Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

Abstract

PDZ domains are ubiquitous small protein domains that are mediators of numerous protein-protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling-transduction complexes. In recent years, PDZ domains have emerged as novel and exciting drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C-terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys/Arg residue in the ligand-binding site of the second PDZ domain of PSD-95, by employing a semisynthetic approach. We generated six semisynthetic PDZ domains comprising different proteogenic and nonproteogenic amino acids representing subtle changes of the conserved Lys/Arg residue. These were tested with four peptide interaction partners, representing the two different binding modes. The results highlight the role of a positively charged amino acid in the β1-β2 loop of PDZ domains, and show subtle differences for canonical and noncanonical interaction partners, thus providing additional insight into the mechanism of PDZ/ligand interaction.

Development of a Potent Cyclic Peptide Inhibitor of the nNOS/PSD-95 Interaction

“…The FP saturation assays showed that the cyclic nNOS peptide bound to both PSD-95-PDZ1 (K d = 3.4 ± 0.3 μM) and PSD-95-PDZ2 (K d = 1.0 ± 0.1 μM) (Figures 1B and S1) while binding with much lower affinity to PSD-95-PDZ3 (K d = 52.0 ± 10.5 μM). These results align with previous binding studies using a cyclic nNOS β-hairpin mimetic peptide 45,46 and the nNOS-PDZ-β-hairpin protein. 53 Additionally, we verified that cyclization was a requirement for the β-hairpin mimetic to bind to PSD-95-PDZ2 by employing isothermal titration calorimetry (ITC).…”

“…This residue was reported to be responsible for binding to the carboxylate terminus of canonical peptides, yet nNOS lacks this carboxylic terminus. 46 PSD-95-PDZ2 containing the F172A mutation in the β-strand B (βB) did not fold according to circular dichroism (CD). Instead, we introduced an F172I mutation, which is the corresponding residue of the syntrophin-1-PDZ domain (Figure S19).…”

“…44 Interestingly, the same cyclic β-hairpin mimetic was used to demonstrate notable differences in the binding mode compared with a canonical PDZ-peptide interaction. 45,46 We herein report the use of SPOT peptide arrays to explore the molecular details of the nNOS β-hairpin binding to PSD-95. This high-throughput approach allows the synthesis of peptides on cellulose support in a parallel and organized manner using standard solid-phase peptide synthesis (SPPS), followed by screening with a labeled target protein.…”

“…Various cyclization strategies were employed, including cyclic disulfide-bridged peptides and peptide “stapling”, to stabilize the β-strand properties of known canonical C-terminal peptides. − Specifically for the cyclic nNOS β-hairpin, the introduction of a type-II turn mimetic in the cyclization scaffold (-G-p-) was employed. , Here, it was shown that the nNOS β-hairpin mimetic based on 23 amino acids (AAs) bound to the syntrophin-1 PDZ domain with a similar binding affinity as the nNOS protein . Interestingly, the same cyclic β-hairpin mimetic was used to demonstrate notable differences in the binding mode compared with a canonical PDZ-peptide interaction. , …”

Photoswitchable peptide-based ‘on-off’ biosensor for electrochemical detection and control of protein-protein interactions

“…We know that the β-finger ligand from the PDZ domain of nNOS forms an extended antiparallel β-sheet which interacts with the binding groove on the α-syntrophin PDZ domain to form a heterodimer (Hillier et al 1999). PDZ protein interaction domains are among the most common protein modules, and in recent years have emerged as novel drug targets for disease (Pedersen et al 2016), thus highlighting their potential in the development of tailored interfaces at the nanoscale.…”