1 Medicinal Chemistry Research, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet2 Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.3 Department of Drug Design and Pharmacology, Faculty of Pharmaceutical Sciences, Københavns Universitet4 Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.5 Medicinal Chemistry Research, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet6 Department of Drug Design and Pharmacology, Faculty of Pharmaceutical Sciences, Københavns Universitet
PDZ domains are scaffolding modules in protein-protein interactions that mediate numerous physiological functions by interacting canonically with the C-terminus or non-canonically with an internal motif of protein ligands. A conserved carboxylate-binding site in the PDZ domain facilitates binding via backbone hydrogen bonds; however, little is known about the role of these hydrogen bonds due to experimental challenges with backbone mutations. Here we address this interaction by generating semisynthetic PDZ domains containing backbone amide-to-ester mutations and evaluating the importance of individual hydrogen bonds for ligand binding. We observe substantial and differential effects upon amide-to-ester mutation in PDZ2 of postsynaptic density protein 95 and other PDZ domains, suggesting that hydrogen bonding at the carboxylate-binding site contributes to both affinity and selectivity. In particular, the hydrogen-bonding pattern is surprisingly different between the non-canonical and canonical interaction. Our data provide a detailed understanding of the role of hydrogen bonds in protein-protein interactions.