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The paralogous multifunctional adaptor proteins YAP and TAZ are the nuclear effectors of the Hippo pathway, a central mechanism of organ size control and stem cell self-renewal. WW domains, mediators of protein-protein interactions, are essential for YAP and TAZ function, enabling interactions with PPxY motifs of numerous partner proteins. YAP has single and double WW domain isoforms (YAP1 and YAP2) whereas only a single WW domain isoform of TAZ has been described to date. Here we identify the first example of a double WW domain isoform of TAZ. Using NMR, we have characterized conformational features and peptide binding of YAP and TAZ tandem WW domains (WW1-WW2). The solution structure of YAP WW2 confirms that it has a canonical three-stranded antiparallel β-sheet WW domain fold. While chemical shift-based analysis indicates that the WW domains in the tandem WW pairs retain the characteristic WW domain fold, 15N relaxation data show that, within the respective WW pairs, YAP WW1 and both WW1 and WW2 of TAZ undergo conformational exchange. 15N relaxation data also indicate that the linker between the WW domains is flexible in both YAP and TAZ. Within both YAP and TAZ tandem WW pairs, WW1 and WW2 bind single PPxY-containing peptide ligand concurrently and noncooperatively with sub-mM affinity. YAP and TAZ WW1-WW2 bind a dual PPxY-containing peptide with approximately 6-fold higher affinity. Our results indicate that both WW domains in YAP and TAZ are functional and capable of enhanced affinity binding to multi-PPxY partner proteins such as LATS1, ErbB4, and AMOT.

TATA box binding protein (TBP)-promoter interaction nucleates assembly of the RNA polymerase II transcription initiation complex. Transcription factor IIA (TFIIA) stabilizes the TBP-promoter complex whereas the N-terminal domain of the largest TAF(II) inhibits TBP-promoter interaction. We have mapped the interaction sites on TBP of Drosophila TAF(II)230 and yeast TFIIA (comprising two subunits, TOA1 and TOA2), using nuclear magnetic resonance (NMR), and also report structural evidence that subdomain II of the TAF(II)230 N-terminal inhibitory domain and TFIIA have overlapping binding sites on the convex surface of TBP. Together with previous mutational and biochemical data, our NMR results indicate that subdomain II augments subdomain I-mediated inhibition of TBP function by blocking TBP-TFIIA interaction.

General transcription factor TFIID consists of TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s), which together play a central role in both positive and negative regulation of transcription. The N-terminal region of the 230 kDa Drosophila TAF(II) (dTAF(II)230) binds directly to TBP and inhibits TBP binding to the TATA box. We report here the solution structure of the complex formed by dTAF(II)230 N-terminal region (residues 11-77) and TBP. dTAF(II)230(11-77) comprises three alpha helices and a beta hairpin, forming a core that occupies the concave DNA-binding surface of TBP. The TBP-binding surface of dTAF(II)230 markedly resembles the minor groove surface of the partially unwound TATA box in the TBP-TATA complex. This protein mimicry of the TATA element surface provides the structural basis of the mechanism by which dTAF(II)230 negatively controls the TATA box-binding activity within the TFIID complex.

Human TFIIB, an essential factor in transcription of protein-coding genes by RNA polymerase II, consists of an amino-terminal zinc binding domain (TFIIBn) connected by a linker of about 60 residues to a carboxy-terminal core domain (TFIIBc). The TFIIB core domain has two internally repeated motifs, each comprising five alpha-helices arranged as in the cyclin box. Compared to the crystal structure of TFIIBc in complex with TBP and a TATA-containing oligonucleotide, the NMR-derived solution structure of free TFIIBc is more compact, with a different repeat-repeat orientation and a significantly shorter first helix in the second repeat. Analysis of backbone 15N relaxation parameters indicates the presence of relatively large amplitude, nanosecond time-scale motions in the TFIIBc interrepeat linker and structural fluctuations throughout the backbone. Interaction of TFIIBc with the acidic activation domain of VP16 or with TFIIBn induces 1H-15N chemical shift and line width changes concentrated in the first repeat, interrepeat linker and the first helix of the second repeat. These results suggest that TFIIB is somewhat pliable and that the conformation of the C-terminal core domain can be modulated by interaction with the N-terminal zinc binding domain. Furthermore, binding of the VP16 activation domain may promote TFIIBc conformations primed for binding to a TBP-DNA complex.

The complementarity of NMR and X-ray crystallography for biomacromolecular studies has been particularly evident in analysis of transcription factor structures and interactions. While X-ray crystallography can be used to tackle relatively complicated structural problems including multicomponent (three and higher) complexes, NMR studies have provided new insights into the nature of protein-DNA and protein-protein interactions that would be difficult to obtain by other biophysical methods. We describe herein some of the novel and important information recently derived from NMR studies of transcription factors.

The transcription factor TFIID is a multimeric protein complex containing the TATA box-binding polypeptide (TBP) and TBP-associated factors. We have previously reported that the N-terminal regions of dTAFII62 and dTAFII42 have sequence similarities with histones H4 and H3. Here, we demonstrate that the histone-homologous regions of dTAFII62 and dTAFII42 form a heteromeric complex both in vitro and in a yeast two-hybrid system. Neither dTAFII62 nor dTAFII42 forms a homomeric complex, in agreement with a nucleosomal histone character. Moreover, circular dichroism measurements show that the heteromeric complex is dominated by alpha-helical secondary structure. These results strongly suggest the existence of a histone-like surface on TFIID.

TFIIB is an essential component of the machinery that transcribes protein-coding genes. The three-dimensional structure of the human TFIIB core domain (TFIIBc) has been determined using multidimensional heteronuclear magnetic resonance spectroscopy. The molecule consists of two direct repeats that adopt similar alpha-helical folds, conferring pseudo-twofold symmetry. An extensive, central basic surface including an amphipathic alpha helix is critical to the function of TFIIB as a bridge between the TBP-promoter complex and RNA polymerase II and associated general and regulatory transcription factors. Similarities between the TFIIBc and cyclin A folds indicate that elements of the eukaryotic cell cycle control apparatus evolved from more fundamental transcriptional control components, demonstrating a link between the transcription and cell cycle molecular machineries.