Studies on the molecular recognition of synthetic methyl β‐lactoside analogs by ricin, a cytotoxic plant lectin

Abstract

The binding of methyl P-lactoside and of all possible monodeoxy derivatives of methyl P-lactoside to the galactose-specific highly cytotoxin lectin ricin, has been investigated. The distribution of low-energy conformers of the disaccharide structures has been first determined using molecular-mechanics calculations and high-resolution NMR spectroscopy. The nuclear Overhauser enhancements and specific deshieldings observed are in agreement with a similar distribution of low-energy conformers for all studied compounds which may be described by a major conformer defined by cp (H1'-C1'-01'-C4) and w (Cl'-Ol'-C4-H4) torsion angles of 49" and 5", respectively, with contribution of conformers with angles cp/w 24"/ -59", 22"/ -32 " and 6"/ -44". Assuming that the disaccharides bind to the lectin in these preferred conformations, the apparent dissociation constants for the ricindisaccharide complexes have been interpreted in terms of specific polar and nonpolar interactions. In agreement with X-ray data, the hydroxyl groups at positions 3, 4 and 6 of the P-D-galactopyranose moiety appear as key polar groups in the interaction with ricin. These results are in contrast to previous results which have established that position 6 is not involved in lectin binding. An important nonpolar interaction involving position 3 of the P-D-glucopyranose moiety, seems to be operative. The distribution of low-energy conformers of these disaccharide structures permits this interaction to take place with the hydroxyl group at this position intramolecularly bonded, thus rendering this region of the molecule more lipophylic in character for acceptance into nonpolar regions of the combining site.The origin of the specificity observed in the molecular recognition of carbohydrates by lectins and antibodies has been suggested to include crucial polar interactions, involving a cluster of two or more hydroxyl groups, and short range multipoint van der Waals interactions between complementary nonpolar surfaces. These nonpolar interactions would play an important role on the setting of the stability of the complex while polar interactions within the combining site would be the key to complex formation [I, 21. On the basis of highly refined X-ray structures of some carbohydrate-protein complexes it has been concluded that both hydrogen bonds and van der Waals contacts are indeed the dominant forces that stabilize carbohydrate-protein interactions, although it has been anticipated that the former would provide the major contributions to the binding [3, 41. Binding studies with synthetic carbohydrate molecules having a range of slightly modified structures have proved extremely fruitful to probe the combining site [I, 2, 5-91, A proper interpretation of the results from these studies together with highly refined crystallographic data may be of paramount importance to achieve an appreciation of the origin of the specific interactions.This paper is concerned with the molecular recognition of synthetic methyl P-lactoside analogs by ricin, the highly Ricin i...

Hydrogen‐bonding pattern of methyl β‐lactoside binding to the Ricinus communis lectins

“…As determined by NMR and molecular mechanics calculations [28] (and unpublished results), the distribution of low-energy conformers for the different methyl p-lactoside analogs studied was very similar. Thus, replacement of the hydroxyl group by hydrogen, fluorine or a methoxy group does not substantially modify the average conformation.…”

“…As determined by NMR and molecular mechanics calculations [28], the preferred conformations of the derivatives were very similar. Therefore, the relative affinity of the lectins for the different structures could be correlated with specific polar and nonpolar interactions.…”

“…The values obtained are given in Table 1. The data previously obtained [28,291 for the eight monodeoxy derivatives of methyl p-lactoside are also included for comparative purposes.…”

“…For all the analogs the plot of the reciprocal of the lectin fraction bound to Sepharose 6B versus the sugar concentration gave a straight line with a main correlation coefficient of 0.988. The apparent dissociation constants were calculated from the intercept and the slope of this line, being K d = (intercept-l)/slope [28,291. The values obtained are given in Table 1.…”

“…The affinity of the lectins for the sugar derivatives was estimated by determining the amount of 12'I-lectin bound to Sepharose 6B in the presence of different concentrations of the sugar, as described previously [28,291. The standard assay was carried out in polypropylene microcentrifuge tubes, in 5 mM sodium phosphate, pH 7.2, containing 0.2 M NaC1.…”