Sengchanthalangsy L L, Datta S, Huang D B, Anderson E, Braswell E H, Ghosh G
Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0359, USA.
J Mol Biol. 1999 Jun 18;289(4):1029-40. doi: 10.1006/jmbi.1999.2823.
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.
Rel/NFκB转录因子家族的二聚体通过五种多肽（p50、p52、p65、cRel和RelB）的组合关联形成，其稳定性各不相同。在此，我们对p50同二聚体的单体 - 二聚体平衡的性质进行了表征。沉降平衡研究表明，p50二聚体解离的平衡常数处于低微摩尔范围内。以p50同二聚体的X射线晶体结构为指导，我们在p50中十个形成二聚体的残基处创建了定点丙氨酸突变，并测量了它们对p50同二聚化的影响。通过一系列化学交联、尺寸排阻色谱和沉降平衡实验对这些丙氨酸突变体进行表征，结果表明，稳定p50二聚体界面的最关键残基是Y267。沉降平衡实验表明，267位的丙氨酸取代使二聚体界面的稳定性降低了2.0千卡/摩尔。另外两个位置L269和V310的丙氨酸取代显著破坏了p50二聚体界面的稳定性。在晶体结构中观察到这两个残基介导关键相互作用。这三个残基共同构成了p50二聚化中蛋白质 - 蛋白质相互作用的“热点”。在二聚体界面的四个带电荷残基R252、D254、E265和D302中，只有D302对p50二聚体稳定性有显著贡献。D254似乎略微破坏了亚基界面的稳定性。尽管残基H304、R305和F307位于界面的疏水核心位置，并且在X射线晶体结构中似乎参与多种相互作用，但这些位置的丙氨酸取代并未显著降低对p50二聚化的亲和力。在评估这些氨基酸残基在p50二聚体界面中的作用后，我们提出少数关键残基的不同贡献决定了Rel/NFκB家族中二聚体形成的选择性。
