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 non‐polar 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 sugar‐binding 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.

Apoptotic cascades may frequently be impaired in tumor cells; therefore, the approaches to circumvent these obstacles emerge as important therapeutic modalities. Our novel derivatives of chlorin e₆, that is, its amide (compound 2) and boronated amide (compound 5) evoked no dark toxicity and demonstrated a significantly higher photosensitizing efficacy than chlorin e₆ against transplanted aggressive tumors such as B16 melanoma and M-1 sarcoma. Compound 5 showed superior therapeutic potency. Illumination with red light of mammalian tumor cells loaded with 0.1 µM of 5 caused rapid (within the initial minutes) necrosis as determined by propidium iodide staining. The laser confocal microscopy-assisted analysis of cell death revealed the following order of events: prior to illumination, 5 accumulated in Golgi cysternae, endoplasmic reticulum and in some (but not all) lysosomes. In response to light, the reactive oxygen species burst was concomitant with the drop of mitochondrial transmembrane electric potential, the dramatic changes of mitochondrial shape and the loss of integrity of mitochondria and lysosomes. Within 3–4 min post illumination, the plasma membrane became permeable for propidium iodide. Compounds 2 and 5 were one order of magnitude more potent than chlorin e₆ in photodamage of artificial liposomes monitored in a dye release assay. The latter effect depended on the content of non-saturated lipids; in liposomes consisting of saturated lipids no photodamage was detectable. The increased therapeutic efficacy of 5 compared with 2 was attributed to a striking difference in the ability of these photosensitizers to permeate through hydrophobic membrane interior as evidenced by measurements of voltage jump-induced relaxation of transmembrane current on planar lipid bilayers. The multimembrane photodestruction and cell necrosis induced by photoactivation of 2 and 5 are directly associated with membrane permeabilization caused by lipid photodamage.