Engineering Peptide Inhibitors To Overcome PDZ Binding Promiscuity

Abstract

Protein-protein interaction domains (PPIDs) are key elements in assembling functional protein complexes and controlling cellular activities. A major class of PPIDs is mediated by PDZ (for PSD-95, Dlg, ZO-1) domains [1][2][3], widespread scaffolding modules essential for regulating the localization and activity of numerous cellular effector proteins. Among the diverse protein interaction domains, PDZ domains are highly conserved in organisms from bacteria to humans [4]. They usually bind the C-terminus of their ligands.Consistent with their structural homology, PDZ domains exhibit overlapping recognition sequences, meaning that a given partner typically can interact with multiple domains. Some years ago, we proposed a general and efficient procedure for profiling PDZ-peptide interactions that provides a picture of specificity and selectivity covering the complete PDZligand sequence space by combining SPOT synthesis and K d prediction [5]. As expected, among the three PDZ domains that were analyzed (AF6, ERBIN and SNA1), the overlap of ligand sequences recognized at K d values between 50-100 μM was substantial. Recent studies have suggested that there is more diversity among PDZ sequence preferences than originally thought [6,7]. Nevertheless, the set of PDZ domains interacting with a given protein necessarily share overlapping binding motifs, and it remains challenging to develop a canonical peptide that will inhibit only a single PDZ domain out of this set.To address this issue, we focused on a set of five PDZ domains known to interact with the Cystic Fibrosis (CF) Transmembrane conductance Regulator (CFTR). The PDZ-containing proteins CAL (CFTR-Associated Ligand) [8,9] and its antagonists NHERF1 and NHERF2 (Na+/H+ Exchanger Regulatory Factor 1/2) [8,10], compete for the binding to CFTR. CAL contains one (CALP) and each NHERF protein two PDZ domains (N1P1, N1P2, N2P1 and N2P2) which control both the activity and the cell surface abundance of CFTR. NHERF family members increase CFTR activity at the apical membrane, whereas CAL promotes its lysosomal degradation. Thus, to explore novel therapeutic strategies for increasing the cellsurface abundance of CFTR, our goal was to design a selective inhibitor of the

Synthetic peptide arrays for investigating protein interaction domains

“…Spot signal intensities can then be correlated to the measured binding affinities, yielding a semi-quantitative binding assay. This approach has been demonstrated with the yeast SH3 interactome [33] and for the CAL PDZ domain interaction [59]. Such follow-up studies are important if peptide arrays are used to develop effective inhibitors or assess the biological relevance of the observed binding events (specific versus unspecific).…”

“…More recently, cystic fibrosis research focused on finding a selective CAL PDZ inhibitory peptide by applying an integrated synthetic peptide array approach designed for PDZ domain screening [59]. The screening discovered a peptide that selectively inhib- its the CAL PDZ domain, which in turn extends the half-life of the DF508-CFTR protein responsible for cystic fibrosis.…”

Hybrid Organic–Inorganic Inhibitors of a PDZ Interaction that Regulates the Endocytic Fate of CFTR

“…[20] We recently combined a screen of inverted peptide arrays with in vitro fluorescence polarization measurements to identify selective CALP inhibitors. [20,21] However, the potency of these inhibitors remains modest, with K i ! 1.3 mm.…”

A Stabilizing Influence: CAL PDZ Inhibition Extends the Half‐Life of ΔF508‐CFTR

“…[13] However, CFTR interacts not only with CAL, but also with the Na + /H + exchanger regulatory factors NHERF1 and NHERF2, which counteract CALs effect, enhancing the activity and the abundance of DF508-CFTR at the apical membrane. [14][15][16] In an accompanying report, [17] we describe a novel strategy that permitted elaboration of the decameric peptide inhibitor iCAL36 10 (iCAL36; ANSRWPTSII). iCAL36 targets the CAL, but not the NHERF, PDZ domains, despite their Figure 1.…”

“…This stereochemical footprint is consistent with observed contributions of N-terminal modifications to peptide affinity and selectivity. [17] We next investigated whether our PDZ domain-based approach predicts peptide interactions with full-length proteins in the context of other cellular factors. Biotinylated (BT-) versions of three peptides were synthesized for pulldown assays: the CFTR C terminus (BT-CFTR), which binds NHERF PDZ domains strongly; the somatostatin receptor type 5 C terminus (BT-SSR5), which binds both NHERF and CAL domains; and BT-iCAL36, which binds CALP with the highest affinity and selectivity.…”