CH-pi Interactions Confer Orientational Flexibility in Protein-Carbohydrate Binding Sites

Protein-carbohydrate binding is essential for biological function and is driven by non-covalent interactions, including hydrogen bonds and CH-pi stacking interactions. CH-pi stacking interactions are favorable and have high orientational flexibility; however, we lack a comprehensive understanding of the roles that they play in protein binding affinity and selectivity. To evaluate the role of CH-pi stacking interactions and their interplay with hydrogen bonds, we curated a set of proteins bound to B-D-galactoside-containing carbohydrates with varied numbers and orientations of CH-pi stacking interactions, numbers of hydrogen bonds, and lengths of carbohydrate ligands. We employ well-tempered metadynamics simulations to obtain binding free energy landscapes. We show that the free energy landscapes of each protein are broad and that the favored CH-pi stacking interaction orientation varies depending on the conformation of the protein binding site. Complexes with extended carbohydrate ligands that form additional hydrogen bonds resulted in more specific orientational dependence. Conversely, complexes with mutations that decreased the number of hydrogen bonds had the opposite effect, broadening the free energy landscape significantly and resulting in the occupation of multiple distinct CH-pi stacking interaction orientations. Thus, hydrogen bonds play a key role in defining the binding orientation. We next show that forming multiple CH-pi stacking interactions facilitates the dynamics necessary for the translocation of polysaccharide ligands within a protein binding site. Our work shows the cooperative nature of hydrogen bonds and CH-pi stacking interactions, demonstrating that tuning the number and positions of these interactions through protein engineering should alter ligand recognition or support ligand movement in protein binding sites.