Ricin A -chain structure, catalysis and immunotoxin synthesis

Abstract

Ricin A-chain is the catalytic monomer of the heterodimeric cytotoxin, Ricin, which depurinates the ribosome at A4324, resulting in ablation of protein translation. The structure of the ribosome in this region is a stem with a GAGA tetra-loop. Short stem-loops with GAGA tetra-loops are also substrates for RTA. Earlier characterization of RTA activity on short stem-loops showed that RNA 10mers (3 base-pair stem) are 10-fold more active as substrates than DNA 10mers. In order to deduce the structural correlates of these phenomena, we kinetically characterized RNA/DNA and RNA/OMeNA hybrid 12mer stem-loops. These replacements resulted in changes of up to 10 4-fold in kcat. K m varied only by 30-fold. RNA/DNA hybrids that bound tighter than A12, the all-RNA stem-loop, were used as templates for transition state inhibitor design and have resulted in the tightest binding one-piece inhibitor known for RTA with an affinity of 30nM. In addition we characterized DNA stem-loops as substrates for RTA. We observed no change in kcat or Km as a function of stem length suggesting that RTA had no catalytic requirement for a stem-loop structure for DNA substrates. Investigation using substrates that were unstructured confirmed that although DNA stem-loop substrates bound tighter to RTA, a stem-loop structure was not required for catalysis of DNA substrates and that RTA's minimal substrate is the DNA 3mer d[GAG]. Characterization of the activity of RNA and RNA-DNA hybrid 4mers, GAGA and GdAGA respectively, showed that change of the reactive residue to DNA increased the activity by 104-fold. The change in the pucker of the reactive residue increases phosphate to phosphate distance allowing RTA binding.