Nucleic Acids Research Advance Access originally published online on February 18, 2007
Nucleic Acids Research 2007 35(5):1660-1672; doi:10.1093/nar/gkm065
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Nucleic Acids Research, 2007, Vol. 35, No. 5 1660-1672
© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses
1Department of Biochemistry, Centre for Protein Science and Crystallography and Molecular Biotechnology Programme, The Chinese University of Hong Kong, Shatin, Hong Kong, China, 2Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China and 3Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, New Territories, Hong Kong, China
*To whom correspondence should be addressed. Tel: 852 2609 8024; Fax: 852 2603 7732; Email: kbwong{at}cuhk.edu.hk Correspondence may also be addressed to Pang-Chui Shaw. Tel: 852 2609 6803; Fax: 852 2603 5123; Email: pcshaw{at}cuhk.edu.hk
Received October 31, 2006. Revised December 21, 2006. Accepted January 22, 2007.
Trichosanthin (TCS) is a type I ribosome-inactivating protein that inactivates ribosome by enzymatically depurinating the A4324 at the 
-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.
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