Involvement of Laser Photo‐CIDNP(Chemically Induced Dynamic Nuclear Polarization)‐Reactive Amino Acid Side Chains in Ligand Binding by Galactoside‐Specific Lectins in Solution

Abstract

For proteins in solution the validity of certain crystallographic parameters can be ascertained by a combination of molecular-dynamics (MD) simulations and NMR spectroscopy. Using the laser photo-CIDNP (chemically induced dynamic nuclear polarization) technique as a measure for surface accessibility of histidine, tyrosine and tryptophan, the spectra of bovine galectin-I and Erythrina corallodendron lectin (EcorL) are readily reconcilable with the crystallographic data for these two proteins. The results emphasise the role of Trp68/Trp69 for carbohydrate binding in bovine galectin-l/chicken galectins and of Trp194 in murine galectin-3. This feature derived from the crystal structure of' bovine galectin-I is maintained in solution for the prototype human homologue, two avian galectins and the chimera-type murine galectin-3, as the spectra corroborate the CIDNP-inferable spatial parameters of the four calculated models for binding-site architecture. In EcorL, Tyr106/Tyr108 are constituents of the extended combining pocket, which can be shielded in solution by ligand presence. Discrepancies between results from modelling and CIDNP measurements concern primarily the lack of reactivity of histidine residues for human and avian prototype galectins and of Tyr82lTyr229 of the plant lectin. Site-directed mutagenesis of EcorL is assumed to provide information on the role of a certain residue for functional aspects. When singlesite mutants of EcorL ([Alal06]EcorL, [Alal08]EcorL, [Ala229]EcorL) were subjected to moleculardynamics (MD) simulations, the apparent surface accessibilities even of spatially separated amino acid side chains could non-uniformly be affected. This conclusion is supported by the assessment of the spectra for the mutant proteins. On the basis of these CIDNP-results modelling of the binding-site architecture of the lectin indicates the occurrence of notable alterations in the orientation of Tyr106/Tyr108 phenyl rings. The implied potential effect of single-site mutations on conformational features of a protein will deserve attention for the interpretation of studies comparing wild-type and mutant proteins.Keywords: galectin ; lectin ; Erythrina agglutinin ; NMR; molecular modeling.X-ray crystallography of proteins provides detailed information on three-dimensional structure. The precision of the derived spatial parameters of the protein in the crystal, however, should not be considered as proof for the solution structure. Among other factors, the often applied non-physiological conditions to favour crystal growth and the intermolecular interactions during packaging may lead to deviations from the solution structure(s) especially for surface group features (Wagner et al., 1992;MacArthur et al., 1994). Consequently, it is desirable to employ a sensitive spectroscopic method which in combination with mo- lecular-dynamics (MD) simulations can verify the applicability of distinct properties determined by crystallographic analysis for the solution structure. With a focus on surface epitopes, whose mobility can...

Galectin-1 Is a Major Receptor for Ganglioside GM1, a Product of the Growth-controlling Activity of a Cell Surface Ganglioside Sialidase, on Human Neuroblastoma Cells in Culture

“…Purification and Sources of the Lectins-The galactoside-binding lectins from V. album L. and galectin-1 from human lung were purified by affinity chromatography on lactosylated Sepharose 4B and further chromatographic steps, as described (35,36). Traces of contaminants such as galactoside-binding immunoglobulin G were removed from galectin-1 by ion exchange chromatography on DEAE-Sepharose CL-6B and by subsequent affinity chromatography on protein A-Sepharose CL-4B.…”

Activity–structure correlations in divergent lectin evolution: fine specificity of chicken galectin CG-14 and computational analysis of flexible ligand docking for CG-14 and the closely related CG-16

“…Modeling initial contact formation between ligand and lectin was guided by the evidence that the CGs maintain the characteristic way a ligand's central galactose unit resides in the vicinity of the Trp moiety of the CRD (Siebert et al 1997;. In detail, the distance restraints for defining the relative positions of the binding partners suitable to drive the simulations were derived from inspection of the mode of interaction between human galectin-1 and galactose as part of lactose in the crystal state (PDB code 1W6M), using the LigPlot program (Wallace et al 1995).…”

“…Evidently, neither the common b-sandwich fold nor the position of the Trp moiety in the central part of the carbohydrate recognition domain (CRD) is impaired by these sequence changes (Siebert et al 1997;Varela et al 1999). Thus, the basic mode of interaction of these two galectins with the b-galactoside core of a glycan is maintained.…”

Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy

“…[11][12][13][14] This member of the family of adhesion/growth-regulatory galectins contains a single Trp residue in position 68 within the carbohydrate recognition domain (CRD). 15,16 It is of interest for a case study as target for inhibitor design, because it is involved in tumor invasion, e.g.…”