Crystal structure at 3 Å of mistletoe lectin I, a dimeric type‐II ribosome‐inactivating protein, complexed with galactose

Abstract

The X-ray structure of mistletoe lectin I (MLI), a type-II ribosome-inactivating protein (RIP), cocrystallized with galactose is described. The model was refined at 3.0 Å resolution to an R-factor of 19.9% using 21 899 reflections, with R free 24.0%. MLI forms a homodimer (A-B) 2 in the crystal, as it does in solution at high concentration. The dimer is formed through contacts between the N-terminal domains of two B-chains involving weak polar and nonpolar interactions. Consequently, the overall arrangement of sugar-binding sites in MLI differs from those in monomeric type-II RIPs: two N-terminal sugar-binding sites are 15 Å apart on one side of the dimer, and two C-terminal sugarbinding sites are 87 Å apart on the other side. Galactose binding is achieved by common hydrogen bonds for the two binding sites via hydroxy groups 3-OH and 4-OH and hydrophobic contact by an aromatic ring. In addition, at the N-terminal site 2-OH forms hydrogen bonds with Asp27 and Lys41, and at the C-terminal site 3-OH and 6-OH undergo water-mediated interactions and C5 has a hydrophobic contact. MLI is a galactose-specific lectin and shows little affinity for N-acetylgalactosamine. The reason for this is discussed. Structural differences among the RIPs investigated in this study (their quaternary structures, location of sugar-binding sites, and fine sugar specificities of their B-chains, which could have diverged through evolution from a two-domain protein) may affect the binding sites, and consequently the cellular transport processes and biological responses of these toxins.

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

“…However, the crystal structures of the lactose complexes with RIPs disclosed only two functionally active sugar-binding sites, each of which did not have any extended sugar binding site (34,36). A comparative analysis of the structures of 1␤-site in HmRIP, ML-I, and ricin revealed that the overall architecture of the site is similar in all of them (Fig.…”

“…8A). The galactose is shifted slightly toward the Trp 34 in a way that the non-polar face makes very strong stacking interactions with the Trp ring, so much so that the electron density of the sugar is continuous with that of the Trp ring even at the 2 cutoff in a ͉2F o Ϫ F c ͉ map. The Asp19 O␦1 and O␦2 form strong H-bonds with galactose O3 and O2 in HmRIP (Fig.…”

“…HmRIP, ML-I, and ebulin have a Thr at this site. In ebulin, the sugar has a direct interaction with the Thr because of its deep seated localization (37); however, in ML-I it is a water-mediated interaction (34). In HmRIP also the interaction is possible through a water molecule, which could not be located because of limitation of the resolution.…”

“…The affinity of ricin for Nacetyl galactosamine has been associated with its proposed binding through Ser 238 from the kink region; however, the substitution of Ala for Ser in ML-I causes steric hindrance, resulting in its reduced affinity for N-acetyl galactosamine (34). The deletion of the corresponding residue in HmRIP has led to the widening of the pocket that provided sufficient space to accommodate the N-acetyl group.…”