Abstract：
The thermodynamics of secondary p53 binding sites on MDM2 and MDMX were evaluated using p53 peptides containing residues 16-29, 17-35, and 1-73. All the peptides had large, negative heat capacity (ΔCp), consistent with the burial of p53 residues F19, W23, and L26 in the primary binding sites of MDM2 and MDMX. MDMX has a higher affinity and more negative ΔCp than MDM2 for p5317-35, which is due to MDMX stabilization and not additional interactions with the secondary binding site. ΔCp measurements show binding to the secondary site is inhibited by the disordered tails of MDM2 for WT p53 but not a more helical mutant where proline 27 is changed to alanine. This result is supported by all-atom molecular dynamics simulations showing that p53 residues 30-35 turn away from the disordered tails of MDM2 in P27A17-35 and make direct contact with this region in p5317-35. Molecular dynamics simulations also suggest that an intramolecular methionine-aromatic motif found in both MDM2 and MDMX structurally adapts to support multiple p53 binding modes with the secondary site. ΔCp measurements also show that tighter binding of the P27A mutant to MDM2 and MDMX is due to increased helicity, which reduces the energetic penalty associated with coupled folding and binding. Our results will facilitate the design of selective p53 inhibitors for MDM2 and MDMX.
摘要：
使用含有残基16-29、17-35和1-73的p53肽评估MDM2和MDMX上的次级p53结合位点的热力学。所有的肽都很大，负热容量(ΔCp)，与MDM2和MDMX的主要结合位点中p53残基F19，W23和L26的埋藏一致。对于p5317-35，MDMX比MDM2具有更高的亲和力和更负的ΔCp，这是由于MDMX稳定而不是与次级结合位点的额外相互作用。ΔCp测量显示，WTp53的MDM2无序尾巴抑制了与次要位点的结合，但脯氨酸27变为丙氨酸的更螺旋的突变体却没有抑制。该结果得到全原子分子动力学模拟的支持，表明p53残基30-35从P27A17-35中MDM2的无序尾部移开，并与p5317-35中的该区域直接接触。分子动力学模拟还表明，在MDM2和MDMX中发现的分子内甲硫氨酸-芳族基序在结构上适应于支持与次要位点的多种p53结合模式。ΔCp测量还表明，P27A突变体与MDM2和MDMX的更紧密结合是由于螺旋度增加，这减少了与耦合折叠和结合相关的能量惩罚。我们的结果将有助于设计MDM2和MDMX的选择性p53抑制剂。
