Mistletoe lectin I forms a double trefoil structure

Abstract

The quaternary structure of mistletoe lectin I (MLI), a type II ribosome inactivating protein, has been determined by X-ray crystallography. A definitive molecular replacement solution was determined for MLI using the co-ordinates of the homologue ricin as a search model. MLI exists as an [AB] P dimer with internal crystallographic two-fold symmetry. Domain I of the B chains is non-covalently associated through interactions involving three looped chains (K K, L L, Q Q) in each molecule of the dimer, forming a double trefoil structure. The ricin molecule which shares 52% sequence homology with MLI has a disulphide bridge between Cys PH and Cys QW in the K K loop. An evolutionary mutation has replaced Cys QW with serine in MLI. This mutation appears to allow the K K loop the flexibility required to take up its place at the dimer interface, and also suggests a rationale for why ricin does not form dimers. Measurement of retention times using FPLC gel filtration confirms that dimerisation also occurs in solution between MLI B chains with an association constant K = 10 T M. z 1998 Federation of European Biochemical Societies.

Small angle neutron scattering as sensitive tool to detect ligand-dependent shape changes in a plant lectin with β-trefoil folding and their dependence on the nature of the solvent

“…In line with literature data [13,20,31] and our gel filtration analysis of aliquots run in parallel the lectin consistently formed (AB) 2 dimers (A = toxin subunit, B = lectin subunit) under these conditions. This confirms that the dimer structure seen in crystals [13,14] was not due to a crystal packing force. The effect of ligand binding on SANS spectra at a VAA concentration of 5.9 mg/mL in PB is shown in Fig.…”

“…Initial evidence was provided for an alteration of the aggregation status of a plant agglutinin/ toxin when using water and the aprotic solvent dimethyl sulfoxide (DMSO) as solvent [12]. Of note, the studied galactoside-specific plant lectin has a β-trefoil folding [13,14]. It thus differs markedly from the galectin with its antiparallel β-strand pattern [15].…”

Mistletoe lectin A‐chain unfolds during the intracellular transport

“…This explains the competitive ELISA data that the high-a¤nity binding with TA72 and TA73 monAbs prevented binding with other monAbs but could not be more than 35% suppressed by any of them. TA7 epitope is 101^105, FTGTT, and also partly exposed on the protein surface [9]. This epitope is not present in RTA sequence.…”

“…The protein loop 93^108 is highly immunogenic and is involved in various epitopes. This loop is not hidden by B-subunit in holotoxin [9]. The region overlaps with the octapeptides binding TA7 monAb (98^108) and weakly binds with TA71, TA76 and TA75 monAbs.…”

“…Thus, hybridoma cell resistance is due to intracellular interaction of toxins with newly synthesized antibody molecules.Antibody speci¢city was assayed with native, denatured and adsorbed on plate MLA, ML, ricin and ricin A-chain (RTA). Ricin and RTA were used as controls, since ricin and ML are similar in intracellular pathway, mechanism of action, amino acid sequence and tertiary structure [9,13]. All monAbs had comparable a¤nities for MLA adsorbed on a plate.…”