Abstract
Both ricin and Shiga holotoxins display no ribosomal activity in their native forms and need to be activated to inhibit translation in a cell-free translation inhibition assay. This is because the ribosome binding site of the ricin A chain (RTA) is blocked by the B subunit in ricin holotoxin. However, it is not clear why Shiga toxin 1 (Stx1) or Shiga toxin 2 (Stx2) holotoxin is not active in a cell-free system. Here, we compare the ribosome binding and depurination activity of Stx1 and Stx2 holotoxins with the A1 subunits of Stx1 and Stx2 using either the ribosome or a 10-mer RNA mimic of the sarcin/ricin loop as substrates. Our results demonstrate that the active sites of Stx1 and Stx2 holotoxins are blocked by the A2 chain and the B subunit, while the ribosome binding sites are exposed to the solvent. Unlike ricin, which is enzymatically active, but cannot interact with the ribosome, Stx1 and Stx2 holotoxins are enzymatically inactive but can interact with the ribosome.
Highlights
Shiga-toxin producing E. coli (STEC) infections can cause life threatening complications such as hemolytic uremic syndrome (HUS) or hemorrhagic colitis (HC), and are the leading cause of death from foodborne bacterial infections in children [1]
Our results demonstrate that the active sites of Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) holotoxins are blocked by the A2 chain and the B subunit, while the ribosome binding sites are exposed to the solvent
Our recent study showed that point mutations at Arg172 or Arg176 located on the opposite face of the active site (Figures 1 and 3) disrupted the at Arg172 or Arg176 located on the opposite face of the active site (Figures 1 and 3) disrupted the ribosome and ribosomal stalk interaction of Stx1A1 and Stx2A1, but did not affect their depurination ribosome and ribosomal stalk interaction of Stx1A1 and Stx2A1, but did not affect their depurination activity on RNA, indicating siteswere were intact were located the opposite activity on RNA, indicatingthat thattheir their active active sites intact andand were located on theon opposite face of face of thethe ribosome
Summary
Shiga-toxin producing E. coli (STEC) infections can cause life threatening complications such as hemolytic uremic syndrome (HUS) or hemorrhagic colitis (HC), and are the leading cause of death from foodborne bacterial infections in children [1]. Shiga toxins (Stxs) are the primary virulence factors of STEC and belong to a group of proteins called type II ribosome inactivating proteins (RIPs). Stx and Stx contain a catalytically active A subunit and five copies of cell binding B subunits [2,3]. Stx and Stx share 55% and 57% amino sequence identity in the A and B subunits, respectively, and have similar molecular structures. The A subunits remove a universally conserved adenine from the sarcin/ricin loop (SRL) of the large ribosomal RNA and inhibit translation
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