Interaction of the transactivation domain of B-Myb with the TAZ2 domain of the coactivator p300: molecular features and properties of the complex.

The transcription factor B-Myb is a key regulator of the cell cycle in vertebrates, with activation of transcription involving the recognition of specific DNA target sites and the recruitment of functional partner proteins, including the coactivators p300 and CBP. Here we report the results of detailed studies of the interaction between the transactivation domain of B-Myb (B-Myb TAD) and the TAZ2 domain of p300. The B-Myb TAD was characterized using circular dichroism, fluorescence and NMR spectroscopy, which revealed that the isolated domain exists as a random coil polypeptide. Pull-down and spectroscopic experiments clearly showed that the B-Myb TAD binds to p300 TAZ2 to form a moderately tight (K(d) ~1.0-10 µM) complex, which results in at least partial folding of the B-Myb TAD. Significant changes in NMR spectra of p300 TAZ2 suggest that the B-Myb TAD binds to a relatively large patch on the surface of the domain (~1200 Å(2)). The apparent B-Myb TAD binding site on p300 TAZ2 shows striking similarity to the surface of CBP TAZ2 involved in binding to the transactivation domain of the transcription factor signal transducer and activator of transcription 1 (STAT1), which suggests that the structure of the B-Myb TAD-p300 TAZ2 complex may share many features with that reported for STAT1 TAD-p300 TAZ2.

Helicobacter pylori NikR's interaction with DNA: a two-tiered mode of recognition.

HPNikR is a prokaryotic nickel binding transcription factor found in the virulent bacterium Helicobacter pylori. HPNikR regulates the expression of multiple genes as an activator or repressor, including those involved in nickel ion homeostasis, acid adaptation, and iron uptake. The target operator sequences of the genes regulated by HPNikR do not contain identifiable symmetrical recognition sites, and the mechanism by which HPNikR distinguishes between the genes it regulates is not understood. Using competitive fluorescence anisotropy (FA) and electrophoretic gel mobility shift (EMSA) assays, the interactions between HPNikR and the target operator sequences of the genes directly regulated (ureA, NixA, NikR, Fur OPI, Fur OPII, Frpb4, FecA3, and exbB) were characterized. These studies revealed that HPNikR utilizes a two-tiered mode of DNA recognition by binding to some genes with high affinity and others with low affinity. The genes that are tightly regulated by HPNikR encode proteins that utilize nickel, while those that are less tightly regulated encode other types of proteins. The affinities of low-affinity metal ions for a second metal binding site were determined to be in the micromolar regime, and a contribution of electrostatics to the HPNikR-DNA binding event was determined. Detailed studies of the role of sequence length and identity for the interaction between HPNikR and ureA revealed a specific length requirement for DNA binding.

Characterization of the Helicobacter pylori NikR-P(ureA) DNA interaction: metal ion requirements and sequence specificity.

HPNikR, a prokaryotic nickel binding transcription factor, is found in Helicobacter pylori where it functions as a regulator of multiple genes, including those involved in acid adaptation and nickel ion homeostasis. Particularly important is HPNikR's role in the regulation of the nickel-dependent enzyme urease which is critical for the organism's survival in the acidic environment of the gastric epithelium. The target operator sequences of the genes regulated by HPNikR do not contain any identifiable palindromes, and the exact mechanism(s) of the HPNikR-DNA recognition event is unknown. HPNikR was expressed and purified as a soluble protein containing mixed alpha/beta secondary structure with evidence of a tertiary fold. A direct and competitive fluorescence anisotropy (FA) assay to probe both the metal ion requirements and sequence specificity of HPNikR for PureA, the operator sequence for the urease gene, was developed. FA studies revealed that apo-HPNikR did not bind to PureA while Ni(II)HPNikR bound PureA with nanomolar affinity, but only in the presence of a second metal ion [magnesium, calcium, or manganese(II)], suggesting that HPNikR contains a second, low-affinity metal binding site. Cu(II)HPNikR also exhibited a requirement for a second metal ion to accomplish PureA binding. Removal of a loosely conserved "putative" palindrome sequence in the PureA operator abrogated HPNikR binding. Together, these results support a model of HPNikR-PureA binding in which specific metal ions must be coordinated to high- and low-affinity sites to modulate binding.

Microbial nickel metalloregulation: NikRs for nickel ions.

Nickel is a required co-factor for several microbial enzymes; however, because of its potential toxicity, nickel import and homeostasis must be tightly controlled. Recent biophysical and biochemical studies have revealed that NikR proteins are a new type of metalloregulatory protein that utilize allostery and coordination geometry to sense nickel ions and regulate transcription of genes involved in nickel import and processing. Nickel import into bacteria occurs through either ABC-type transporters (NikABCDE) or HoxN type permeases (NixA). Recent structural evidence suggests that nickel is transported through NikABCDE as a metallophore (akin to a siderophore). Nickel storage is accomplished via the HPN protein, a histidine-rich protein similar to metallothionein.