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 A(1) 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 A(1) 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 A(1) 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 A(1) chain, whereas P0, lacking this common C terminus, still binds to the A(1) 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 A(1) 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 A(1) 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 A(1) 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.
McCluskey
Andrew J
AJ
Department of Pharmaceutical Sciences, University of Toronto, Ontario, Canada.
Poon
Gregory M K
GM
Bolewska-Pedyczak
Eleonora
E
Srikumar
Tharan
T
Jeram
Stanley M
SM
Raught
Brian
B
Gariépy
Jean
J
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002457:Cell Extracts / Q000737:chemistry; D017124:Conserved Sequence; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D054683:Ribosome Subunits, Large, Eukaryotic / Q000378:metabolism; D012276:Ricin / Q000378:metabolism; D022622:Shiga Toxin 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D020798:Two-Hybrid System Techniques
D002457:Cell Extracts; D010750:Phosphoproteins; D012269:Ribosomal Proteins; D022622:Shiga Toxin 1; C031937:phosphoprotein P2, ribosomal; C035690:ribosomal phosphoprotein P1; C054675:ribosomal protein P0; D012276:Ricin