Characterization of the dimer interface of transcription factor NFkappaB p50 homodimer

Dimers of the Rel/NFkappaB transcription factor family form with differential stabilities through the combinatorial association of five polypeptides: p50, p52, p65, cRel, and RelB. Here, we have characterized the nature of the monomer-dimer equilibrium of the p50 homodimer. Sedimentation equilibrium studies show that the equilibrium constant for p50 dimer dissociation is in the low micromolar range. Using the X-ray crystal structure of the p50 homodimer as a guide, we have created site-directed alanine mutations at ten dimer-forming residues in p50 and measured their effects on p50 homodimerization. Characterization of these alanine mutants by a series of chemical crosslinking, size-exclusion chromatography, and sedimentation equilibrium experiments shows that the most critical residue in stabilizing the p50 dimer interface is Y267. Sedimentation equilibrium experiments show that an alanine substitution at position 267 destabilizes the dimer interface by 2.0 kcal/mol. Alanine substitutions at two other positions, L269 and V310, significantly destabilize the p50 dimer interface. These two residues are observed to mediate critical interactions in the crystal structure. Together, these three residues constitute the "hot-spot" of protein-protein interaction in p50 dimerization. Of the four charged residues in the dimer interface, R252, D254, E265, and D302, only D302 contributes significantly to p50 dimer stability. D254 appears to slightly destabilize the subunit interface. Although residues H304, R305, and F307 occupy positions at the hydrophobic core of the interface and appear to be involved in multiple interactions in the X-ray crystal structure, alanine substitutions at these positions do not significantly reduce the affinity for p50 dimerization. Upon evaluating the roles of these amino acid residues at the p50 dimer interface, we propose that differential contributions of a few key residues dictate the selectivity of dimer formation within the Rel/NFkappaB family.