Probing backbone hydrogen bonding in PDZ/ligand interactions by protein amide-to-ester mutations

Abstract

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.

The Role of Backbone Hydrogen Bonds in the Transition State for Protein Folding of a PDZ Domain

“…We designed and synthesized five different amide-to-ester mutants of PSD-95 PDZ2, L170λ, G171γ, F172φ, I174ι and G176γ as described [21] (see Powers et al [24] for nomenclature of amide-to-ester mutations). In general, such backbone mutations remove a hydrogen bond formed by the NH of the amide and destabilize the hydrogen bond formed by the amide carbonyl oxygen [3], [24].…”

“…Thus, the pseudo wild type in the folding experiments in this paper is the double mutant of PSD-95 PDZ2, V178C/Y190W. Semisynthesis of the PSD-95 PDZ2 V178C/Y190W amide-to-ester mutants was performed as previously described [21].…”

Design and synthesis of triazole-based peptidomimetics of a PSD-95 PDZ domain inhibitor

“…1A and B). [18][19][20] Truncation studies of peptide ligands targeting PSD-95 have shown that pentapeptides display affinities similar to longer peptides, while tetra-and tripeptides show a stepwise reduction in affinity. 15 pentapeptides have subsequently been derivatized into dimeric inhibitors with nanomolar potency.…”

“…The C-terminal carboxylic acid of the peptide ligand is crucial for binding 17 and is accommodated by the conserved PDZ 'GLGF' loop. 18 Peptides binding to PDZ domains form an anti-parallel β-sheet with the PDZ βB strand via amide backbone hydrogen bonds. 14,18,19 Structure-activity relationship (SAR) investigations of both peptide and protein backbone have revealed that not all amide atoms of the peptide engage directly with the PDZ domain.…”

“…18 Peptides binding to PDZ domains form an anti-parallel β-sheet with the PDZ βB strand via amide backbone hydrogen bonds. 14,18,19 Structure-activity relationship (SAR) investigations of both peptide and protein backbone have revealed that not all amide atoms of the peptide engage directly with the PDZ domain. Especially, the P −1 amino acid does not interact with PDZ, but seem to act more as a spacer that ensures the right positioning of the essential P 0 and P −2 residues (Fig.…”