2.8‐Å crystal structure of a nontoxic type‐II ribosome‐inactivating protein, ebulin l

Abstract

Ebulin l is a type-II ribosome-inactivating protein (RIP) isolated from the leaves of Sambucus ebulus L. As with other type-II RIP, ebulin is a disulfide-linked heterodimer composed of a toxic A chain and a galactoside-specific lectin B chain. A normal level of ribosome-inactivating N-glycosidase activity, characteristic of the A chain of type-II RIP, has been demonstrated for ebulin l. However, ebulin is considered a nontoxic type-II RIP due to a reduced cytotoxicity on whole cells and animals as compared with other toxic type-II RIP like ricin. The molecular cloning, amino acid sequence, and the crystal structure of ebulin l are presented and compared with ricin. Ebulin l is shown to bind an A-chain substrate analogue, pteroic acid, in the same manner as ricin. The galactoside-binding ability of ebulin l is demonstrated crystallographically with a complex of the B chain with galactose and with lactose. The negligible cytotoxicity of ebulin l is apparently due to a reduced affinity for galactosides. An altered mode of galactoside binding in the 2gamma subdomain of the lectin B chain primarily causes the reduced affinity.

Structure-Function Analysis and Insights into the Reduced Toxicity of Abrus precatorius Agglutinin I in Relation to Abrin

“…abrin (8), ricin (9), mistletoe lectin (16), ebulin (17), and Himalayan RIP (18). The complete amino acid sequence of APA-I has been determined (19) .…”

Crystal Structure of Himalayan Mistletoe Ribosome-inactivating Protein Reveals the Presence of a Natural Inhibitor and a New Functionally Active Sugar-binding Site

“…The respective nitrogen interacts with O4 in ML-I and ebulin and O4 and O6 in ricin. N of Lys 37 , which comes from the ␤I-␤II segment, makes strong H-bonds with galactose O2 in HmRIP and O2 and O3 in ricin and ML-I. The Lys 37 also makes important interactions with Asn 43 and stabilizes the conformation of this key sugarbinding residue.…”

“…The Lys 37 also makes important interactions with Asn 43 and stabilizes the conformation of this key sugarbinding residue. The corresponding Lys is mutated to Gly in ebulin (37). In addition, the deletion of two residues shortens the ␤I-␤II segment in ebulin.…”

“…As discussed, three structural features have been proposed to date as being essential for a functionally active sugar-binding site (36,37). A detailed analysis of the active sugar-binding sites of RIPs suggests the importance of another structural feature pertaining to the ␤I-␤II segment connecting the two ␤-sheets of the subdomain.…”

Structure and Function of Carbohydrate-Binding Module Families 13 and 42 of Glycoside Hydrolases, Comprising a β-Trefoil Fold

“…The ricin B lectin family is also referred to as a ricin-type or R-type lectin family, or as a (Gln-X-Trp) 3 -domain family, 27) with consensus motif Gly-X-X-X-Gln-X-Trp(Tyr). 28) Several lectins with a -trefoil fold, such as abrin and eburin, specifically bind D-galactose (hereafter galactose) or galactose-derivatives, [29][30][31][32][33][34][35][36][37][38] whereas other lectins preferentially bind other carbohydrates. 39,40) Nevertheless, the CBM13 domains have not yet been described in lectins.…”