No abstract

The enzymatic subunit of Shiga toxin (StxA1) is a member of the ribosome-inactivating protein (RIP) family, which includes the ricin A chain as well as other examples of plant toxins. StxA1 catalytically depurinates a well-conserved GAGA tetra-loop of 28S rRNA which lies in the acceptor site of eukaryotic ribosomes. The specific activities of native StxA1, as well as mutated forms of the enzyme with substitutions in catalytic site residues, were measured by an in vitro translation assay. Electroporation was developed as an alternative method for the delivery of purified A1 polypeptides into Vero cells. Site-directed mutagenesis coupled with N-bromosuccinimide modification indicated that the sole tryptophan residue of StxA1 is required for binding it to the 28S rRNA backbone. Northern analysis established that the catalytic site substitutions reduced enzymatic activity by specifically interfering with the capacity of StxA1 to depurinate 28S rRNA. Ribosomes were protected from StxA1 by molar excesses of tRNA and free adenine, indicating that RIPs have the capacity to enter the acceptor site groove prior to binding and depurinating the GAGA tetra-loop.Shiga toxin (STX) is a multimeric molecule consisting of a single 32-kDa enzymatic A subunit (StxA) noncovalently associated with a ring of five 7.7-kDa B subunits (StxB) (17). The StxB subunits mediate the binding of toxin to its receptor on eukaryotic membranes, and the entire STX molecule enters cells via endocytosis from coated pits (28). StxA is processed in the trans-Golgi network by the cellular protease furin (11) to release StxA1, which possesses the enzymatic activity, and StxA2, which acts as a bridge between StxA1 and the B pentamer. StxA1 is an N-glycosidase which catalytically removes a specific adenine from a well-conserved tetra-loop of 28S rRNA and thus inhibits protein synthesis by preventing the binding of aminoacyl tRNAs to the ribosome and halting protein elongation (7). StxA1 is a member of the ribosome-inactivating protein (RIP) family of enzymes, which are a large group of protein synthesis inhibitors primarily found in the leaves, seeds, and roots of dicotyledonous plants (5,7,30). The only RIPs identified so far which are not produced by plants are the members of the STX family of toxins.The catalytic sites of StxA1 have been identified by sequence comparison of RIP family members and site-specific mutagenesis. Amino acid substitutions in three regions of StxA1 which are highly conserved among the RIPs identified tyrosine 77, glutamic acid 167, arginine 170, and tryptophan 203 as important for catalytic activity (2,16,33). Corresponding mutations in another RIP, the A chain of the plant lectin ricin (RTA), corroborated the findings with StxA1 (24, 29).The X-ray crystal structures of StxA1 and RTA have been solved at 2.5 Å (10, 20). Comparison of these structures revealed an active site cleft which contains all of the catalytic site residues previously identified by site-specific mutagenesis. Xray crystallographic analysis of StxA1 and RTA als...