Crystal structures of ebulin, a non-toxic ribosome -inactivating protein.

摘要

Ebulin is a 56 kilodalton, type II ribosome-inactivating protein (RIP) isolated from the leaves of the dwarf elder (Scanbucus ebulus). RIPs are N-glycosidases that act on a specific adenine of 28S ribosomal RNA. Removing this adenine prevents the ribosome from associating with elongation factor II. This action terminates protein synthesis, and cell death occurs. Type II RIPS are two-chain cytotoxins: an N-glycosidase A chain that is disulfide-linked to a lectin B chain that enhances cell uptake by a strong association with cell surface glycoproteins. Some endocytosed toxin is delivered to the cytosol, where the A chain inactivates the ribosome. Type II RIPS are some of the most potent cytotoxins known to man. The extreme cytotoxicity of these proteins has been harnessed in two notable yet contradictory ways: as therapeutic immunotoxins and as agents of murder by poisoning.;Despite showing N-glycosidase activity comparable to other RIPS, ebulin is essentially nontoxic to animals and whole cells and is classified as a non-toxic, type II RIP. The negligible toxicity of ebulin is surprising given that the protein appears to have the required attributes of a functional type II RIP: N-glycosidase activity and binding of cell surface galactosides. A comparison of the three-dimensional structure of ebulin with other type II RIPs was undertaken to better understand its behavior and to better understand RIP function in general.;Two crystal structures of ebulin are presented: one based on an orthorhombic crystal form, and one based on a trigonal crystal form. The trigonal crystal form is shown bound to pteroic acid, an A chain substrate analog. The mode of galactoside binding is demonstrated with complexes of the trigonal crystal form with lactose and galactose. These structures are compared to the structure of ricin, the archetype of the toxic, type II RIP protein family. Ricin and ebulin's amity for a dense matrix of lactose is also compared. The negligible cytotoxicity of ebulin is apparently due to a reduced amity for complex saccharides as compared to ricin. The reduction in affinity is caused by slight alterations in the B chain of ebulin in an area that is a known galactoside-binding domain in ricin.