Date: Thursday, June 12, 2025
Time: 2:00 – 3:00 PM
Room: 66-110
CSB Ph.D. Candidate: Allison Keys
Supervisor: Heather Kulik (ChemE) & Laura Kiessling (Chemistry)
TDC Members:
Professors Amy Keating, Adam Willard, and Yu-Shan Lin (External)
Title: The Role of CH–π Interactions in Protein-Carbohydrate Binding
Abstract: Protein-carbohydrate binding is essential for biological processes, including cellular recognition and immune signaling. Binding is driven by several types of non-covalent interactions: hydrogen bonding, metal ion coordination, and the less well-understood CH–π interactions. CH–π interactions are pervasive in protein-carbohydrate binding sites and have emerged as critical drivers of protein–carbohydrate recognition; however, the energetics of CH–π stacking interactions, their orientational landscapes, and their interplay with other non-covalent interactions have been unclear.
In this thesis, I identified carbohydrate-aromatic CH–π stacking interactions from crystallographic structures in the Protein Data Bank. I performed quantum mechanical calculations to quantify interaction energies and found that CH–π stacking interactions can be more favorable than hydrogen bonds. Using atomistic simulations, I also demonstrated that CH–π stacking interactions are necessary for human galectin-3 binding to lactose. To assess the orientational landscape of CH–π stacking interactions, I evaluated the orientations of CH–π stacking interactions formed by β-D-galactose and found that numerous orientations are highly favorable. I then identified carbon atom distances that define an orientational landscape for these interactions. To assess the interplay between non-covalent interactions in protein-carbohydrate binding sites, I used CH–π distance features to bias metadynamics simulations of a curated set of protein–β-D-galactoside complexes. From these simulations, I found that while bound carbohydrates sample many CH–π stacking orientations, the hydrogen bonds in the protein binding site drive the optimal orientation of each ligand. Longer carbohydrate ligands with more hydrogen bonding constraints have more specific orientational dependence, while ligands in binding sites with a reduced number of hydrogen bonds occupy a broader range of orientations. Unlike hydrogen bonds, CH–π stacking interactions confer orientational flexibility: enzymes can exploit multiple CH–π stacking interactions to facilitate the translocation of polysaccharide substrates. Extending this analysis to other carbohydrates, I showed that carbohydrate stereochemistry drives the orientational preferences of CH–π stacking interactions; however, there is also a tradeoff between the presence of hydrogen bonds to charged amino acids and the CH–π interaction strength for each carbohydrate. Overall, this thesis demonstrates that CH–π interactions are favorable and confer high orientational flexibility and that hydrogen bonds act in concert with CH–π interactions to stabilize protein-carbohydrate binding. Tuning the number and positions of these interactions through protein engineering should alter protein selectivity and ligand movement in protein binding sites.
