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Binding of the nuclear factor-Y complex (NF-Y) to the inverted CCAAT-box interferes with transcription activation through nucleosome reorganization. The three homologous proteins forming the zinc-fingers and homeoboxes (ZHX) family interact with the activation domain of NF-Ya to repress transcription. Each ZHX-protein contains two generic C2H2 zinc-fingers (ZNF1 and ZNF2) followed by five homeodomains. Although the proteins have been related to the occurrence of certain cancers, the function and structure of the individual ZHX domains are still unknown. Here, we determined the structure of the tandem zinc-finger region of human ZHX1. Folding and secondary structure predictions combined with expression screening revealed that the C-terminal extension (E) to ZNF2 could form a single domain with the two hZHX1 zinc-fingers. We therefore decided to determine the solution structure of the zinc-fingers followed by this extension. We show that both zinc-fingers adopt canonical betabetaalpha-folds in which a zinc ion is coordinated by two cysteine and two histidine residues. The C-terminal extension to ZNF2 forms two beta-strands to make a beta-sheet with the beta-strands of this zinc-finger. The ZNF1 and ZNF2-E domains do not show evident contacts and their mutual orientation seems variable. The high degree of sequence conservation among ZHX family members permitted us to prepare homology models for ZHX2 and ZHX3, revealing distinct surface characteristics for each family member. Implications of these structural features for ZHX-functioning in transcription regulation are discussed.

Proteins frequently contain unstructured regions apart from a functionally important and well-conserved structured domain. Functional and structural aspects for these regions are frequently less clear. The general human positive cofactor 4 (PC4), has such a domain organization and can interact with various DNA substrates, transcriptional activators, and basal transcription factors. While essential for the cofactor function, structural and functional knowledge about these interactions is limited. Using biochemical, nuclear magnetic resonance (NMR), and docking experiments, we show that the carboxy-terminal structured core domain (PC4ctd) is required and sufficient for binding to single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and the herpes simplex virion protein 16 (VP16) activation domain (VP16ad). We determined the interaction surfaces within PC4 and showed that VP16 and DNA binding are mutually exclusive. Although the amino-terminal domain of PC4 (PC4ntd) alone is devoid of any bioactivity, it increases the interaction with VP16ad. While it decreases the ssDNA-binding and DNA-unwinding activity, it does not influence dsDNA binding. Structural characterization of this domain showed that it is highly flexible and mostly unstructured both in the free form and in the complex. NMR titration experiments using various protein and DNA substrates of the individual domains and the full-length PC4 revealed local conformational or environmental changes in both the structured and unstructured subdomains, which are interpreted to be caused by inter- and intramolecular interactions. We propose that the unstructured PC4ntd regulates the PC4 cofactor function by specific interactions with the activator and through modulation and/or shielding of the interaction surface in the structured core of PC4ctd.

Herpes simplex virion protein 16 (VP16) contains two strong activation regions that can independently and cooperatively activate transcription in vivo. We have identified the regions and residues involved in the interaction with the human transcriptional coactivator positive cofactor 4 (PC4) and the general transcription factor TFIIB. NMR and biochemical experiments revealed that both VP16 activation regions are required for the interaction and undergo a conformational transition from random coil to alpha-helix upon binding to its target PC4. The interaction is strongly electrostatically driven and the binding to PC4 is enhanced by the presence of its amino-terminal domain. We propose models for binding of VP16 to the core domains of PC4 and TFIIB that are based on two independent docking approaches using NMR chemical shift changes observed in titration experiments. The models are consistent with results from site-directed mutagenesis and provide an explanation for the contribution of both acidic and hydrophobic residues for transcriptional activation by VP16. Both intrinsically unstructured activation domains are attracted to their interaction partner by electrostatic interactions, and adopt an alpha-helical conformation around the important hydrophobic residues. The models showed multiple distinct binding surfaces upon interaction with various partners, providing an explanation for the promiscuous properties, cooperativity, and the high activity of this activation domain.

The retinoid X receptor (RXR) is a prominent member of the nuclear receptor family of ligand-inducible transcription factors. Many proteins of this family exert their function as heterodimers with RXR as a common upstream partner. Studies of the DNA-binding domains of several nuclear receptors reveal differences in structure and dynamics, both between the different proteins and between the free- and DNA-bound receptor DBDs. We investigated the differences in dynamics between RXR free in solution and in complex with a 14 base-pair oligonucleotide, using (1)H and (15)N relaxation studies. Nano- to picosecond dynamics were probed on (15)N, employing Lipari-Szabo analysis with an axially symmetric tumbling model to estimate the exchange contributions to the transverse relaxation rates. Furthermore, milli- to microsecond dynamics were estimated qualitatively for (1)H and (15)N, using CPMG-HSQC and CPMG-T(2) measurements with differential pulse spacing. RXR shows hardly any nano- to picosecond time-scale internal motion. Upon DNA binding, the order parameters show a tiny increase. Dynamics in the milli- to microsecond time scale is more prevalent. It is localized in the first and second zinc fingers of the free RXR. Upon DNA-binding, exchange associated with specific/aspecific DNA-binding of RXR is observed throughout the sequence, whereas conformational flexibility of the D-box and the second zinc finger of RXR is greatly reduced. Since this DNA-binding induced folding transition occurs remote from the DNA in a region which is involved in protein-protein interactions, it may very well be related to the cooperativity of dimeric DNA binding.