INSIGHTS INTO SPECIFICITY OF CLEAVAGE AND MECHANISM OF CELL ENTRY FROM THE CRYSTAL-STRUCTURE OF THE HIGHLY SPECIFIC ASPERGILLUS RIBOTOXIN, RESTRICTOCIN

Background: Restrictocin, a highly specific ribotoxin made by the fung us Aspergillus restrictus, cleaves a single phosphodiester bond in the 28S RNA of eukaryotic ribosomes, inhibiting protein synthesis, The se quence around this cleavage site is a binding site for elongation fact ors, and is conserved in all cytoplasmic ribosomes. The catalytic mech anism of restrictocin and the reasons for its high substrate specifici ty are unknown, No structure has been determined for any other member of the Aspergillus ribotoxin family, Results: The crystal structure of restrictocin was determined at 2.1 Angstrom resolution by single isom orphous replacement and anomalous scattering techniques, and refined t o 1,7 Angstrom resolution using synchrotron Laue data, The structural core of the protein, in which a three-turn a helix is packed against a five-stranded antiparallel beta sheet, can be well aligned with that of ribonuclease T1. Large positively charged peripheral loops near the active site construct a platform with a concave surface for RNA bindi ng, Conclusions: Restrictocin appears to combine the catalytic compone nts of T1 ribonucleases with the base recognition components of Sa rib onucleases. Modeling studies using an NMR structure of an RNA substrat e analog suggest that the tertiary structure of the substrate RNA is i mportant in protein-RNA recognition, fitting closely into the concavit y of the presumed binding site. We speculate that the large 39-residue loop L3, which has similarities to loops found in lectin sugar-bindin g domains, may be responsible for restrictocin's ability to cross cell membranes.