Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication

Abstract

Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by β-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.

Figure 1. Thermofluor assays.

(a) Normalized thermal denaturation curves for galectin-4, galectin-4N and galectin-4C. Measured apparent unfolding temperatures were 55.92 ± 0.05 °C for galectin-4, 56.8 ± 0.1 °C for galectin-4N and 68.12 ± 0.05 °C for galectin-4C. (b) Evaluation of thermal shift profile for galectin-4, galectin-4N and galectin-4C at different categories of additives. Bars show all additives that contribute to interpretable transitions with positive and/or negative thermal shift for the three proteins. Compounds and the respective thermal shift values are listed in Supplementary Table S1. (c) Thermal shift profile as function of lactose concentration.

Figure 2. Crystal structures of galectin-4N and galectin-4C.

(a) Overall β-sandwich fold of galectin-4N (blue) and galectin-4C (pink) structures. The antiparallel β-sheets are shown in blue (F0-F5) and cyan (S1-S6a/b) for galectin-4N, and pink (F0′-F5′) and light pink (S1′-S6a′) for galectin-4C. (b) Electrostatic potential surface for both the galectin-4N and galectin-4C structures. Front view (β-sheet S1-S6/S1′-S6′) and back view (β-sheet F0-F5/F0′-F5′). The circle marks the canonical binding site. (c) Canonical (pink) and extended (yellow) binding sites of galectin-4 domains. The main residues involved in binding interactions are represented as sticks. (d) Sequence alignment of galectin-4N and galectin-4C showing secondary structures elements. Marked in bold are the conserved residues. Highlighted in pink are the residues of canonical carbohydrate-binding site; the star is the only conservative substitution in the binding site residues between both domains. In yellow are the extended binding site residues.

Figure 3. Model of full-length galectin-4.

(a) Cartoon representation of the initial model for full-length protein (b) Overall fold of galectin-4 model after equilibrium dynamics and geometry optimization. (c) Representation of inter-domain interactions mediated by hydrogen bonds.

Figure 4. Solution conformation of full-length galectin-4 examined by X-ray scattering.

(a) The experimental scattering of galectin-4 in the absence of ligand (gray) is well fit by the theoretical scattering of the full-length model in Fig. 3b (solid line), confirming that the two CRDs associate in solution. In contrast, a comparison of the experimental scattering to the theoretical scattering of the model found in Fig. 3a in which the CRDs are non-associating (dotted), shows a poor fit. (b) An ab initio shape reconstruction generated from ligand-free galectin-4 scattering data also shows good agreement with the full-length model. (c) Addition of lactose leads to a subtle expansion in the width of the pair-distance distribution function, P(r), and a slight increase in radius of gyration.

Figure 5. RMSD plots for molecular dynamics simulation with (+lactose) and without (−lactose) lactose, 150 ns trajectories.

RMSD by domains structure (a) (−lactose) and (b) (+lactose). (c) RMSF box chart for MD simulation without and with lactose and cartoon putty representation of mobility through trajectory (inset); the blue-white-magenta scale calculated B-factor from 0 to 250 Ų. Porcupine plot of the first eigenvector generated through principal component analysis of the representative structure with lactose in (d) front view and (e) bottom view. The vectors, represented as blue arrows, show the tendency of movement. Plot of atomic correlations of MD without lactose (f) with lactose (g). The correlated movements are shown in pink and anticorrelated movements in blue scale bar. The bars indicate the portion of the graph relating to each domain, white for galectin-4N, light gray for linker and dark gray for galectin-4C.