Origins of PDZ Domain Ligand Specificity

Abstract

The LAP (leucine-rich repeat and PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis of both the ligand and PDZ domain side chains responsible for binding. C-terminal phage display has been used to identify preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW ؊4 ETW ؊1 V; IC 50 ‫؍‬ ϳ0.15 M) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable interactions with Trp ؊1 are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ domains), whereas the ␤2-␤3 loop provides a binding site for the Trp ؊4 side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation for the importance of at least five ligand C-terminal side chains in determining PDZ domain binding energy and highlight the mechanisms of ligand discrimination among the several hundred PDZ domains present in the human genome.

Comparative Structural Analysis of the Erbin PDZ Domain and the First PDZ Domain of ZO-1

“…In contrast with earlier studies that emphasized the importance of site 0 and site Ϫ2 (22)(23)(24), more recent studies, including the work described above, make it clear that PDZ domains recognize ligands through interactions that extend across the entire surface of the binding groove (25)(26)(27)(28)(29)(30)(31)(32)(33). According to the established PDZ domain classification system based on site 0 and site…”

“…Protein Purification-Erbin-PDZ (residues 1314 -1422) was purified as described previously (29). For ZO1-PDZ1, a DNA fragment encoding for residues 18 -110 of human ZO-1 was cloned into the NdeI/BamHI sites of the pET22d expression vector, and this created an open reading frame encoding for ZO1-PDZ1 with an N-terminal His tag and a thrombin cleavage site.…”

“…We solved the unliganded crystal structures of both domains, as well as high resolution structures of ZO1-PDZ1 in complex with two different phage-derived ligands designed to investigate the broadened specificity of ZO1-PDZ1. Together with a previously reported structure of Erbin-PDZ bound to an optimal ligand (29), these structures allow for a detailed comparison of the ligand-binding sites of the two domains. Remarkably, the analysis reveals that the differences in the site Ϫ1 and site Ϫ3 specificities of Erbin-PDZ and ZO1-PDZ1 are likely caused by a single amino acid difference between the two domains, and furthermore, the differences in site 0 can be accounted for by one additional difference.…”

“…Ϫ2 of Erbin-PDZ and ZO1-PDZ1-To better understand the molecular basis for ligand recognition and selectivity by PDZ domains, we compared the liganded structures of ZO1-PDZ1 bound to peptides WRRTTWV COOH or WRRTTYL COOH with an NMR structure of Erbin-PDZ bound to a high affinity peptide (TGWETWV COOH ) (29). In each structure, the PDZ domain forms extensive contacts with the bound ligand.…”

In Vitro and in Vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B, Type I (SR-BI), to the PDZ1 Domain of Its Adaptor Protein PDZK1

“…structures of PDZ domains with target peptides from PSD95 (PDZ3), Erbin, and PTP-1E (Protein Data Bank code 3LNY) (2, 37)) have suggested that the positive side chain at this position would, via one or two intermediate water molecules, participate in stabilization of the buried negative charge of the C-terminal carboxylate group of the target peptide. Indeed, mutagenesis analysis of Erbin by Skelton et al (43) suggests that the equivalent Lys 1287 (CBL) in Erbin significantly contributes to target peptide binding. The structure of the PDZ1-SR-BI peptide interaction suggests an attractive explanation of the essentially normal binding for the K14A mutant (Fig.…”