Vikas Bhat

Biophysical basis of the binding of WWOX tumor suppressor to WBP1 and WBP2 adaptors.

The WW-containing oxidoreductase (WWOX) tumor suppressor participates in a diverse array of cellular activities by virtue of its ability to recognize WW-binding protein 1 (WBP1) and WW-binding protein 2 (WBP2) signaling adaptors among a wide variety of other ligands. Herein, using a multitude of biophysical techniques, we provide evidence that while the WW1 domain of WWOX binds to PPXY motifs within WBP1 and WBP2 in a physiologically relevant manner, the WW2 domain exhibits no affinity toward any of these PPXY motifs. Importantly, our data suggest that while R25/W44 residues located within the binding pocket of a triple-stranded β-fold of WW1 domain are critical for the recognition of PPXY ligands, they are replaced by the chemically distinct E66/Y85 duo at structurally equivalent positions within the WW2 domain, thereby accounting for its failure to bind PPXY ligands. Predictably, not only does the introduction of E66R/Y85W double substitution within the WW2 domain result in gain of function but the resulting engineered domain, hereinafter referred to as WW2_RW, also appears to be a much stronger binding partner of WBP1 and WBP2 than the wild-type WW1 domain. We also show that while the WW1 domain is structurally disordered and folds upon ligand binding, the WW2 domain not only adopts a fully structured conformation but also aids stabilization and ligand binding to WW1 domain. This salient observation implies that the WW2 domain likely serves as a chaperone to augment the physiological function of WW1 domain within WWOX. Collectively, our study lays the groundwork for understanding the molecular basis of a key protein-protein interaction pertinent to human health and disease.

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.

Molecular origin of the binding of WWOX tumor suppressor to ErbB4 receptor tyrosine kinase.

The ability of WWOX tumor suppressor to physically associate with the intracellular domain (ICD) of ErbB4 receptor tyrosine kinase is believed to play a central role in downregulating the transcriptional function of the latter. Herein, using various biophysical methods, we show that while the WW1 domain of WWOX binds to PPXY motifs located within the ICD of ErbB4 in a physiologically relevant manner, the WW2 domain does not. Importantly, while the WW1 domain absolutely requires the integrity of the PPXY consensus sequence, nonconsensus residues within and flanking this motif do not appear to be critical for binding. This strongly suggests that the WW1 domain of WWOX is rather promiscuous toward its cellular partners. We also provide evidence that the lack of binding of the WW2 domain of WWOX to PPXY motifs is due to the replacement of a signature tryptophan, lining the hydrophobic ligand binding groove, with tyrosine (Y85). Consistent with this notion, the Y85W substitution within the WW2 domain exquisitely restores its binding to PPXY motifs in a manner akin to the binding of the WW1 domain of WWOX. Of particular significance is the observation that the WW2 domain augments the binding of the WW1 domain to ErbB4, implying that the former serves as a chaperone within the context of the WW1-WW2 tandem module of WWOX in agreement with our findings reported previously. Altogether, our study sheds new light on the molecular basis of an important WW-ligand interaction involved in mediating a plethora of cellular processes.

Binding of the ERα 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.

pH modulates the binding of early growth response protein 1 transcription factor to DNA

The transcription factor early growth response protein (EGR)1 orchestrates a plethora of signaling cascades involved in cellular homeostasis, and its downregulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with an increase in pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as His382 by virtue of the fact that its replacement by nonionizable residues abolishes the pH dependence of the binding of EGR1 to DNA. Notably, His382 inserts into the major groove of DNA, and stabilizes the EGR1–DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, His382 is mainly conserved across other members of the EGR family, implying that histidine protonation–deprotonation may serve as a molecular switch for modulating the protein–DNA interactions that are central to this family of transcription factors. Collectively, our findings reveal an unexpected but a key step in the molecular recognition of the EGR family of transcription factors, and suggest that they may act as sensors of pH within the intracellular environment.

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.

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.

Molecular basis of the binding of YAP transcriptional regulator to the ErbB4 receptor tyrosine kinase

The newly discovered transactivation function of ErbB4 receptor tyrosine kinase is believed to be mediated by virtue of the ability of its proteolytically-cleaved intracellular domain (ICD) to physically associate with YAP2 transcriptional regulator. In an effort to unearth the molecular basis of YAP2-ErbB4 interaction, we have conducted a detailed biophysical analysis of the binding of WW domains of YAP2 to PPXY motifs located within the ICD of ErbB4. Our data show that the WW1 domain of YAP2 binds to PPXY motifs within the ICD in a differential manner and that this behavior is by and large replicated by the WW2 domain. Remarkably, while both WW domains absolutely require the integrity of the PPXY consensus sequence, non-consensus residues within and flanking this motif do not appear to be critical for binding. In spite of this shared mode of binding, the WW domains of YAP2 display distinct conformational dynamics in complex with PPXY motifs derived from ErbB4. Collectively, our study lends new insights into the molecular basis of a key protein-protein interaction involved in a diverse array of cellular processes.