Abstract
The design of allosteric modulators to control protein function is a key objective in drug discovery programs. Altering functionally essential allosteric residue networks provides unique protein family subtype specificity, minimizes unwanted off-target effects, and helps avert resistance acquisition typically plaguing drugs that target orthosteric sites. In this work, we used protein engineering and dimer interface mutations to positively and negatively modulate the immunosuppressive activity of the proapoptotic human galectin-7 (GAL-7). Using the PoPMuSiC and BeAtMuSiC algorithms, mutational sites and residue identity were computationally probed and predicted to either alter or stabilize the GAL-7 dimer interface. By designing a covalent disulfide bridge between protomers to control homodimer strength and stability, we demonstrate the importance of dimer interface perturbations on the allosteric network bridging the two opposite glycan-binding sites on GAL-7, resulting in control of induced apoptosis in Jurkat T cells. Molecular investigation of G16X GAL-7 variants using X-ray crystallography, biophysical, and computational characterization illuminates residues involved in dimer stability and allosteric communication, along with discrete long-range dynamic behaviors involving loops 1, 3, and 5. We show that perturbing the protein–protein interface between GAL-7 protomers can modulate its biological function, even when the overall structure and ligand-binding affinity remains unaltered. This study highlights new avenues for the design of galectin-specific modulators influencing both glycan-dependent and glycan-independent interactions.
Highlights
Human galectins (GAL) are homodimeric β-galactosidase-binding lectins assembled from small (~15 kDa) protomeric carbohydrate recognition domains (CRD)
Some reports have alluded to the potential importance of interface residues involved in dimer formation and stability, in addition to proposing the existence of allosteric networks connecting the two distant glycan binding sites (GBS) sites on opposite GAL-7 protomers [35]
As a counterpart to destabilizing mutations, we 5.2-6.7 μM). These results suggest that residue Gly16 is searched for mutational predictions that favored stabiliza- directly involved in monomer-dimer stabilization and/or tion of the GAL-7 homodimer rather than its monomeric allosteric communication between protomers in GAL-7
Summary
Human galectins (GAL) are homodimeric β-galactosidase-binding lectins assembled from small (~15 kDa) protomeric carbohydrate recognition domains (CRD). An increasing number of studies have confirmed the importance of GBS-independent activities modulated by galectins [22,23,24], including potentially relevant hetero-oligomeric galectin architectures, modular designs, and valence variability [25,26,27,28] This should come as no surprise, as it has been known for a while that lectins can bind non-carbohydrate compounds, often exhibiting higher affinities than their ‘natural’ saccharide ligands [29]. Targeting non-GBS regions in galectins would offer means to develop new generations of galectin inhibitors that modulate glycan-independent functions in the cell, a therapeutic strategy that remains marginally represented In support of this avenue, galectins have been shown to undergo evolutionary pressure that stabilizes their quaternary oligomeric architecture to improve ligand affinity and biological function [33]. Biophysical, structural, and computational characterization of G16X variants provides a clearer view of the allosteric network governing molecular function in GAL-7
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