Allostery mediates ligand binding to Grb2 adaptor in a mutually exclusive manner.

Allostery plays a key role in dictating the stoichiometry and thermodynamics of multi-protein complexes driving a plethora of cellular processes central to health and disease. Herein, using various biophysical tools, we demonstrate that although Sos1 nucleotide exchange factor and Gab1 docking protein recognize two non-overlapping sites within the Grb2 adaptor, allostery promotes the formation of two distinct pools of Grb2-Sos1 and Grb2-Gab1 binary signaling complexes in concert in lieu of a composite Sos1-Grb2-Gab1 ternary complex. Of particular interest is the observation that the binding of Sos1 to the nSH3 domain within Grb2 sterically blocks the binding of Gab1 to the cSH3 domain and vice versa in a mutually exclusive manner. Importantly, the formation of both the Grb2-Sos1 and Grb2-Gab1 binary complexes is governed by a stoichiometry of 2:1, whereby the respective SH3 domains within Grb2 homodimer bind to Sos1 and Gab1 via multivalent interactions. Collectively, our study sheds new light on the role of allostery in mediating cellular signaling machinery.

Multivalent binding and facilitated diffusion account for the formation of the Grb2-Sos1 signaling complex in a cooperative manner.

Despite its key role in driving cellular growth and proliferation through receptor tyrosine kinase (RTK) signaling, the Grb2-Sos1 macromolecular interaction remains poorly understood in mechanistic terms. Herein, using an array of biophysical methods, we provide evidence that although the Grb2 adaptor can potentially bind to all four PXψPXR motifs (designated herein S1-S4) located within the Sos1 guanine nucleotide exchange factor, the formation of the Grb2-Sos1 signaling complex occurs with a 2:1 stoichiometry. Strikingly, such bivalent binding appears to be driven by the association of the Grb2 homodimer to only two of four potential PXψPXR motifs within Sos1 at any one time. Of particular interest is the observation that of a possible six pairwise combinations in which S1-S4 motifs may act in concert for the docking of the Grb2 homodimer through bivalent binding, only S1 and S3, S1 and S4, S2 and S4, and S3 and S4 do so, while pairwise combinations of sites S1 and S2 and sites S2 and S3 appear to afford only monovalent binding. This salient observation implicates the role of local physical constraints in fine-tuning the conformational heterogeneity of the Grb2-Sos1 signaling complex. Importantly, the presence of multiple binding sites within Sos1 appears to provide a physical route for Grb2 to hop in a flip-flop manner from one site to the next through facilitated diffusion, and such rapid exchange forms the basis of positive cooperativity driving the bivalent binding of Grb2 to Sos1 with high affinity. Collectively, our study sheds new light on the assembly of a key macromolecular signaling complex central to cellular machinery in health and disease.

Acidic pH promotes oligomerization and membrane insertion of the BclXL apoptotic repressor.

Solution pH is believed to serve as an intricate regulatory switch in the induction of apoptosis central to embryonic development and cellular homeostasis. Herein, using an array of biophysical techniques, we provide evidence that acidic pH promotes the assembly of BclXL apoptotic repressor into a megadalton oligomer with a plume-like appearance and harboring structural features characteristic of a molten globule. Strikingly, our data reveal that pH tightly modulates not only oligomerization but also ligand binding and membrane insertion of BclXL in a highly subtle manner. Thus, while oligomerization and the accompanying molten globular content of BclXL is least favorable at pH 6, both of these structural features become more pronounced under acidic and alkaline conditions. However, membrane insertion of BclXL appears to be predominantly favored under acidic conditions. In a remarkable contrast, while ligand binding to BclXL optimally occurs at pH 6, it is diminished by an order of magnitude at lower and higher pH. This reciprocal relationship between BclXL oligomerization and ligand binding lends new insights into how pH modulates functional versatility of a key apoptotic regulator and strongly argues that the molten globule may serve as an intermediate primed for membrane insertion in response to apoptotic cues.

Biophysical analysis of binding of WW domains of the YAP2 transcriptional regulator to PPXY motifs within WBP1 and WBP2 adaptors.

The YAP2 transcriptional regulator mediates a plethora of cellular functions, including the newly discovered Hippo tumor suppressor pathway, by virtue of its ability to recognize WBP1 and WBP2 signaling adaptors among a wide variety of other ligands. Herein, using isothermal titration calorimery and circular dichroism in combination with molecular modeling and molecular dynamics, we provide evidence that the WW1 and WW2 domains of YAP2 recognize various PPXY motifs within WBP1 and WBP2 in a highly promiscuous and subtle manner. Thus, although both WW domains strictly require the integrity of the consensus PPXY sequence, nonconsensus residues within and flanking this motif are not critical for high-affinity binding, implying that they most likely play a role in stabilizing the polyproline type II helical conformation of the PPXY ligands. Of particular interest is the observation that both WW domains bind to a PPXYXG motif with highest affinity, implicating a preference for a nonbulky and flexible glycine one residue to the C-terminal side of the consensus tyrosine. Importantly, a large set of residues within both WW domains and the PPXY motifs appear to undergo rapid fluctuations on a nanosecond time scale, suggesting that WW-ligand interactions are highly dynamic and that such conformational entropy may be an integral part of the reversible and temporal nature of cellular signaling cascades. Collectively, our study sheds light on the molecular determinants of a key WW-ligand interaction pertinent to cellular functions in health and disease.

Binding of the cSH3 domain of Grb2 adaptor to two distinct RXXK motifs within Gab1 docker employs differential mechanisms.

A ubiquitous component of cellular signaling machinery, Gab1 docker plays a pivotal role in routing extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. Here, using isothermal titration calorimetry (ITC) in combination with macromolecular modeling (MM), we show that although Gab1 contains four distinct RXXK motifs, designated G1, G2, G3, and G4, only G1 and G2 motifs bind to the cSH3 domain of Grb2 adaptor and do so with distinct mechanisms. Thus, while the G1 motif strictly requires the PPRPPKP consensus sequence for high-affinity binding to the cSH3 domain, the G2 motif displays preference for the PXVXRXLKPXR consensus. Such sequential differences in the binding of G1 and G2 motifs arise from their ability to adopt distinct polyproline type II (PPII)- and 3(10) -helical conformations upon binding to the cSH3 domain, respectively. Collectively, our study provides detailed biophysical insights into a key protein-protein interaction involved in a diverse array of signaling cascades central to health and disease.

Structural and thermodynamic consequences of the replacement of zinc with environmental metals on estrogen receptor α-DNA interactions.

Estrogen receptor α (ERα) acts as a transcription factor by virtue of the ability of its DNA-binding (DB) domain, comprised of a tandem pair of zinc fingers, to recognize the estrogen response element within the promoters of target genes. Herein, using an array of biophysical methods, we probe the structural consequences of the replacement of zinc within the DB domain of ERα with various environmental metals and their effects on the thermodynamics of binding to DNA. Our data reveal that whereas the DB domain reconstituted with divalent ions of zinc, cadmium, mercury, and cobalt binds to DNA with affinities in the nanomolar range, divalent ions of barium, copper, iron, lead, manganese, nickel, and tin are unable to regenerate DB domain with DNA-binding potential, although they can compete with zinc for coordinating the cysteine ligands within the zinc fingers. We also show that the metal-free DB domain is a homodimer in solution and that the binding of various metals only results in subtle secondary and tertiary structural changes, implying that metal coordination may only be essential for binding to DNA. Collectively, our findings provide mechanistic insights into how environmental metals may modulate the physiological function of a key nuclear receptor involved in mediating a plethora of cellular functions central to human health and disease.

Genetic variations within the ERE motif modulate plasticity and energetics of binding of DNA to the ERα nuclear receptor.

Upon binding to estrogens, the ERα nuclear receptor acts as a transcription factor and mediates a multitude of cellular functions central to health and disease. Herein, using isothermal titration calorimetry (ITC) and circular dichroism (CD) in conjunction with molecular modeling (MM), we analyze the effect of symmetric introduction of single nucleotide variations within each half-site of the estrogen response element (ERE) on the binding of ERα nuclear receptor. Our data reveal that ERα exudes remarkable tolerance and binds to all genetic variants in the physiologically relevant nanomolar-micromolar range with the consensus ERE motif affording the highest affinity. We provide rationale for how genetic variations within the ERE motif may reduce its affinity for ERα by orders of magnitude at atomic level. Our data also suggest that the introduction of genetic variations within the ERE motif allows it to sample a much greater conformational space. Surprisingly, ERα displays no preference for binding to ERE variants with higher AT content, implying that any advantage due to DNA plasticity may be largely compensated by unfavorable entropic factors. Collectively, our study bears important consequences for how genetic variations within DNA promoter elements may fine-tune the physiological action of ERα and other nuclear receptors.

Binding of the ERalpha nuclear receptor to DNA is coupled to proton uptake.

Nuclear receptors act as ligand-modulated transcription factors and orchestrate a plethora of cellular functions central to health and disease. Although studied for more than half a century, many mysteries surrounding the mechanism of action of nuclear receptors remain unresolved. Herein, using isothermal titration calorimetry (ITC) in conjunction with macromolecular modeling (MM), we provide evidence that the binding of the ERalpha nuclear receptor to its DNA response element is coupled to proton uptake by two ionizable residues, H196 and E203, located at the protein-DNA interface. Alanine substitution of these ionizable residues decouples protonation and hampers the binding of ERalpha to DNA by nearly 1 order of magnitude. Remarkably, H196 and E203 are predominantly conserved across approximately 50 members of the nuclear receptor family, implying that proton-coupled equilibrium may serve as a key regulatory switch for modulating protein-DNA interactions central to nuclear receptor function and regulation. Taken together, our findings unearth an unexpected but critical step in the molecular action of nuclear receptors and suggest that they may act as sensors of intracellular pH.

Dissecting the role of leucine zippers in the binding of bZIP domains of Jun transcription factor to DNA.

Leucine zippers, structural motifs typically comprised of five successive heptads of amino acids with a signature leucine at every seventh position, play a central role in the dimerization of bZIP family of transcription factors and their subsequent binding to the DNA promoter regions of target genes. Herein, using analytical laser scattering (ALS) in combination with isothermal titration calorimetry (ITC), we study the effect of successive C-terminal truncation of leucine zippers on the dimerization and energetics of binding of bZIP domains of Jun transcription factor to its DNA response element. Our data show that all five heptads are critical for the dimerization of bZIP domains and that the successive C-terminal truncation of residues leading up to each signature leucine significantly compromises the binding of bZIP domains to DNA. Taken together, our study provides novel insights into the energetic contributions of leucine zippers to the binding of bZIP domains of Jun transcription factor to DNA.

Assembly of the Sos1-Grb2-Gab1 ternary signaling complex is under allosteric control.

Allostery has evolved as a form of local communication between interacting protein partners allowing them to quickly sense changes in their immediate vicinity in response to external cues. Herein, using isothermal titration calorimetry (ITC) in conjunction with circular dichroism (CD) and macromolecular modeling (MM), we show that the binding of Grb2 adaptor--a key signaling molecule involved in the activation of Ras GTPase--to its downstream partners Sos1 guanine nucleotide exchange factor and Gab1 docker is under tight allosteric regulation. Specifically, our findings reveal that the binding of one molecule of Sos1 to the nSH3 domain allosterically induces a conformational change within Grb2 such that the loading of a second molecule of Sos1 onto the cSH3 domain is blocked and, in so doing, allows Gab1 access to the cSH3 domain in an exclusively non-competitive manner to generate the Sos1-Grb2-Gab1 ternary signaling complex.

Single nucleotide variants of the TGACTCA motif modulate energetics and orientation of binding of the Jun-Fos heterodimeric transcription factor.

The Jun-Fos heterodimeric transcription factor is 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. Here, using isothermal titration calorimetry, we report detailed thermodynamics of the binding of bZIP domains of Jun-Fos heterodimer to synthetic dsDNA oligos containing the TGACTCA cis element and all possible single nucleotide variants thereof encountered widely within the promoters of a diverse array of genes. Our data show that Jun-Fos heterodimer tolerates single nucleotide substitutions and binds to TGACTCA variants with affinities in the physiologically relevant micromolar to submicromolar range. The energetics of binding are richly favored by enthalpic forces and opposed by entropic changes across the entire spectrum of TGACTCA variants in agreement with the notion that protein-DNA interactions are largely driven by electrostatic interactions and intermolecular hydrogen bonding. Of particular interest is the observation that the Jun-Fos heterodimer binds to specific TGACTCA variants in a preferred orientation. Our 3D atomic models reveal that such orientational preference results from asymmetric binding and may in part be attributable to chemically distinct but structurally equivalent residues R263 and K148 located within the basic regions of Jun and Fos, respectively. Taken together, our data suggest that the single nucleotide variants of the TGACTCA motif modulate energetics and orientation of binding of the Jun-Fos heterodimer and that such behavior may be a critical determinant of differential regulation of specific genes under the control of this transcription factor. Our study also bears important consequences for the occurrence of single nucleotide polymorphisms within the TGACTCA cis element at specific gene promoters between different individuals.

SH3 domains of Grb2 adaptor bind to PXpsiPXR motifs within the Sos1 nucleotide exchange factor in a discriminate manner.

Ubiquitously encountered in a wide variety of cellular processes, the Grb2-Sos1 interaction is mediated through the combinatorial binding of nSH3 and cSH3 domains of Grb2 to various sites containing PXpsiPXR motifs within Sos1. Here, using isothermal titration calorimetry, we demonstrate that while the nSH3 domain binds with affinities in the physiological range to all four sites containing PXpsiPXR motifs, designated S1, S2, S3, and S4, the cSH3 domain can only do so at the S1 site. Further scrutiny of these sites yields rationale for the recognition of various PXpsiPXR motifs by the SH3 domains in a discriminate manner. Unlike the PXpsiPXR motifs at S2, S3, and S4 sites, the PXpsiPXR motif at the S1 site is flanked at its C-terminus with two additional arginine residues that are absolutely required for high-affinity binding of the cSH3 domain. In striking contrast, these two additional arginine residues augment the binding of the nSH3 domain to the S1 site, but their role is not critical for the recognition of S2, S3, and S4 sites. Site-directed mutagenesis suggests that the two additional arginine residues flanking the PXpsiPXR motif at the S1 site contribute to free energy of binding via the formation of salt bridges with specific acidic residues in SH3 domains. Molecular modeling is employed to project these novel findings into the 3D structures of SH3 domains in complex with a peptide containing the PXpsiPXR motif and flanking arginine residues at the S1 site. Taken together, this study furthers our understanding of the assembly of a key signaling complex central to cellular machinery.

DNA plasticity is a key determinant of the energetics of binding of Jun-Fos heterodimeric transcription factor to genetic variants of TGACGTCA motif.

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 to 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 correlates with the overall free energy of binding in agreement with the 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.

Spine Stereotactic Radiosurgery for Metastatic Pheochromocytoma.

PURPOSE:  Despite aggressive primary treatment, up to 13.5% of patients diagnosed with pheochromocytoma may develop metastases, most often affecting the axial skeleton. Given that systemic therapy options are often inadequate, local therapy remains the cornerstone of palliation for these patients. Historically poor responses to standard fractionated radiotherapy have led to the consideration of stereotactic radiosurgery as an option to overcome potential radioresistance and provide durable local control of these tumors. Here we report our institutional experience in treating spine metastases from pheochromocytoma with spine stereotactic radiosurgery (SSRS). METHODS AND MATERIALS: Our clinical databases were retrospectively reviewed for patients with metastatic pheochromocytoma treated with SSRS from 2000-2017. Seven patients with 16 treated metastatic spinal lesions were identified. Local control was evaluated using magnetic resonance imaging (MRI). Pain and symptom data were assessed to evaluate toxicity using Common Terminology Criteria for Adverse Events (CTCAE) v4.03. The Kaplan-Meier method was used to assess local control and overall survival (OS). RESULTS: Median follow-up for treated lesions was 11 months (range 2.2 - 70.8). Most lesions were treated to a dose of 27 Gy in three fractions (62.5%). Other fractionation schemes included 24 Gy in one fraction (25%), 16 Gy in one fraction (6.3%), and 18 Gy in three fractions (6.3%). Treatment sites included the cervical spine (18.8%), thoracic spine (37.5%), lumbar spine (31.3%), and sacrum (12.5%). The crude local control rate was 93.7%, with one thoracic spine lesion progressing 20.7 months after treatment with 24 Gy in one fraction. Kaplan-Meier OS rates at 1 and 2 years after SSRS were 71.4% and 42.9%, respectively. Most common toxicities included acute grade 1-2 pain and fatigue. There was one case of vertebral fracture in a cervical spine lesion treated to 27 Gy in three fractions, which was managed non-surgically. CONCLUSION: Very few studies have explored the use of SSRS in metastatic pheochromocytoma. Our data suggest this modern radiation modality is effective, safe, and provides durable local control to palliate symptoms and potentially limit further metastatic seeding. Larger patient numbers and longer follow-up will further define the role of SSRS as a treatment option in these patients.

Low incidence of late failure and toxicity after spine stereotactic radiosurgery: Secondary analysis of phase I/II trials with long-term follow-up.

BACKGROUND AND PURPOSE: To characterize local control and late toxicity in long-term survivors prospectively-treated with spine stereotactic radiosurgery (SSRS). MATERIALS AND METHODS: From 2002 to 2011, 228 patients were prospectively-treated on protocol for metastatic disease of 261 vertebral sites. A subset of 52 patients surviving >4 years following treatment were collectively treated for 58 sites (encompassing 69 vertebrae) and underwent secondary analysis. Of all sites, 9% received prior radiation, and 16% encompassed multiple contiguous vertebrae. Radiation prescriptions were most commonly 24 Gy in 1 and 27 Gy in 3 fractions. Outcomes were evaluated via Kaplan-Meier, and associations analyzed via logistic regression. RESULTS: Median follow-up was 6.7 years (range: 49-142 months). Five-year local control by site was 91%, with late failures (>2 years) occurring in 3%. Overall and Grade ≥3 late toxicities (>2 years) were observed in 5% and 2% of sites. The last known neurologic event (grade 2 radiculopathy) was noted 2.1 years post-treatment, while the last documented fracture occurred at 4.1 years. No Grade ≥3 events were witnessed after 3.1 years post-SSRS, and no toxicities were noted after 4.1 years through end of follow-up. Re-irradiation, number of segments treated per site (1 vs. 2-3), and fractionation (1 vs. 3-5) were not associated with failure or toxicity. CONCLUSION: SSRS maintains excellent disease control and a favorable late toxicity profile even among long-term survivors, with very few failures or toxicities after 2 years in this prospectively-treated population. Overall, these data support the durable control and long-term safety of SSRS with extended follow-up.

Thermodynamic analysis of the heterodimerization of leucine zippers of Jun and Fos transcription factors.

Jun and Fos are components of the AP1 family of transcription factors and bind to the promoters of a diverse multitude of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryonic development and cancer. Here, using the powerful technique of isothermal titration calorimetry, we characterize the thermodynamics of heterodimerization of leucine zippers of Jun and Fos. Our data suggest that the heterodimerization of leucine zippers is driven by enthalpic forces with unfavorable entropy change at physiological temperatures. Furthermore, the basic regions appear to modulate the heterodimerization of leucine zippers and may undergo at least partial folding upon heterodimerization. Large negative heat capacity changes accompanying the heterodimerization of leucine zippers are consistent with the view that leucine zippers do not retain alpha-helical conformations in isolation and that the formation of the native coiled-coil alpha-helical dimer is attained through a coupled folding-dimerization mechanism.

Structural basis of the differential binding of the SH3 domains of Grb2 adaptor to the guanine nucleotide exchange factor Sos1.

Grb2-Sos1 interaction, mediated by the canonical binding of N-terminal SH3 (nSH3) and C-terminal SH3 (cSH3) domains of Grb2 to a proline-rich sequence in Sos1, provides a key regulatory switch that relays signaling from activated receptor tyrosine kinases to downstream effector molecules such as Ras. Here, using isothermal titration calorimetry in combination with site-directed mutagenesis, we show that the nSH3 domain binds to a Sos1-derived peptide containing the proline-rich consensus motif PPVPPR with an affinity that is nearly threefold greater than that observed for the binding of cSH3 domain. We further demonstrate that such differential binding of nSH3 domain relative to the cSH3 domain is largely due to the requirement of a specific acidic residue in the RT loop of the beta-barrel fold to engage in the formation of a salt bridge with the arginine residue in the consensus motif PPVPPR. While this role is fulfilled by an optimally positioned D15 in the nSH3 domain, the chemically distinct and structurally non-equivalent E171 substitutes in the case of the cSH3 domain. Additionally, our data suggest that salt tightly modulates the binding of both SH3 domains to Sos1 in a thermodynamically distinct manner. Our data further reveal that, while binding of both SH3 domains to Sos1 is under enthalpic control, the nSH3 binding suffers from entropic penalty in contrast to entropic gain accompanying the binding of cSH3, implying that the two domains employ differential thermodynamic mechanisms for Sos1 recognition. Our new findings are rationalized in the context of 3D structural models of SH3 domains in complex with the Sos1 peptide. Taken together, our study provides structural basis of the differential binding of SH3 domains of Grb2 to Sos1 and a detailed thermodynamic profile of this key protein-protein interaction pertinent to cellular signaling and cancer.

Coupling of folding and DNA-binding in the bZIP domains of Jun-Fos heterodimeric transcription factor.

In response to mitogenic stimuli, the heterodimeric transcription factor Jun-Fos binds to the promoters of a diverse array of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryogenic development and cancer. In so doing, Jun-Fos heterodimer regulates gene expression central to physiology and pathology of the cell in a specific and timely manner. Here, using the technique of isothermal titration calorimetry (ITC), we report detailed thermodynamics of the bZIP domains of Jun-Fos heterodimer to synthetic dsDNA oligos containing the TRE and CRE consensus promoter elements. Our data suggest that binding of the bZIP domains to both TRE and CRE is under enthalpic control and accompanied by entropic penalty at physiological temperatures. Although the bZIP domains bind to both TRE and CRE with very similar affinities, the enthalpic contributions to the free energy of binding to CRE are more favorable than TRE, while the entropic penalty to the free energy of binding to TRE is smaller than CRE. Despite such differences in their thermodynamic signatures, enthalpy and entropy of binding of the bZIP domains to both TRE and CRE are highly temperature-dependent and largely compensate each other resulting in negligible effect of temperature on the free energy of binding. From the plot of enthalpy change versus temperature, the magnitude of heat capacity change determined is much larger than that expected from the direct association of bZIP domains with DNA. This observation is interpreted to suggest that the basic regions in the bZIP domains are largely unstructured in the absence of DNA and only become structured upon interaction with DNA in a coupled folding and binding manner. Our new findings are rationalized in the context of 3D structural models of bZIP domains of Jun-Fos heterodimer in complex with dsDNA oligos containing the TRE and CRE consensus sequences. Taken together, our study demonstrates that enthalpy is the major driving force for a key protein-DNA interaction pertinent to cellular signaling and that protein-DNA interactions with similar binding affinities may be accompanied by differential thermodynamic signatures. Our data corroborate the notion that the DNA-induced protein structural changes are a general feature of the bZIP family of transcription factors.

Evidence that the bZIP domains of the Jun transcription factor bind to DNA as monomers prior to folding and homodimerization.

The Jun oncoprotein belongs to the AP1 family of transcription factors that is collectively engaged in diverse cellular processes by virtue of their ability to bind to the promoters of a wide spectrum of genes in a DNA sequence-dependent manner. Here, using isothermal titration calorimetry, we report detailed thermodynamics of the binding of bZIP domain of Jun to synthetic dsDNA oligos containing the TRE and CRE consensus promoter elements. Our data suggest that binding of Jun to both sites occurs with indistinguishable affinities but with distinct thermodynamic signatures comprised of favorable enthalpic contributions accompanied by entropic penalty at physiological temperatures. Furthermore, anomalously large negative heat capacity changes observed provoke a model in which Jun loads onto DNA as unfolded monomers coupled with subsequent folding and homodimerization upon association. Taken together, our data provide novel insights into the energetics of a key protein-DNA interaction pertinent to cellular signaling and cancer. Our study underscores the notion that the folding and dimerization of transcription factors upon association with DNA may be a more general mechanism employed in protein-DNA interactions and that the conventional school of thought may need to be re-evaluated.

Grb2 adaptor undergoes conformational change upon dimerization.

Grb2 is an adaptor protein that couples activated receptor tyrosine kinases to downstream effector molecules such as Ras and Akt. Despite being a central player in mitogenic signaling and a target for therapeutic intervention, the role of Grb2 oligomerization in cellular signaling is not well understood. Here, using the techniques of size-exclusion chromatography, mass spectrometry, analytical ultra-centrifugation and isothermal titration calorimetry, we demonstrate that Grb2 exists in monomer-dimer equilibrium in solution and that the dissociation of dimer into monomers is entropically-driven without an unfavorable enthalpic change at physiological temperatures. Our data indicate that enthalpy and entropy of dimer dissociation are highly temperature-dependent and largely compensate each other resulting in negligible effect of temperature on the overall free energy. From the plot of enthalpy change versus temperature, the magnitude of heat capacity change derived is much smaller than that expected from the rather large molecular surfaces becoming solvent-occluded upon Grb2 dimerization, implying that Grb2 monomers undergo conformational rearrangement upon dimerization. 3D structural models of Grb2 dimer and monomers suggest strongly that such conformational rearrangement upon dimerization may arise from domain swapping. Taken together, our study provides novel insights into the role of Grb2 as an adaptor in cellular signaling circuitry and how Grb2 dimerization may impart high fidelity in signal transduction as well as lead to rapid signal amplification upon receptor stimulation.