domain-specific interrogation of hydration with salt, which probed the charged axial surfaces of oligomeric DNA preferentially over the uncharged termini. Minor groove recognition by DB1976, therefore, generates dynamically distinct domains that can make favorable contributions to hydration release in both high- and low-affinity binding. Because ligand binding at internal sites of DNA oligomers modulates dynamics at the termini, the results suggest both short- and long-range dynamic effects along the DNA target that can influence their effectiveness as low-MW competitors of protein binding.

DNA base damage arises frequently in living cells and needs to be removed by base excision repair (BER) to prevent mutagenesis and genome instability. Both the formation and repair of base damage occur in chromatin and are conceivably affected by DNA-binding proteins such as transcription factors (TFs). However, to what extent TF binding affects base damage distribution and BER in cells is unclear. Here, we used a genome-wide damage mapping method, N-methylpurine-sequencing (NMP-seq), to characterize alkylation damage distribution and BER at TF binding sites in yeast cells treated with the alkylating agent methyl methanesulfonate (MMS). Our data shows that alkylation damage formation was mainly suppressed at the binding sites of yeast TFs Abf1 and Reb1, but individual hotspots with elevated damage levels were also found. Additionally, Abf1 and Reb1 binding strongly inhibits BER in vivo and in vitro, causing slow repair both within the core motif and its adjacent DNA. The observed effects are caused by the TF-DNA interaction, because damage formation and BER can be restored by depletion of Abf1 or Reb1 protein from the nucleus. Thus, our data reveal that TF binding significantly modulates alkylation base damage formation and inhibits repair by the BER pathway. The interplay between base damage formation and BER may play an important role in affecting mutation frequency in gene regulatory regions.

Somatic mutations in DNA-binding sites for CCCTC-binding factor (CTCF) are significantly elevated in many cancers. Prior analysis has suggested that elevated mutation rates at CTCF-binding sites in skin cancers are a consequence of the CTCF-cohesin complex inhibiting repair of UV damage. Here, we show that CTCF binding modulates the formation of UV damage to induce mutation hot spots. Analysis of genome-wide CPD-seq data in UV-irradiated human cells indicates that formation of UV-induced cyclobutane pyrimidine dimers (CPDs) is primarily suppressed by CTCF binding but elevated at specific locations within the CTCF motif. Locations of CPD hot spots in the CTCF-binding motif coincide with mutation hot spots in melanoma. A similar pattern of damage formation is observed at CTCF-binding sites in vitro, indicating that UV damage modulation is a direct consequence of CTCF binding. We show that CTCF interacts with binding sites containing UV damage and inhibits repair by a model repair enzyme in vitro. Structural analysis and molecular dynamic simulations reveal the molecular mechanism for how CTCF binding modulates CPD formation.

Hairpin polyamides are synthetic small molecules that bind DNA minor groove sequence-selectively and, in many sequences, induce widening of the minor groove and compression of the major groove. The structural distortion of DNA caused by polyamides has enhanced our understanding of the regulation of DNA-binding proteins via polyamides. Polyamides have DNA binding affinities that are comparable to those proteins, therefore, can potentially be used as therapeutic agents to treat diseases caused by aberrant gene expression. In fact, many diseases are characterized by over- or under-expressed genes. PU.1 is a transcription factor that regulates many immune system genes. Aberrant expression of PU.1 has been associated with the development of acute myeloid leukemia (AML). We have, therefore, designed and synthesized ten hairpin polyamides to investigate their capacity in controlling the PU.1-DNA interaction. Our results showed that nine of the polyamides disrupt PU.1-DNA binding and the inhibition capacity strongly correlates with binding affinity. One molecule, FH1024, was observed forming a FH1024-PU.1-DNA ternary complex instead of inhibiting PU.1-DNA binding. This is the first report of a small molecule that is potentially a weak agonist that recruits PU.1 to DNA. This finding sheds light on the design of polyamides that exhibit novel regulatory mechanisms on protein-DNA binding.

Functional evidence increasingly implicates low-affinity DNA recognition by transcription factors as a general mechanism for the spatiotemporal control of developmental genes. Although the DNA sequence requirements for affinity are well-defined, the dynamic mechanisms that execute cognate recognition are much less resolved. To address this gap, here we examined ETS1, a paradigm developmental transcription factor, as a model for which cognate discrimination remains enigmatic. Using molecular dynamics simulations, we interrogated the DNA-binding domain of murine ETS1 alone and when bound to high-and low-affinity cognate sites or to nonspecific DNA. The results of our analyses revealed collective backbone and side-chain motions that distinguished cognate versus nonspecific as well as high- versus low-affinity cognate DNA binding. Combined with binding experiments with site-directed ETS1 mutants, the molecular dynamics data disclosed a triad of residues that respond specifically to low-affinity cognate DNA. We found that a DNA-contacting residue (Gln-336) specifically recognizes low-affinity DNA and triggers the loss of a distal salt bridge (Glu-343/Arg-378) via a large side-chain motion that compromises the hydrophobic packing of two core helices. As an intact Glu-343/Arg-378 bridge is the default state in unbound ETS1 and maintained in high-affinity and nonspecific complexes, the low-affinity complex represents a unique conformational adaptation to the suboptimization of developmental enhancers.

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs.

The understanding of sequence-specific DNA minor groove interactions has recently made major steps forward and as a result, the goal of development of compounds that target the minor groove is an active research area. In an effort to develop biologically active minor groove agents, we are preparing and exploring the DNA interactions of diverse diamidine derivatives with a 5’-GAATTC-3’ binding site using a powerful array of methods including, biosensor-SPR methods, and X-ray crystallography. The benzimidazole-thiophene module provides an excellent minor groove recognition component. A central thiophene in a benzimidazole-thiophene-phenyl aromatic system provides essentially optimum curvature for matching the shape of the minor groove. Comparison of that structure to one with the benzimidazole replaced with an indole shows that the two structures are very similar, but have some interesting and important differences in electrostatic potential maps, the DNA minor groove binding structure based on x-ray crystallographic analysis, and inhibition of the major groove binding PU.1 transcription factor complex. The binding K(D) for both compounds is under 10 nM and both form amidine H-bonds to DNA bases. They both have bifurcated H-bonds from the benzimidazole or indole groups to bases at the center of the -AATT- binding site. Analysis of the comparative results provides an excellent understanding of how thiophene compounds recognize the minor groove and can act as transcription factor inhibitors.

The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro–B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro– to pre–B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1’s critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.

Eukaryotic transcription factors are versatile mediators of specificity in gene regulation. This versatility is achieved through mutual specification by context-specific DNA binding on the one hand, and identity-specific protein-protein partnerships on the other. This interactivity, known as combinatorial control, enables a repertoire of complex transcriptional outputs that are qualitatively disjoint, or non-continuum, with respect to binding affinity. This feature contrasts starkly with prokaryotic gene regulators, whose activities in general vary quantitatively in step with binding affinity. Biophysical studies on prokaryotic model systems and more recent investigations on transcription factors highlight an important role for folded state dynamics and molecular hydration in protein/DNA recognition. Analysis of molecular models of combinatorial control and recent literature in low-affinity gene regulation suggest that transcription factors harbor unique conformational dynamics that are inaccessible or unused by prokaryotic DNA-binding proteins. Thus, understanding the intrinsic dynamics involved in DNA binding and co-regulator recruitment appears to be a key to understanding how transcription factors mediate non-continuum outcomes in eukaryotic gene expression, and how such capability might have evolved from ancient, structurally conserved counterparts.

Distinct dimeric forms of PU.1 mutually antagonize to control the transcriptionally active dose via a disordered PEST domain., Transcription factors comprise a major reservoir of conformational disorder in the eukaryotic proteome. The hematopoietic master regulator PU.1 presents a well-defined model of the most common configuration of intrinsically disordered regions (IDRs) in transcription factors. We report that the structured DNA binding domain (DBD) of PU.1 regulates gene expression via antagonistic dimeric states that are reciprocally controlled by cognate DNA on the one hand and by its proximal anionic IDR on the other. The two conformers are mediated by distinct regions of the DBD without structured contributions from the tethered IDRs. Unlike DNA-bound complexes, the unbound dimer is markedly destabilized. Dimerization without DNA is promoted by progressive phosphomimetic substitutions of IDR residues that are phosphorylated in immune activation and stimulated by anionic crowding agents. These results suggest a previously unidentified, nonstructural role for charged IDRs in conformational control by mitigating electrostatic penalties that would mask the interactions of highly cationic DBDs.

To extend the value of biosensor-SPR in the characterization of DNA recognition by nucleoproteins, we report a comparative analysis of DNA-facilitated target search by two ETS-family transcription factors: Elk1 and ETV6. ETS domains represent an attractive system for developing biosensor-based techniques due to a broad range of physicochemical properties encoded within a highly conserved DNA-binding motif. Building on a biosensor approach in which the protein is quantitatively sequestered and presented to immobilized cognate DNA as nonspecific complexes, we assessed the impact of intrinsic cognate and nonspecific affinities on long-range (intersegmental) target search. The equilibrium constants of DNA-facilitated binding were sensitive to the intrinsic binding properties of the proteins such that their relative specificity for cognate DNA were reinforced when binding occurred by transfer vs. without nonspecific DNA. Direct measurement of association and dissociation kinetics revealed ionic features of the activated complex that evidenced DNA-facilitated dissociation, even though Elk1 and ETV6 harbor only a single DNA-binding surface. At salt concentrations that masked the effects of nonspecific pre-binding at equilibrium, the dissociation kinetics of cognate binding were nevertheless distinct from conditions under which nonspecific DNA was absent. These results further strengthen the significance of long-range DNA-facilitated translocation in the physiologic environment.

Previous investigations of sequence-specific DNA binding by model minor groove-binding compounds showed that the ligand/DNA complex was destabilized in the presence of compatible co-solutes. Inhibition was interpreted in terms of osmotic stress theory as the uptake of significant numbers of excess water molecules from bulk solvent upon complex formation. Here, we interrogated the AT-specific DNA complex formed with the symmetric heterocyclic diamidine DB1976 as a model for minor groove DNA recognition using both ionic (NaCl) and non-ionic cosolutes (ethylene glycol, glycine betaine, maltose, nicotinamide, urea). While the non-ionic cosolutes all destabilized the ligand/DNA complex, their quantitative effects were heterogeneous in a cosolute- and salt-dependent manner. Perturbation with NaCl in the absence of non-ionic cosolute showed that preferential hydration water was released upon formation of the DB1976/DNA complex. As salt probes counter-ion release from charged groups such as the DNA backbone, we propose that the preferential hydration uptake in DB1976/DNA binding observed in the presence of osmolytes reflects the exchange of preferentially bound cosolute with hydration water in the environs of the bound DNA, rather than a net uptake of hydration waters by the complex.

The eponymous DNA-binding domain of ETS (E26 transformation specific) transcription factors binds a single sequence-specific site as a monomer over a single helical turn. Following our previous observation by titration calorimetry that the ETS member PU.1 dimerizes sequentially at a single sequence-specific DNA binding site to form a 2:1 complex, we have carried out an extensive spectroscopic and biochemical characterization of site-specific PU.1 ETS complexes. While 10 bp of DNA was sufficient to support PU.1 binding as a monomer, additional flanking bases were required to invoke sequential dimerization of the bound protein. NMR spectroscopy revealed a marked loss of signal intensity in the 2:1 complex, and mutational analysis implicated the distal surface away from the bound DNA as the dimerization interface. Hydroxyl radical DNA footprinting indicated that the site-specifically bound PU.1 dimers occupied an extended DNA interface downstream from the 5'-GGAA-3' core consensus relative to its 1:1 counterpart, thus explaining the apparent site size requirement for sequential dimerization. The site-specifically bound PU.1 dimer resisted competition from nonspecific DNA and showed with affinities similar to other functionally significant PU.1 interactions. As sequential dimerization did not occur with the ETS domain of Ets-1, a close structural homolog of PU.1, 2:1 complex formation may represent an alternative auto-inhibitory mechanism in the ETS family at the protein-DNA level.

The ETS family of transcription factors is a functionally heterogeneous group of gene regulators that share a structurally conserved, eponymous DNA-binding domain. Unlike other ETS homologues, such as Ets-1, DNA recognition by PU.1 is highly sensitive to its osmotic environment due to excess interfacial hydration in the complex. To investigate interfacial hydration in the two homologues, we mutated a conserved tyrosine residue, which is exclusively engaged in coordinating a well-defined water contact between the protein and DNA among ETS proteins, to phenylalanine. The loss of this water-mediated contact blunted the osmotic sensitivity of PU.1/DNA binding, but did not alter binding under normo-osmotic conditions, suggesting that PU.1 has evolved to maximize osmotic sensitivity. The homologous mutation in Ets-1, which was minimally sensitive to osmotic stress due to a sparsely hydrated interface, reduced DNA-binding affinity at normal osmolality but the complex became stabilized by osmotic stress. Molecular dynamics simulations of wildtype and mutant PU.1 and Ets-1 in their free and DNA-bound states, which recapitulated experimental features of the proteins, showed that abrogation of this tyrosine-mediated water contact perturbed the Ets-1/DNA complex not through disruption of interfacial hydration, but by inhibiting local dynamics induced specifically in the bound state. Thus, a configurationally identical water-mediated contact plays mechanistically distinct roles in mediating DNA recognition by structurally homologous ETS transcription factors.

The ETS family of transcription factors is a functionally heterogeneous group of gene regulators that share a structurally conserved, eponymous DNA-binding domain. DNA target specificity derives from combinatorial interactions with other proteins as well as intrinsic heterogeneity among ETS domains. Emerging evidence suggests molecular hydration as a fundamental feature that defines the intrinsic heterogeneity in DNA target selection and susceptibility to epigenetic DNA modification. This perspective invokes novel hypotheses in the regulation of ETS proteins in physiologic osmotic stress, their pioneering potential in heterochromatin, and the effects of passive and pharmacologic DNA demethylation on ETS regulation.

The ends of nucleic acids oligomers alter the statistics of interior nonspecific ligand binding and act as binding sites with altered properties. While the former aspect of “end effects” has received much theoretical attention in the literature, the physical nature of end-binding, and hence its potential impact on a wide range of studies with oligomers, remains poorly known. Here, we report for the first time end-binding to DNA using a model helix-turn-helix motif, the DNA-binding domain of ETV6, as a function of DNA sequence length. Spectral analysis of ETV6 intrinsic tryptophan fluorescence by singular value decomposition showed that end-binding to nonspecific fragments was negligible at >0.2 kbp and accumulated to 8% of total binding to 23-bp oligomers. The affinity for end-binding was insensitive to salt but tracked the affinity of interior binding, suggesting translocation from interior sites rather than free solution as its mechanism. As the presence of a cognate site in the 23-bp oligomer suppressed end-binding, neglect of end-binding to the short cognate DNA does not introduce significant error. However, the same applies to nonspecific DNA only if longer fragments (>0.2 kbp) are used.

Transcription factors recognize specific genomic sequences to regulate complex gene-expression programs. Although it is well-established that transcription factors bind to specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood1,2. Many DNA-binding proteins induce changes in the structure of the DNA outside the intrinsic B-DNA envelope. However, how the energetic cost that is associated with distorting the DNA contributes to recognition has proven difficult to study, because the distorted DNA exists in low abundance in the unbound ensemble3-9. Here we use a high-throughput assay that we term SaMBA (saturation mismatch-binding assay) to investigate the role of DNA conformational penalties in transcription factor-DNA recognition. In SaMBA, mismatched base pairs are introduced to pre-induce structural distortions in the DNA that are much larger than those induced by changes in the Watson-Crick sequence. Notably, approximately 10% of mismatches increased transcription factor binding, and for each of the 22 transcription factors that were examined, at least one mismatch was found that increased the binding affinity. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into 'super sites' that exhibit stronger affinity than any known canonical binding site. Determination of high-resolution X-ray structures, combined with nuclear magnetic resonance measurements and structural analyses, showed that many of the DNA mismatches that increase binding induce distortions that are similar to those induced by protein binding-thus prepaying some of the energetic cost incurred from deforming the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit transcription factors and thus modulate replication and repair activities in the cell10,11.

Purpose To model the quantitative probabilistic features of multiple choice question (MCQ) style assessments, with specific focus on controlling the false-positive rate ( α ) of wrongly passing a student, and to examine and summarize MCQ-writing tips to minimize key errors in MCQ test design. Method We generated Monte Carlo simulations and binomial probability distributions for different MCQ test structures, varying in number of questions, choices per question, and pass mark. Educated guessing and student blunder (incorrect response despite knowing the material) were modeled. Knowledge levels associated with α Results Pass marks designed to detect failing levels of knowledge at Conclusion Pass marks need to address MCQ test structure and the probabilistic nature of MCQ testing to accurately discriminate learner competency.

Transactivation by the ETS family of transcription factors, whose members share structurally conserved DNA-binding domains, is variably sensitive to methylation of their target genes. The mechanism by which DNA methylation controls ETS proteins remains poorly understood. Uncertainly also pervades the effects of hemi-methylated DNA, which occurs following DNA replication and in response to hypomethylating agents, on site recognition by ETS proteins. To address these questions, we measured the affinities of two sequence-divergent ETS homologs, PU.1 and Ets-1, to DNA sites harboring a hemi- and fully methylated CpG dinucleotide. While the two proteins bound unmethylated DNA with indistinguishable affinity, their affinities to methylated DNA are markedly heterogeneous and exhibit major energetic coupling between the two CpG methylcytosines. Analysis of simulated DNA and existing co-crystal structures revealed that hemi-methylation induced non-local backbone and groove geometries that were not conserved in the fully methylated state. Indirect readout of these perturbations was differentially achieved by the two ETS homologs, with the distinctive interfacial hydration in PU.1/DNA binding moderating the inhibitory effects of DNA methylation on binding. This data established a biophysical basis for the pioneering properties associated with PU.1, which robustly bound fully methylated DNA, but not Ets-1, which was substantially inhibited.

Heterocyclic dications are receiving increasing attention as targeted inhibitors of transcription factors. While many dications act as purely competitive inhibitors, some fail to displace protein efficiently at drug concentrations expected to saturate their DNA target. To achieve a mechanistic understanding of these non-competitive effects, we used a combination of dications, which are intrinsically fluorescent and spectrally-separated fluorescently labeled DNA to dissect complex interactions in multi-component drug/DNA/protein systems. Specifically, we interrogated site-specific binding by the transcription factor PU.1 and its perturbation by DB270, a furan-bisbenzimidazole-diamidine that strongly targets PU.1 binding sites yet poorly inhibits PU.1/DNA complexes. By titrating DB270 and/or cyanine-labeled DNA with protein or unlabeled DNA, and following the changes in their fluorescence polarization, we found direct evidence that DB270 bound protein independently of their mutual affinities for sequence-specific DNA. Each of the three species competed for the other two, and this interplay of mutually dependent equilibria abrogated DB270's inhibitory activity, which was substantively restored under conditions that attenuated DB270/PU.1 binding. PU.1 binding was consistent with DB270's poor inhibitory efficacy of PU.1 in vivo, while its isosteric selenophene analog (DB1976), which did not bind PU.1 and strongly inhibited the PU.1/DNA complex in vitro, fully antagonized PU.1-dependent transactivation in vivo.

Hydration of interfaces is a major determinant of target specificity in protein/DNA interactions. Interfacial hydration is a highly variable feature in DNA recognition by ETS transcription factors and functionally relates to cellular responses to osmotic stress. To understand how hydration is mediated in the conserved ETS/DNA binding interface, secondary structures comprising the DNA contact surface of the strongly hydrated ETS member PU.1 were substituted, one at a time, with corresponding elements from its sparsely hydrated relative Ets-1. The resultant PU.1/Ets-1 chimeras exhibited variably reduced sensitivity to osmotic pressure, indicative of a distributed pattern of interfacial hydration in wildt-ype PU.1. With the exception of the recognition helix H3, the chimeras retained substantially high affinities. Ets-1 residues could therefore offset the loss of favorable hydration contributions in PU.1 via low-water interactions, but at the cost of decreased selectivity at base positions flanking the 5′-GGA-3′ core consensus. Substitutions within H3 alone, which contacts the core consensus, impaired binding affinity and PU.1 transactivation in accordance with the evolutionary separation of the chimeric residues involved. The combined biophysical, bioinformatics and functional data therefore supports hydration as an evolved specificity determinant that endows PU.1 with more stringent sequence selection over its ancestral relative Ets-1.

Recurrent mutations are frequently associated with transcription factor (TF) binding sites (TFBS) in melanoma, but the mechanism driving mutagenesis at TFBS is unclear. Here, we use a method called CPD-seq to map the distribution of UV-induced cyclobutane pyrimidine dimers (CPDs) across the human genome at single nucleotide resolution. Our results indicate that CPD lesions are elevated at active TFBS, an effect that is primarily due to E26 transformation-specific (ETS) TFs. We show that ETS TFs induce a unique signature of CPD hotspots that are highly correlated with recurrent mutations in melanomas, despite high repair activity at these sites. ETS1 protein renders its DNA binding targets extremely susceptible to UV damage in vitro, due to binding-induced perturbations in the DNA structure that favor CPD formation. These findings define a mechanism responsible for recurrent mutations in melanoma and reveal that DNA binding by ETS TFs is inherently mutagenic in UV-exposed cells., Many factors contribute to mutation hotspots in cancer cells. Here the authors map UV damage at single-nucleotide resolution across the human genome and find that binding sites of ETS transcription factors are especially prone to forming UV lesions, leading to mutation hotspots in melanoma.

The ETS family of transcription factors exemplifies current uncertainty in how eukaryotic genetic regulators with overlapping DNA sequence preferences achieve target site specificity. PU.1 and Ets-1 represent archetypes for studying site discrimination by ETS proteins because their DNA-binding domains are the most divergent in sequence, yet they share remarkably superimposable DNA-bound structures. To gain insight into the contrasting thermodynamics and kinetics of DNA recognition by these two proteins, we investigated the structure and dynamics of site discrimination by their DNA-binding domains. Electrophoretic mobilities of complexes formed by the two homologs with circularly permuted binding sites showed significant dynamic differences only for DNA complexes of PU.1. Free solution measurements by dynamic light scattering showed PU.1 to be more dynamic than Ets-1; moreover, dynamic changes are strongly coupled to site discrimination by PU.1, but not Ets-1. Interrogation of the protein/DNA interface by DNA footprinting showed similar accessibility to dimethyl sulfate for PU.1/DNA and Ets-1/DNA complexes, indicating that the dynamics of PU.1/DNA complexes reside primarily outside that interface. An information-based analysis of the two homologs' binding motifs suggests a role for dynamic coupling in PU.1's ability to enforce a more stringent sequence preference than Ets-1 and its proximal sequence homologs.

To find their DNA target sites in complex solution environments containing excess heterogeneous DNA, sequence-specific DNA-binding proteins execute various translocation mechanisms known collectively as facilitated diffusion. For proteins harboring a single DNA contact surface, long-range translocation occurs by jumping between widely spaced DNA segments. We have configured biosensor-based surface plasmon resonance to directly measure the affinity and kinetics of this intersegmental jumping by the ETS-family transcription factor ETS variant 6 (ETV6). To isolate intersegmental target binding in a functionally defined manner, we pre-equilibrated ETV6 with excess salmon sperm DNA, a heterogeneous polymer, before exposing the nonspecifically bound protein to immobilized oligomeric DNA harboring a high-affinity ETV6 site. In this way, the mechanism of ETV6-target association could be toggled electrostatically through varying NaCl concentration in the bulk solution. Direct measurements of association and dissociation kinetics of the site-specific complex indicated that 1) freely diffusive binding by ETV6 proceeds through a nonspecific-like intermediate, 2) intersegmental jumping is rate-limited by dissociation from the nonspecific polymer, and 3) dissociation of the specific complex is independent of the history of complex formation. These results show that target searches by proteins with an ETS domain, such as ETV6, whose single DNA-binding domain cannot contact both source and destination sites simultaneously, are nonetheless strongly modulated by intersegmental jumping in heterogeneous site environments. Our findings establish biosensors as a general technique for directly and specifically measuring target site search by DNA-binding proteins via intersegmental translocation.

ETS family transcription factors regulate diverse genes through binding at cognate DNA sites that overlap substantially in sequence. The DNA-binding domains of ETS proteins (ETS domains) are highly conserved structurally yet share limited amino acid homology. To define the mechanistic implications of sequence diversity within the ETS family, we characterized the thermodynamics and kinetics of DNA site recognition by the ETS domains of Ets-1 and PU.1, which represent the extremes in amino acid divergence among ETS proteins. Even though the two ETS domains bind their optimal sites with similar affinities under physiologic conditions, their nature of site recognition differs strikingly in terms of the role of hydration and counter ion release. The data suggest two distinct mechanisms wherein Ets-1 follows a "dry" mechanism that rapidly parses sites through electrostatic interactions and direct protein-DNA contacts, whereas PU.1 utilizes hydration to interrogate sequence-specific sites and form a long-lived complex relative to the Ets-1 counterpart. The kinetic persistence of the high affinity PU.1 · DNA complex may be relevant to an emerging role of PU.1, but not Ets-1, as a pioneer transcription factor in vivo. In addition, PU.1 activity is critical to the development and function of macrophages and lymphocytes, which present osmotically variable environments, and hydration-dependent specificity may represent an important regulatory mechanism in vivo, a hypothesis that finds support in gene expression profiles of primary murine macrophages.

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤10(5)M(-)(1)s(-)(1)), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>10(7)M(-)(1)s(-)(1)). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes.

Transcription factors with structurally homologous DNA-binding domains are conventionally thought to operate similar mechanisms of DNA site recognition, and instead derive their biological specificity from extrinsic interactions such as recruitment of partner proteins and post-translational modifications. The ETS superfamily of transcriptional factors, all of which share structurally conserved, eponymous DNA-binding domains but nonetheless regulate non-redundant genetic networks, exemplify this paradigmic view of eukaryotic gene regulation. Recent genomic studies have defined a hierarchy within the ETS family based on “pioneering” properties: the ability of some ETS-family homologs such as PU.1, but not others such as Ets-1, to autonomously bind nucleosomal and methylated DNA in vivo. To better understand this heterogeneity at a mechanistic level, we used fluorescence anisotropy to explicitly interrogate the recognition by the DNA-binding domains of these two ETS homologs for a single cognate DNA site harboring a defined CpG dinucleotide in which one, the other, or both cytosines have been methylated. We observed that while both ETS domains bind the unmethylated sequence with equally high affinity, they are differentially inhibited at mono-methylated sites in a position-dependent manner. Moreover, whereas PU.1 binds the di-methylated DNA site, Ets-1 is strongly inhibited. Our data indicate that sensitivity to methylated DNA is an intrinsic property of the ETS domain and, more generally, can be encoded into a structurally conserved DNA-binding domain without external control.

Isothermal titration calorimetry (ITC) produces a differential heat signal with respect to the total titrant concentration. This feature gives ITC excellent sensitivity for studying the thermodynamics of complex biomolecular interactions in solution. Currently, numerical methods for data fitting are based primarily on indirect approaches rooted in the usual practice of formulating biochemical models in terms of integrated variables. Here, a direct approach is presented wherein ITC models are formulated and solved as numerical initial value problems for data fitting and simulation purposes. To do so, the ITC signal is cast explicitly as a first-order ordinary differential equation (ODE) with total titrant concentration as independent variable and the concentration of a bound or free ligand species as dependent variable. This approach was applied to four ligand-receptor binding and homotropic dissociation models. Qualitative analysis of the explicit ODEs offers insights into the behavior of the models that would be inaccessible to indirect methods of analysis. Numerical ODEs are also highly compatible with regression analysis. Since solutions to numerical initial value problems are straightforward to implement on common computing platforms in the biochemical laboratory, this method is expected to facilitate the development of ITC models tailored to any experimental system of interest.

The formation of oligomeric proteins proceeds at a major cost of reducing the translational and rotational entropy for their subunits in order to form the stabilizing interactions found in the oligomeric state. Unlike site-directed mutations, covalent linkage of subunits represents a generically applicable strategy for enhancing oligomeric stability by reducing the entropic driving force for dissociation. Although this can be realized by introducing de novo disulfide cross-links between subunits, issues with irreversible aggregation limit the utility of this approach. In contrast, tandem linkage of subunits in a single polypeptide chain offers a universal method of pre-paying the entropic cost of oligomer formation. In the present paper, thermodynamic, structural and experimental aspects of designing and characterizing tandem-linked oligomers are discussed with reference to engineering a stabilized tetramer of the oligomerization domain of the human p53 tumour-suppressor protein by tandem dimerization.

Polycationic macromolecules and cationic peptides acting as PTDs (protein transduction domains) and CPPs (cell-penetrating peptides) represent important classes of agents used for the import and delivery of a wide range of molecular cargoes into cells. Their entry into cells is typically initiated through interaction with cell-surface HS (heparan sulfate) molecules via electrostatic interactions, followed by endocytosis of the resulting complexes. However, the endocytic mechanism employed (clathrin-mediated endocytosis, caveolar uptake or macropinocytosis), defining the migration of these peptides into cells, depends on parameters such as the nature of the cationic agent itself and complex formation with cargo, as well as the nature and distribution of proteoglycans expressed on the cell surface. Moreover, a survey of the literature suggests that endocytic pathways should not be considered as mutually exclusive, as more than one entry mechanism may be operational for a given cationic complex in a particular cell type. Specifically, the observed import may best be explained by the distribution and uptake of cell-surface HSPGs (heparan sulfate proteoglycans), such as syndecans and glypicans, which have been shown to mediate the uptake of many ligands besides cationic polymers. A brief overview of the roles of HSPGs in ligand internalization is presented, as well as mechanistic hypotheses based on the known properties of these cell-surface markers. The identification and investigation of interactions made by glycosaminoglycans and core proteins of HSPGs with PTDs and cationic polymers will be crucial in defining their uptake by cells.

Biosensor-surface plasmon resonance (SPR) technology has emerged as a powerful label-free approach for the study of nucleic acid interactions in real time. The method provides simultaneous equilibrium and kinetic characterization for biomolecular interactions with minimal materials and without an external probe. A detailed and practical guide for protein-DNA interaction analyses using biosensor-SPR methods is presented. Details of the SPR technology and basic fundamentals are described with recommendations on the preparation of the SPR instrument, sensor chips and samples, as well as extensive information on experimental design, quantitative and qualitative data analyses and presentation. A specific example of the interaction of a transcription factor with DNA is shown with results evaluated by both kinetic and steady-state SPR methods.

Nucleic acid-amino acid interactions are fundamental to understanding higher-order interactions made by nucleic acid-binding proteins. Here we employ electrophoresis to investigate DNA-amino acid interactions by using a set of amino acids (Ala, Gln, Gly, Met, Phe, Val, bicine and tricine) as trailing ions in a discontinuous buffer, and monitoring their interactions with duplex (from 12 to 3000 bp) and frayed wire [a set of self-assembled superstructures arising from d(A(15)G(15)) oligodeoxyribonucleotides] DNA by the change in their ionic mobility (in terms of %R(f)) as a function of amino acid concentration in a polyacrylamide matrix. By titration of the pH of Tris-HCl polyacrylamide gels (from 7 to 10), a span of steady-state amino acid concentrations and extents of ionization can be maintained. We found that with a decrease in pH (thereby increasing amino acid concentrations and the extent of ionization of the alpha-amino group), both the %R(f) and stacking limit were increased, but the extent varied among the trailing ions, resulting in an induced dispersion of %R(f) values for a given analyte. Using singular-value analysis to take into account the %R(f) dependence on fragment size (i.e., the %R(f) distribution), the degree of dispersion was found to be positively correlated with the accumulation of N-protonated trailing ions in the resolving phase. These results indicate that the modification of %R(f) of DNA is a mass-action effect involving DNA-amino acid interactions under essentially aqueous conditions.

In systems biology, molecular titration is a mechanism for introducing sensitivity of genetic networks to their regulatory proteins. Since most eukaryotic transcription factors lack chemical control by low-molecular-weight hormones or metabolites, transcription factors have evolved various strategies, such as cooperative DNA binding and sequestering partner proteins, to generate sensitivity in the expression of the genes that they regulate. The ETS superfamily of transcription factors, which are broadly distributed in the animal kingdom, regulate target genes in an active monomeric state. On the one hand, many ETS members, such as the master transcription factor PU.1, are involved in critical cell-fate decisions and signaling pathways that require highly sensitive output responses, yet no coherent mechanism exists for generating the required sensitivity. On the other hand, dimerization of ETS proteins in both DNA-bound and free states via their DNA-binding domain has been reported, but its purpose and biophysical nature remain obscure. We propose that dimerization serves as a sensitivity-generating mechanism by molecular self-titration: self-association sequesters free ETS proteins in inactive dimers, while dimerization in the DNA-bound state inhibits transactivation by the active protein/DNA complex. With the positive auto-regulation of the PU.1 (Spi1) gene generating self-amplification of PU.1, homodimerization would provide the minimal negative feedback needed to prevent explosion and maintain stable dynamics of PU.1 target genes in the absence of additional interactions. We are defining the thermodynamic and structural parameters of the PU.1 homodimer in the free and DNA-bound states. The data show that dimerization involves previously unexplored molecular surfaces that are distinct from the “orthosteric” protein/DNA contact interface and offer insight into how molecular self-titration would participate in the dynamics of PU.1-dependent gene expression.

This year marks the 50th anniversary of the proposal of a double helical structure for DNA by James Watson and Francis Crick. The place of this proposal in the history and development of molecular biology is discussed. Several other discoveries that occurred in the middle of the twentieth century were perhaps equally important to our understanding of cellular processes; however, none of these captured the attention and imagination of the public to the same extent as the double helix. The existence of multiple forms of DNA and the uses of DNA in biological technologies is presented. DNA is also finding increasing use as a material due to its rather unusual structural and physical characteristics as well as its ready availability.

Functionally critical decreases in levels or activity of the ETS family transcription factor PU.1 are present in approximately 2/3 of patients with acute myeloid leukemia (AML), across different AML subtypes (Sive, Leukemia 2016) including at the stem cell level (Steidl, Nat Genet 2006; Will, Nat Med 2015). Thus, targeting PU.1 could be an appealing option for treatment. As complete loss of PU.1 leads to stem cell failure (Iwasaki, Blood 2005), we hypothesized that PU.1 inhibition could eradicate leukemic cells harboring already low levels of PU.1, with modest effects on normal cells. We initially tested this hypothesis using 3 different shRNAs, and found that PU.1 inhibition led to a significant decrease in proliferation and clonogenicity, and increased apoptosis of mouse and human leukemic cell lines with low PU.1 levels, as well as the majority of primary human AML cells tested. We demonstrated that these effects were indeed due to decreased PU.1 levels by retroviral add-back experiments. The direct pharmacologic targeting of transcription factors has proven challenging in the past. Besides the core ETS binding motif (GGAA) in the DNA major groove, PU.1 binding to chromatin depends on additional minor groove contacts enriched for AT nucleotides upstream of the ETS motif, which determine selectivity for PU.1. Using an integrated screening strategy utilizing biosensor surface plasmon resonance, DNA footprinting, and cell-based dual-color PU.1 reporter assays, we developed novel small molecules of the heterocyclic diamidine family acting as first-in-class PU.1 inhibitors. Targeted occupancy by our compounds in the minor groove induces perturbations in DNA conformation that are transmitted to the PU.1 site in the major groove and thus inhibits PU.1 binding via an allosteric mechanism. Consistent with this, the inhibitory effects were selective for PU.1 versus other ETS transcription factors. Treatment with 3 different compounds led to cell growth inhibitory effect with respect to PU.1 level and preferentially affects PU.1low AML cells. Similarly to what we observed with shRNAs, treatment with our novel inhibitors led to decreased proliferation and colony forming capacity, increased apoptosis, and disrupted serial replating capacity of PU.1low AML cells and a majority of primary AML cell samples. Targeted ChIP and expression analysis showed that the compounds disrupt PU.1-promoter interaction and lead to downregulation of canonical PU.1 transcriptional targets in AML cells, confirming on-target activity in AML cells. Genome-wide analysis showed highly significant enrichment of known transcriptional targets of PU.1, and selectivity over genes regulated by other ETS family members. Comparison with published transcriptomic and PU.1 ChIP-seq data sets, as well as ARACNe analysis of the PU.1 regulon in primary AML cells, demonstrated that the inhibitors antagonize PU.1-regulated pathways at a genome-wide level. ChIP-seq performed in PU.1low AML cells confirmed a genome-wide decrease of PU.1 peaks after treatment and provides novel insight into the molecular mechanisms mediating the anti-leukemic effects of pharmacological PU.1 inhibition. To test the effects of PU.1 inhibition on normal hematopoiesis, we treated normal hematopoietic stem/progenitors cells (HSPC) in colony forming assays and saw decreased production of mature granulo-monocytic cells, consistent with PU.1's known role in this lineage. However, this effect was reversible upon drug removal, and serial replating capacity was not affected suggesting no significant effects on more immature HSPC. Congenic transplantation assays of treated normal bone marrow cells led to no change in myeloid and T-cells and only a modest decrease in B-cell numbers. Lastly, in vivo treatment with PU.1 inhibitors in mouse and human AML (xeno)transplantation models significantly decreased tumor burden and increased survival. To conclude, our study provides proof-of-principle for PU.1 inhibition as a novel therapeutic strategy in AML. Furthermore, we present the development of first-in-class PU.1 inhibitors acting via an allosteric minor groove-mediated mechanism. Our work shows that the specific pharmacological targeting of the DNA interaction of transcription factors such as PU.1 is feasible in principle, and may open the way for targeting of other transcription factors through minor groove-directed approaches. Disclosures Will: Novartis Pharmaceuticals: Consultancy, Research Funding. Steidl: Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding; Bayer Healthcare: Consultancy.

PU.1 belongs to the ETS family of transcription factors whose DNA-binding domains recognize purine-rich sequences containing the consensus 5'-GGAA/T-3'. We have characterized the sequence-specific association of the ETS domain of murine PU.1 to the lambdaB site of the Ig(lambda)2-4 enhancer as a function of temperature and pH by electrophoretic mobility shift, filter binding, and CD spectroscopy. From 0 to 25 degrees C, the dissociation equilibrium constant KD is, within experimental uncertainty, insensitive to temperature, and is only a weak function of temperature from 25 to 52 degrees C. van't Hoff analysis yielded a small value of DeltaCp = -2.1 kJ x mol(-1) x K(-1) in phosphate buffer, pH 7.4, containing 250 mM Na+. KD also shows a weak dependence at 25 degrees C on pH from 6.7 to 9.0 in phosphate, cacodylate, and Tris buffers that have disparate heats of ionization. The CD spectrum of the protein-DNA complex could be accounted for by a simple linear combination of the spectra of the free components throughout the binding temperature range. Structural calculations indicate that dehydration of solvent-accessible contact surfaces on the protein and DNA accounts for up to DeltaCp approximately -1 kJ x mol(-1) x K(-1). Taken together, these observations suggest that the hydrophobic effect and, in particular, coupled folding do not contribute significantly to the energetics of sequence-specific association. This is unusual with respect to other sequence-specific protein-DNA interactions for which significant enthalpic contributions and large negative heat capacities are commonly observed.

Prodrugs are widely used in the targeted delivery of cytotoxic compounds to cancer cells. To date, targeted prodrugs for cancer therapy have achieved great diversity in terms of target selection, activation chemistry, as well as size and physicochemical nature of the prodrug. Macromolecular prodrugs such as antibody-drug conjugates, targeted polymer-drug conjugates and other conjugates that self-assemble to form liposomal and micellar nanoparticles currently represent a major trend in prodrug development for cancer therapy. In this review, we explore a unified view of cancer-targeted prodrugs and highlight several examples from recombinant technology that exemplify the prodrug concept but are not identified as such. Recombinant “prodrugs” such as engineered anthrax toxin show promise in biological specificity through the conditionally targeting of multiple cellular markers. Conditional targeting is achieved by structural complementation, the spontaneous assembly of engineered inactive subunits or fragments to reconstitute functional activity. These complementing systems can be readily adapted to achieve conditionally bispecific targeting of enzymes that are used to activate low-molecular weight prodrugs. By leveraging strengths from medicinal chemistry, polymer science, and recombinant technology, prodrugs are poised to remain a core component of highly focused and tailored strategies aimed at conditionally attacking complex molecular phenotypes in clinically relevant cancer.

ETS transcription factors mediate a wide array of cellular functions and are attractive targets for pharmacological control of gene regulation. We report the inhibition of the ETS-family member PU.1 with a panel of novel heterocyclic diamidines. These diamidines are derivatives of furamidine (DB75) in which the central furan has been replaced with selenophene and/or one or both of the bridging phenyl has been replaced with benzimidazole. Like all ETS proteins, PU.1 binds sequence specifically to 10-bp sites by inserting a recognition helix into the major groove of a 5'-GGAA-3' consensus, accompanied by contacts with the flanking minor groove. We showed that diamidines target the minor groove of AT-rich sequences on one or both sides of the consensus and disrupt PU.1 binding. Although all of the diamidines bind to one or both of the expected sequences within the binding site, considerable heterogeneity exists in terms of stoichiometry, site-site interactions and induced DNA conformation. We also showed that these compounds accumulate in live cell nuclei and inhibit PU.1-dependent gene transactivation. This study demonstrates that heterocyclic diamidines are capable of inhibiting PU.1 by targeting the flanking sequences and supports future efforts to develop agents for inhibiting specific members of the ETS family.

The ETS family of transcription factors is widely distributed among the metazoan phyla and regulates the expression of a wide range of genes. Despite their functional diversity, all ETS proteins share a structurally conserved DNA-binding (or ETS) domain. Given the highly overlapping sequence preferences among ETS members, it is as yet unclear how ETS proteins achieve functional specificity, a problem known as the “specificity conundrum.” Compounding this problem is a current lack of understanding of the biophysical mechanism of sequence selectivity among ETS binding sites. We hypothesize that the structural conservation among ETS domains disguises physicochemical heterogeneity in their mechanisms of sequence recognition. We have previously demonstrated that the ETS-family member PU.1 (Spi-1) recruits a cooperative network of water-mediated contacts along the protein-DNA interface for high-affinity binding. We have now compared the thermodynamics and kinetics of sequence recognition between the ETS domains of PU.1 and ETS1 which represent extremes of sequence divergence (∼30% homology) in the ETS family. We found that the thermodynamics and kinetics between the two structurally conserved ETS domains are highly differentiated under physiological conditions. More precisely, whereas high-affinity PU.1 ETS-DNA binding is enthalpically driven against an entropic penalty, ETS1 ETS-DNA binding is entropically driven. Kinetically, whereas ETS1 ETS associates rapidly with a high-affinity cognate site (ka > 107 M−1 s−1), PU.1 ETS is strikingly slow (ka ∼104 M−1 s−1). This profound difference in association rate constants means that the high-affinity PU.1 ETS-DNA complex, despite being somewhat thermodynamically less stable than the corresponding complex with ETS1, is significantly longer-lived. If these differences that underlie the intrinsic heterogeneity in site recognition by ETS proteins extend to protein-protein and domain-domain interactions, they offer one potential biophysical resolution to the specificity conundrum.

Designed ligands that self-assemble noncovalently via an independent oligomerization domain have demonstrated enhancement in affinity for a variety of chemical and biological targets. To better understand the thermodynamic linkage between enhanced receptor binding and self-assembly, we have developed linkage models for the three commonly encountered types of noncovalently oligomeric ligands: homofunctional oligomeric ligands, heterodimeric ligands that target a single receptor, and bispecific ligands that crosslink noninteracting receptors. Expressions and numerical approaches for exact analysis as a function of total ligand concentrations are provided. We apply the linkage models to the binding data for two published noncovalently oligomeric ligands: one targeting a small molecule (phosphocholine) and the other targeting a soluble protein (tumor necrosis factor α). The linkage models provide a quantitative measure of the potential and realized enhancement in affinity that could inform and guide design optimization efforts, and they reveal physical insight that would elude model-free analysis. Incorporation of the linkage models, therefore, is expected to be valuable in the rational engineering of noncovalently oligomeric ligands.