Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses.
Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses.
复制标题
通过化学移动扰动和诱变分析，滴虫，核糖体灭活蛋白与核糖体茎蛋白P2之间的相互作用。
DOI：
10.1093/nar/gkm065
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发表时间：
2007
影响因子：
14.9
通讯作者：
Wong, Kam-Bo
中科院分区：
文献类型：
--
作者：
Chan, Denise S B;Chu, Lai-On;Lee, Ka-Ming;Too, Priscilla H M;Ma, Kit-Wan;Sze, Kong-Hung;Zhu, Guang;Shaw, Pang-Chui;Wong, Kam-Bo
天花粉蛋白是一种I型核糖体失活蛋白，它通过酶促28SRRNA的α-sarcin/ricin环上的A4324来灭活核糖体。我们在这项研究和以前的研究中已经证明TCS与人的酸性核糖体蛋白P0、P1和P2相互作用，这些蛋白构成了真核核糖体的侧柄。缺失诱变表明，TCS与P2的C-末端相互作用，其序列在P0、P1和P2中保守。通过化学位移微扰实验，将TCS上的P2结合位点映射到C-末端结构域。扫描电荷对丙氨酸的诱变表明，TCS C-末端结构域的K173、R174和K177参与了与P2的相互作用，可能是通过与P2的C-末端保守的DDD基序形成电荷相互作用。TCS的三个丙氨酸突变体K173A/R174A/K177A在体外不能与P2和核糖体茎结合，但在兔网织红细胞裂解物中抑制翻译的活性降低了18倍，这表明TCS需要与P蛋白相互作用才能发挥充分的活性。与茎蛋白在结合延伸因子中的作用类似，我们认为与酸性核糖体茎蛋白的相互作用有助于TCS定位其RNA底物。
Trichosanthin (TCS) is a type I ribosome-inactivating protein that inactivates ribosome by enzymatically depurinating the A4324 at the α-sarcin/ricin loop of 28S rRNA. We have shown in this and previous studies that TCS interacts with human acidic ribosomal proteins P0, P1 and P2, which constitute the lateral stalk of eukaryotic ribosome. Deletion mutagenesis showed that TCS interacts with the C-terminal tail of P2, the sequences of which are conserved in P0, P1 and P2. The P2-binding site on TCS was mapped to the C-terminal domain by chemical shift perturbation experiments. Scanning charge-to-alanine mutagenesis has shown that K173, R174 and K177 in the C-terminal domain of TCS are involved in interacting with the P2, presumably through forming charge–charge interactions to the conserved DDD motif at the C-terminal tail of P2. A triple-alanine variant K173A/R174A/K177A of TCS, which fails to bind P2 and ribosomal stalk in vitro, was found to be 18-fold less active in inhibiting translation in rabbit reticulocyte lysate, suggesting that interaction with P-proteins is required for full activity of TCS. In an analogy to the role of stalk proteins in binding elongation factors, we propose that interaction with acidic ribosomal stalk proteins help TCS to locate its RNA substrate.