Ribosome-inactivating proteins (RIPs) such as Shiga-like toxin 1 (SLT-1) halt protein synthesis in eukaryotic cells by depurinating a single adenine base in the sarcin-ricin loop of 28S rRNA. The molecular details involved in the ER lumenal escape and subsequent site-specific depurination are lacking, despite a general understanding of the biochemical basis of SLT-1 toxicity. Using a combination of yeast-2-hybrid and HeLa lysate pull-down followed by LC-MS/MS we have discovered yeast and human proteins that interact with the catalytic A1 chain of SLT-1. Yeast-2-hybrid library screens followed by the expression of full-length protein candidates and pull-down experiments yielded Cue2 as the only yeast cellular component that binds to the SLT-1 A1 chain. Further truncational analysis revealed that the known protein domains (two Cue domains and a Smr domain) within the primary sequence of Cue 2 were not essential for the interaction. Cue2 is a yeast monoubiquitin binding protein of no known function that is structurally homologous to the human ubiquitin-associated domain which has been implicated in intracellular routing and ER-associated degradation. Pull-down experiments indicated that the mechanism by which the catalytic domain of RIPs cleaves its substrate involves initial docking interactions with the ribosomal stalk by virtue of a conserved acidic C-terminal peptide domain common to all three stalk proteins P0, P1, and P2. The A1 chain of SLT-1 transiently binds to this peptide with a modest binding constant and rapid on and off rates. Mutagenesis of charged residues within the A1 chain identified a cationic surface that interacts with the peptide motif. In addition, phage-display was used to rapidly probe the importance of each residue within this C-terminal ribosomal peptide. The analysis revealed a complementary acidic surface and an additional hydrophobic motif involved in the interaction. Moreover, deletion mutagenesis performed on the ribosomal protein P0 revealed that the A1 chain binds to an alternate site on P0 in proximity to the contact sites for P1/P2 heterodimers. These results demonstrate that the catalytic chain of RIPs such as SLT-1 dock on ribosomes using two classes of binding sites located within the ribosomal stalk which may aid in orienting their catalytic domain in close proximity to the depurination site.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/32046 |
| Date | 18 January 2012 |
| Creators | McCluskey, Andrew |
| Contributors | Gariepy, Jean |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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