Physical and functional interactions between the prostate suppressor homeoprotein NKX3.1 and serum response factor.

The NKX3.1 transcription factor is an NK family homeodomain protein and a tumor suppressor gene that is haploinsufficient and down-regulated in the early phases of prostate cancer. Like its cardiac homolog, NKX2.5, NKX3.1 acts synergistically with serum response factor (SRF) to activate expression from the smooth muscle gamma-actin (SMGA) gene promoter. Using NMR spectroscopy, three conserved motifs in a construct containing the N-terminal region and homeodomain of NKX3.1 were observed to interact with the MADS box domain of SRF. These motifs interacted both in the absence of DNA and when both proteins were bound to a SMGA promoter DNA sequence. No significant interaction was seen between the homeodomain and SRF MADS box. One of the SRF-interacting regions was the tinman (TN) or engrailed homology-1 motif (EH-1), residues 29-35 (FLIQDIL), which for other NK proteins is the site of interaction with the repressor protein Groucho. A second hydrophobic interacting region was designated the SRF-interacting (SI) motif and included residues 99-105 (LGSYLLD). A third interacting motif was the acidic region adjacent to the SI motif including residues 88-96 (ETLAETEPE). The acidic domain (AD) motif signals also showed strengthening upon the NKX3.1 homeodomain binding to DNA in the absence of SRF, consistent with the acidic region weakly interacting with the homeodomain in the unbound state. The importance of these linear motifs in the transcriptional interaction of NKX3.1 and SRF was demonstrated by targeted mutagenesis of an NKX3.1 expression vector in a SMGA reporter assay. The results implicate the NKX3.1 N-terminal region in regulation of transcriptional activity of this tumor suppressor.

Cardiac-specific Nkx2.5 homeodomain: conformational stability and specific DNA binding of Nkx2.5(C56S).

The cardiac-specific Nkx2.5 homeodomain has been expressed as a 79-residue protein with the oxidizable Cys(56) replaced with Ser. The Nkx2.5 or Nkx2.5(C56S) homeodomain is 73% identical in sequence to and has the same NMR structure as the vnd (ventral nervous system defective)/NK-2 homeodomain of Drosophila when bound to the same specific DNA. The thermal unfolding of Nkx2.5(C56S) at pH 6.0 or 7.4 is a reversible, two-state process with unit cooperativity, as measured by differential scanning calorimetry (DSC) and far-UV circular dichroism. Adding 100 mM NaCl to Nkx2.5(C56S) at pH 7.4 increases T(m) from 44 to 54 +/- 0.2 degrees C and DeltaH from 34 to 45 +/- 2 kcal/mol (giving a DeltaC(p) of approximately 1.2 kcal K(-)(1) mol(-)(1) for homeodomain unfolding). DSC profiles of Nkx2.5 indicate fluctuating nativelike structures at <37 degrees C. Titrations of specific 18 bp DNA with Nkx2.5(C56S) in buffer at pH 7.4 with 100 mM NaCl yield binding constants of 2-6 x 10(8) M(-)(1) from 10 to 37 degrees C and a stoichiometry of 1:1 for homeodomain binding DNA, using isothermal titration calorimetry. The DNA binding reaction of Nkx2.5 is enthalpically controlled, and the temperature dependence of DeltaH gives a DeltaC(p) of -0.18 +/- 0.01 kcal K(-)(1) mol(-)(1). This corresponds to 648 +/- 36 A(2) of buried apolar surface upon Nkx2.5(C56S) binding duplex B-DNA. Thermodynamic parameters differ for Nkx2.5 and vnd/NK-2 homeodomains binding specific DNA. Unbound NK-2 is more flexible than Nkx2.5.

Mutations that affect the ability of the vnd/NK-2 homeoprotein to regulate gene expression: transgenic alterations and tertiary structure.

The importance in downstream target regulation of tertiary structure and DNA binding specificity of the protein encoded by the vndNK-2 homeobox gene is analyzed. The ectopic expression patterns of WT and four mutant vndNK-2 genes are analyzed together with expression of two downstream target genes, ind and msh, which are down-regulated by vndNK-2. Three mutants are deletions of conserved regions (i.e., tinman motif, acidic motif, and NK-2 box), and the fourth, Y54M vndNK-2, corresponds to a single amino acid residue replacement in the homeodomain. Of the four ectopically expressed mutant genes examined, only the Y54M mutation inactivates the ability of the vndNK-2 homeodomain protein to repress ind and msh. The acidic motif deletion mutant slightly reduced the ability of the protein to repress ind and msh. By contrast, both tinman and NK-2 box deletion mutants behaved as functional vndNK-2 genes in their ability to repress ind and msh. The NMR-determined tertiary structures of the Y54M vndNK-2 homeodomain, both free and bound to DNA, are compared with the WT analog. The only structural difference observed for the mutant homeodomain is in the complex with DNA and involved closer interaction of the methionine-54 with A2, rather than with C3 of the (-) strand of the DNA. This subtle change in the homeodomain-DNA complex resulted in modifications of binding affinities to DNA. These changes resulting from a single amino acid residue replacement constitute the molecular basis for the phenotypic alterations observed on ectopic expression of the Y54M vndNK-2 gene during embryogenesis.