Stereochemical Preferences Modulate Affinity and Selectivity among Five PDZ Domains that Bind CFTR: Comparative Structural and Sequence Analyses

Abstract

Summary PDZ domain interactions are involved in signaling and trafficking pathways that coordinate crucial cellular processes. Alignment-based PDZ binding motifs identify the few most favorable residues at certain positions along the peptide backbone. However, sequences that bind the CAL (CFTR-Associated Ligand) PDZ domain reveal only a degenerate motif that overpredicts the true number of high affinity interactors. Here, we combine extended peptide-array motif analysis with biochemical techniques to show that non-motif ‘modulator’ residues influence CAL binding. The crystallographic structures of 13 new CAL:peptide complexes reveal defined, but accommodating stereochemical environments at non-motif positions, which are reflected in modulator preferences uncovered by multi-sequence substitutional arrays. These preferences facilitate the identification of new high-affinity CAL binding sequences and differentially affect CAL and NHERF PDZ binding. As a result, they also help determine the specificity of a PDZ domain network that regulates the trafficking of CFTR at the apical membrane.

Chemically Modified Peptide Scaffolds Target the CFTR-Associated Ligand PDZ Domain

“…In particular, our structural studies confirm that even modified peptides bind to the CAL PDZ domain in a common conformation, such that each peptide side chain interacts with a defined stereochemical environment [17]. As a result, our acetylated lysine peptide structures provide templates for future screening efforts.…”

Optimization of the process of inverted peptides (PIPEPLUS) to screen PDZ domain ligands

“…CALP displayed strong preferences for I/L/V and S/T at position 0 (P 0 ) and position −2 (P −2 ), respectively, where P 0 is defined as the most C-terminal residue. These preferences are in excellent agreement with the assignment of CALP as a bona fide class I PDZ domain 31 , and with our previous studies 27,30,32 . At positions P −6 through P −9 , the wt amino acids can be replaced by any other, which is typical of the relatively non-specific interactions of N-terminal residues with the PDZ domain 14 .…”

“…Using this method, we synthesized peptide arrays on a PUC membrane, at a density of 200 nmol.cm −2 . To assess the quality of the protocol with a more diverse set of sequences, we synthesized a substitutional analysis (SubAna) array for a peptide (ANSRWPTSII, also called iCAL36) that has been extensively studied by our group 27–30 . This peptide has been shown to modulate the trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR), by inhibiting its interaction with the PDZ domain of the CFTR-associated ligand (CALP).…”

“…Interesting sequences could be selected such as ANSρWPTSII [norvaline (1.7)] to inhibit arginine cleavage by endopeptidases or ANSRWPTSIτ [2-aminobutyric acid (3.1)] to inhibit exopeptidase-mediated degradation. Crystallographic evidence shows that CAL PDZ has additional space for binding at P −5 that is not fully occupied by tryptophan 30 . Thus, replacement with the voluminous 1-naphthylalanine (which has the highest binding signal: 4.5) could generate a ligand with more potency for CAL and higher specificity versus other PDZ domains.…”

A Phosphosite within the SH2 Domain of Lck Regulates Its Activation by CD45

“…FITC conjugated peptides corresponding to the phosphorylated C-terminal tail of Lck and Hck and variants were synthesized and purified to >90% by HPLC (Biomatik). FP assays were conducted as described previously (Amacher et al, 2014), using the following buffer: 25 mM Tris pH 8.0, 50 mM NaCl, 10% ( w/v ) glycerol, 0.5 mM TCEP. Briefly, K d values were determined by adding 30 nM fluorescein-labeled peptide to a 3-fold dilution series of WT or Y209E Hck SH2 protein.…”