Probing the Role of Backbone Hydrogen Bonds in Protein–Peptide
Interactions by Amide-to-Ester Mutations
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Abstract
One of the most frequent
protein–protein interaction modules
in mammalian cells is the postsynaptic density 95/discs large/zonula
occludens 1 (PDZ) domain, involved in scaffolding and signaling and
emerging as an important drug target for several diseases. Like many
other protein–protein interactions, those of the PDZ domain
family involve formation of intermolecular hydrogen bonds: C-termini
or internal linear motifs of proteins bind as β-strands to form
an extended antiparallel β-sheet with the PDZ domain. Whereas
extensive work has focused on the importance of the amino acid side
chains of the protein ligand, the role of the backbone hydrogen bonds
in the binding reaction is not known. Using amide-to-ester substitutions
to perturb the backbone hydrogen-bonding pattern, we have systematically
probed putative backbone hydrogen bonds between four different PDZ
domains and peptides corresponding to natural protein ligands. Amide-to-ester
mutations of the three C-terminal amides of the peptide ligand severely
affected the affinity with the PDZ domain, demonstrating that hydrogen
bonds contribute significantly to ligand binding (apparent changes
in binding energy, ΔΔ<i>G</i> = 1.3 to >3.8
kcal mol<sup>–1</sup>). This decrease in affinity was mainly
due to an increase in the dissociation rate constant, but a significant
decrease in the association rate constant was found for some amide-to-ester
mutations suggesting that native hydrogen bonds have begun to form
in the transition state of the binding reaction. This study provides
a general framework for studying the role of backbone hydrogen bonds
in protein–peptide interactions and for the first time specifically
addresses these for PDZ domain–peptide interactions