Department of Biochemistry and Molecular Biology, Indiana University School of Medicine-Simon Cancer Center, Indianapolis, Indiana 46032.
Department of Pathology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461.
J Biol Chem. 2011 Nov 4;286(44):38264-38274. doi: 10.1074/jbc.M111.277012. Epub 2011 Sep 8.
Ribosomal protein L11 (RPL11) has been shown to activate p53 by binding to MDM2 and negating its p53 suppression activity in response to ribosomal stress. Although a mutation at Cys-305 within the zinc finger domain of MDM2 has been shown to drastically impair MDM2 interaction with RPL11 and thus escapes the inhibition by this ribosomal protein, it still remains elusive whether RPL11 inactivates MDM2 via direct action on this zinc finger domain and what is the chemical nature of this specific interaction. To define the roles of the MDM2 zinc finger in association with RPL11, we conducted hydrogen-deuterium exchange mass spectrometry, computational modeling, circular dichroism, and mutational analyses of the zinc finger domain of MDM2 and human RPL11. Our study reveals that RPL11 forms a stable complex with MDM2 in vitro through direct contact with its zinc finger. This binding is disrupted by single mutations of non-cysteine amino acids within the zinc finger domain of MDM2. Basic residues in RPL11 are crucial for the stable binding and RPL11 suppression of MDM2 activity toward p53. These results provide the first line of evidence for the specific interaction between RPL11 and the zinc finger of MDM2 via hydrophilic residues as well as a molecular foundation for better understanding RPL11 inhibition of MDM2 function.
核糖体蛋白 L11（RPL11）已被证明通过与 MDM2 结合并消除其对核糖体应激的 p53 抑制活性来激活 p53。虽然已经表明 MDM2 锌指结构域内的 Cys-305 突变会严重损害 MDM2 与 RPL11 的相互作用，从而逃避这种核糖体蛋白的抑制，但仍然不清楚 RPL11 是否通过直接作用于该锌指结构域使 MDM2 失活，以及这种特定相互作用的化学性质是什么。为了确定 MDM2 锌指在与 RPL11 相关联中的作用，我们进行了氢氘交换质谱、计算建模、圆二色性和 MDM2 及人 RPL11 锌指结构域的突变分析。我们的研究表明，RPL11 通过与锌指直接接触，在体外与 MDM2 形成稳定的复合物。这种结合会被锌指结构域中 MDM2 的非半胱氨酸氨基酸的单个突变所破坏。RPL11 中的碱性残基对于稳定结合和 RPL11 抑制 MDM2 对 p53 的活性至关重要。这些结果为 RPL11 与 MDM2 的锌指之间通过亲水性残基的特异性相互作用提供了第一手证据，也为更好地理解 RPL11 抑制 MDM2 功能提供了分子基础。
