Rajamohan F, Mao C, Uckun F M
Biotherapy Program, Parker Hughes Cancer Center, and the Departments of Protein Engineering, Structural Biology, and Virology, Parker Hughes Institute, St. Paul, Minnesota 55113, USA.
J Biol Chem. 2001 Jun 29;276(26):24075-81. doi: 10.1074/jbc.M011406200. Epub 2001 Apr 19.
Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that catalytically cleaves a specific adenine base from the highly conserved alpha-sarcin/ricin loop of the large ribosomal RNA, thereby inhibiting protein synthesis at the elongation step. Recently, we discovered that alanine substitutions of the active center cleft residues significantly impair the depurinating and ribosome inhibitory activity of PAP. Here we employed site-directed mutagenesis combined with standard filter binding assays, equilibrium binding assays with Scatchard analyses, and surface plasmon resonance technology to elucidate the putative role of the PAP active center cleft in the binding of PAP to the alpha-sarcin/ricin stem loop of rRNA. Our findings presented herein provide experimental evidence that besides the catalytic site, the active center cleft also participates in the binding of PAP to the target tetraloop structure of rRNA. These results extend our recent modeling studies, which predicted that the residues of the active center cleft could, via electrostatic interactions, contribute to both the correct orientation and stable binding of the substrate RNA molecules in PAP active site pocket. The insights gained from this study also explain why and how the conserved charged and polar side chains located at the active center cleft of PAP and certain catalytic site residues, that do not directly participate in the catalytic deadenylation of ribosomal RNA, play a critical role in the catalytic removal of the adenine base from target rRNA substrates by affecting the binding interactions between PAP and rRNA.
商陆抗病毒蛋白（PAP）是一种核糖体失活蛋白，它能催化从大核糖体RNA高度保守的α-肌动蛋白/蓖麻毒素环上切割下一个特定的腺嘌呤碱基，从而在延伸步骤抑制蛋白质合成。最近，我们发现活性中心裂隙残基的丙氨酸取代显著损害了PAP的脱嘌呤和核糖体抑制活性。在此，我们采用定点诱变结合标准滤膜结合试验、基于Scatchard分析的平衡结合试验以及表面等离子体共振技术，以阐明PAP活性中心裂隙在PAP与rRNA的α-肌动蛋白/蓖麻毒素茎环结合中的假定作用。本文所呈现的研究结果提供了实验证据，表明除了催化位点外，活性中心裂隙也参与PAP与rRNA靶四环结构的结合。这些结果扩展了我们最近的建模研究，该研究预测活性中心裂隙的残基可通过静电相互作用，有助于底物RNA分子在PAP活性位点口袋中的正确定向和稳定结合。从这项研究中获得的见解还解释了为什么以及PAP活性中心裂隙处保守的带电和极性侧链以及某些不直接参与核糖体RNA催化去腺苷化的催化位点残基如何通过影响PAP与rRNA之间的结合相互作用，在从靶rRNA底物催化去除腺嘌呤碱基中发挥关键作用。
