Probing backbone hydrogen bonding in PDZ/ligand interactions by protein amide-to-ester mutations.
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.
Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Søren W
SW
Pedersen
Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Stine B
SB
Pedersen
Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Louise
L
Anker
Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
Greta
G
Hultqvist
Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Anders S
AS
Kristensen
Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden.
Per
P
Jemth
Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Kristian
K
Strømgaard
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D000076122:Disks Large Homolog 4 Protein; D004952:Esters / Q000737:chemistry; D006860:Hydrogen Bonding; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D054731:PDZ Domains; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry
D000577:Amides; C501444:DLG4 protein, human; D000076122:Disks Large Homolog 4 Protein; D004952:Esters; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C121001:NR2B NMDA receptor; D011506:Proteins; D016194:Receptors, N-Methyl-D-Aspartate