The Catalytic Subunit of Shiga-like Toxin 1 Interacts with Ribosomal Stalk Proteins and is Inhibited by Their Conserved C-Terminal Domain
Shiga-like toxin 1 (SLT-1) is a type II ribosome-inactivating protein; its A1 domain blocks protein synthesis in eukaryotic cells by catalyzing the depurination of a single adenine base in 28 S rRNA. The molecular mechanism leading to this site-specific depurination event is thought to involve inter...
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Veröffentlicht in: | Journal of molecular biology 2008-04, Vol.378 (2), p.375-386 |
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Sprache: | eng |
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Zusammenfassung: | Shiga-like toxin 1 (SLT-1) is a type II ribosome-inactivating protein; its A1 domain blocks protein synthesis in eukaryotic cells by catalyzing the depurination of a single adenine base in 28 S rRNA. The molecular mechanism leading to this site-specific depurination event is thought to involve interactions with eukaryotic ribosomal proteins. Here, we present evidence that the A1 chain of SLT-1 binds to the ribosomal proteins P0, P1, and P2. These proteins were identified from a HeLa cell lysate by tandem mass spectrometry, and subsequently confirmed to bind to SLT-1 A1 chain by yeast-two-hybrid and pull-down experiments using candidate full-length proteins. Moreover, the removal of the last 17 amino acids of either protein P1 or P2 abolishes the interaction with the A1 chain, whereas P0, lacking this common C terminus, still binds to the A1 domain. In vitro pull-down experiments using fusion protein-tagged C-terminal peptides corresponding to the common 7, 11, and 17 terminal residues of P1 and P2 confirmed that the A1 chain of SLT-1 as well as the A chain of ricin bind to this shared C-terminal peptide motif. More importantly, a synthetic peptide corresponding to the 17 amino acid C terminus of P1 and P2 was shown to inhibit the ribosome-inactivating function of the A1 chain of SLT-1 in an in vitro transcription and translation-coupled assay. These results suggest a role for the ribosomal stalk in aiding the A1 chain of SLT-1 and other type II ribosome-inactivating proteins in localizing its catalytic domain near the site of depurination in the 28 S rRNA. |
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ISSN: | 0022-2836 1089-8638 |
DOI: | 10.1016/j.jmb.2008.02.014 |