Fine-tuning of prototype chicken galectins: structure of CG-2 and structure–activity correlations

Abstract

The comparatively small number of members of the family of adhesion/growth-regulatory galectins in chicken predestines this system as an attractive model to study the divergence of these lectins after gene duplication. Expression profiling of the three homodimeric (prototype) chicken galectins (CG-1A, CG-1B and CG-2) has raised evidence of distinct functionalities, explaining the interest in a detailed crystallographic analysis of CG-2. As revealed here, marked differences are found in the ligand-binding site and in the contact pattern within the homodimer interface, underlying a characteristic orientation of the two subunits. Notably, a distinctive trimer of dimers that is unique in all galectin crystal structures reported to date forms the core unit of the crystallographic assembly. Combination with spectroscopic and thermodynamic measurements, and comparisons with CG-1A and CG-1B, identify differential changes in the circular-dichroism spectra in the presence of lactose, reflecting the far-reaching impact of the ligand on hydrodynamic behaviour, and inter-galectin differences in both the entropy and the enthalpy of binding. This structural information is a salient step to complete the analysis of the full set of galectins from this model organism.

Chicken GRIFIN: Structural characterization in crystals and in solution

“…It is about 1.3 for C-GRIFIN and CG-1A but 1.63 for CG-2 [36]. Of note for CG-2, heterogeneity in size distribution had been observed for this protein at 4 mg/mL, with the acquisition of the quaternary structure as trimer of dimers [37]. We proceeded to determine the quaternary structure of C-GRIFIN at concentrations up to 9.0 mg/mL.…”

Galectin-related protein: An integral member of the network of chicken galectins 1. From strong sequence conservation of the gene confined to vertebrates to biochemical characteristics of the chicken protein and its crystal structure

“…Such a sensitivity of quaternary structure on the environment had been reported in the case of human galectin-1 and its appearance as dimer of dimers in an aprotic solvent [61]. In terms of the root-mean-square difference (rmsd) for positions of the Cα atoms, values between 1.07 Å and 1.54 Å relative to the structures of CG-8N (1.07 Å; [62]), CG-2 (1.33 Å; [63]), CG-1A (1.42 Å; [64]) and CG-1B (1.54 Å; [65]) underscore the close relationship. The superimpositions of the C-GRP-C fold with that of the listed canonical CGs, however, disclosed notable differences in several regions involving loops that are between adjacent β-strands in the concave face of the groove, the site of accommodation of lactose in canonical CGs (Fig.…”

“…Looking at the equivalent of the contact site for lactose in canonical CGs, the sequence signature for operative binding (His45, Asn47, Arg49, Asn58, Trp65, Glu68 and Arg70 for example in CG-2 [63]) is turned into Glu49, Lys51, Val53, Asn62, Trp69, Glu72 and Ser74 in C-GRP-C. Only three of seven positions are thus maintained (Fig. 7).…”

Combining Crystallography and Hydrogen–Deuterium Exchange to Study Galectin–Ligand Complexes

“…The five canonical chicken galectins( CGs), classified into homodimeric (prototype) (CG-1A, CG-1B, CG-2), chimera-type (CG-3), and tandem-repeat-type (CG-8) proteins, meet this requirement, [6][7][8][9][10] and crystallographic analysiso ft he prototype proteins has already been achieved. [11][12][13] The next step is the study of CG-8 with its two carbohydrate recognitiond omains separatedb yal inker of either 9o r2 8a minoa cids, physiologically an antagonisto fC G-1A in patterning of precartilage condensations in the developing limb. [9,14] Because homodimeric galectins respond to binding lactose with ac hange in the gyration radius (first measured for human Gal-1 by small-angle neutron scattering [15] )o rd iffusion constant (detected for humanG al-1 and CG-1B/2 by fluorescence correlation spectroscopy [16,17] ), we combined crystallographic analysis with the sensorc apacity of hydrogen-deuterium exchange (HDX) in solution,i ntroducingi ts application to lectin research.…”