FC0054
NK2 homeodomain (NKX3.1)  -  DNA

Biological function
NKX3.1 is a homeodomain (HD) protein belonging to the NK-2 family, a class of transcription factors critical for the development of many organs. NKX3.1 is a prostate tumor suppressor.

Domain organization/sequence features
NKX3.1 possesses a stretch of 10-15 residues enriched in acidic amino acids, the acidic domain (AD), in the flexible, disordered region N-terminal to the HD. Interactions between the N-terminal region of NKX3.1 and its homeodomain affect protein stability and DNA binding.

Structural evidence
NMR signals from a region of NKX3.1 preceding the HD intensify upon binding of NKX3.1 to DNA, an effect most likely due to this region becoming more flexible and mobile. This region, which contains the acidic domain (AD) and SRF interacting (SI) motifs, interacts with the HD but becomes displaced when the HD binds to DNA. The NMR signals for the entire N-terminal region preceding the HD, including the AD and SI motifs, exhibit poor chemical shift dispersion, have strong intensities, and have C^R and C^β chemical shift values typical of a flexible, disordered peptide structure. The AD and SI regions appear to be fully flexible by NMR, even when interacting with the homeodomain.

Biochemical evidence
AD and SI motif interactions might be responsible for the weaker DNA binding previously observed for the N-terminal NKX3.1 (1-184) construct. Consequently, the transcription factors could recruit NKX3.1 via favorable AD or SI interactions, competing with their homeodomain interactions, and thus could increase the strength of binding of NKX3.1 to adjacent DNA sites. The observed interactions were weak, with effective binding constants no stronger than 0.5 mM; however, because the interacting domains are covalently linked, their effective concentration for interaction is predicted to be in the millimolar range. Based on polymer theory, effective concentrations for interactions between domains connected by flexible linkers are predicted to be in the millimolar range, and thus, the weak intramolecular interactions observed here could conceivably modulate or compete with stronger, intermolecular interactions with the NKX3.1 HD.

Mechanism category
competitive binding

Posttranslational modification
Phosphorylation of two threonines in the AD by CK2 was found to affect NKX3.1 protein half-life and blocking CK2 led to proteasomal degradation of NKX3.1. NMR spectroscopy was used to measure and map the interaction of the HD with phosphorylated and non-phosphorylated forms of the AD peptide. The interaction with the phosphorylated AD peptide was considerably stronger (K_d = 0.5 ± 0.2 mM), resulting in large chemical shift perturbations for residues Ser150 and Arg175 in the HD, as well as increase in the HD thermal stability compared to that of the non-phosphorylated form.

Significance
Fuzziness enables combinatorial usage of AD and Si interactions. The implication is that transcription factors could recruit NKX3.1 via favorable AD or SI interactions, competing with their homeodomain interactions, and thus could increase the strength of binding of NKX3.1 to adjacent DNA sites. Because of their high effective concentrations, these weak intra- molecular interactions could effectively compete with sub- micromolar binding constants and concentrations typical of inter- molecular interactions inside cells.