Erbin is a recently described member of the LAP (leucine-rich repeat and PDZ domain) protein family. We used a C-terminally displayed phage peptide library to identify optimal ligands for the Erbin PDZ domain. Phage-selected peptides were type 1 PDZ ligands that bound with high affinity and specificity to the Erbin PDZ domain in vitro. These peptides most closely resembled the C-terminal PDZ domain-binding motifs of three p120-related catenins: ␦-catenin, ARVCF, and p0071 (DSWV-COOH). Analysis of the interactions of the Erbin PDZ domain with synthetic peptides matching the C termini of ARVCF or ␦-catenin also demonstrated specific high affinity binding. We characterized the interactions between the Erbin PDZ domain and both ARVCF and ␦-catenin in vitro and in vivo. The Erbin PDZ domain co-localized and coprecipitated with ARVCF or ␦-catenin complexed with ␤-catenin and E/N-cadherin. Mutagenesis and peptide competition experiments showed that the association of Erbin with the cadherincatenin complex was mediated by the interaction of its PDZ domain with the C-terminal PDZ domain-binding motifs (DSWV-COOH) of ARVCF and ␦-catenin. Finally, we showed that endogenous ␦-catenin and Erbin co-localized in and co-immunoprecipitated from neurons. These results suggest that ␦-catenin and ARVCF may function to mediate the association of Erbin with the junctional cadherin-catenin complex. They also demonstrate that C-terminal phage-display technology can be used to predict physiologically relevant ligands for PDZ domains.PDZ 1 domains are 80 -100-amino acid compact globular motifs that are usually embedded in larger multidomain scaffolding proteins (1-3). PDZ domains predominantly mediate protein/protein interactions by recognizing the C termini of various intracellular and cell-surface proteins. Type 1 PDZ domains interact with the C-terminal consensus sequence X(S/ T)X(V/I/L)-COOH, whereas type 2 domains bind to the C-terminal consensus sequence X-hydrophobe-X-hydrophobe-COOH (3-5). Structural analyses of peptides bound to PDZ domains suggest necessary interactions at both positions 0 and Ϫ2 (6-8). However, among type 1 PDZ ligands, these residues are relatively invariant, indicating that other residues within the C terminus likely contribute to the specificity of PDZ domain/ ligand interactions (3). For example, two previous studies (9 -11), as well as those presented here, demonstrated the importance of residues Ϫ1 and Ϫ3 for binding specificity and affinity for some PDZ domain/ligand interactions.Genetic evidence supports a role for several families of PDZ domain-containing proteins as scaffolding molecules that target signaling complexes to various subcellular locations. For example, the multi-PDZ domain protein INAD was found to assemble components of the Drosophila visual transduction system to allow for efficient signaling in response to light (12, 13). Another study in the nematode Caenorhabditis elegans demonstrated that the basolateral localization of the LET-23 receptor tyrosine kinase is dependent upon direct b...

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We report the 1.7 Å crystal structure of ribosomal protein S4 from Bacillus stearothermophilus. To facilitate the crystallization, 41 apparently flexible residues at the N-terminus of the protein have been deleted (S4Δ41). S4Δ41 has two domains; domain 1 is completely α-helical and domain 2 comprises a fivestranded antiparallel β-sheet with three α-helices packed on one side. Domain 2 is an insertion within domain 1, and it shows significant structural homology to the ETS domain of eukaryotic transcription factors. A phylogenetic analysis of the S4 primary structure shows that the likely RNA interaction surface is predominantly on one side of the protein. The surface is extensive and highly positively charged, and is centered on a distinctive canyon at the domain interface. The latter feature contains two arginines that are totally conserved in all known species of S4 including eukaryotes, and are probably crucial in binding RNA. As has been shown for other ribosomal proteins, mutations within S4 that affect ribosome function appear to disrupt the RNA-binding sites. The structure provides a framework with which to probe the RNA-binding properties of S4 by site-directed mutagenesis.

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Protein S4 is essential for bacterial small ribosomal subunit assembly and recognizes the 5' domain (approximately 500 nt) of small subunit rRNA. This study characterizes the thermodynamics of forming the S4-5' domain rRNA complex from a thermophile, Bacillus stearothermophilus, and points out unexpected differences from the homologous Escherichia coli complex. Upon incubation of the protein and RNA at temperatures between 35 and 50 degrees C under ribosome reconstitution conditions [350 mM KCl, 8 mM MgCl2, and 30 mM Tris (pH 7.5)], a complex with an association constant of > or = 10(9) M(-1) was observed, more than an order of magnitude tighter than previously found for the homologous E. coli complex under similar conditions. This high-affinity complex was shown to be stoichiometric, in equilibrium, and formed at rates on the order of magnitude expected for diffusion-controlled reactions ( approximately 10(7) M(-1) x s(-1)), though at low temperatures the complex became kinetically trapped. Heterologous binding experiments with E. coli S4 and 5' domain RNA suggest that it is the B. stearothermophilus S4, not the rRNA, that is activated by higher temperatures; the E. coli S4 is able to bind 5' domain rRNA equally well at 0 and 37 degrees C. Tight complex formation requires a low Mg ion concentration (1-2 mM) and is very sensitive to KCl concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 9.3]. The protein has an unusually strong nonspecific binding affinity of 3-5 x 10(6) M(-1), detected as a binding of one or two additional proteins to the target 5' domain RNA or two to three proteins binding a noncognate 23S rRNA fragment of the approximately same size. This binding is not as sensitive to monovalent ion concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 6.3] as specific binding and does not require Mg ion. These findings are consistent with S4 stabilizing a compact form of the rRNA 5' domain.