Distinct Binding Specificity of the Multiple PDZ Domains of INADL, a Human Protein with Homology to INAD from Drosophila melanogaster

Abstract

PDZ domains are protein-protein interaction modules that typically bind to short peptide sequences at the carboxyl terminus of target proteins. Proteins containing multiple PDZ domains often bind to different transmembrane and intracellular proteins, playing a central role as organizers of multimeric complexes. To characterize the rules underlying the binding specificity of different PDZ domains, we have assembled a novel repertoire of random peptides that are displayed at high density at the carboxyl terminus of the capsid D protein of bacteriophage . We have exploited this combinatorial library to determine the peptide binding preference of the seven PDZ domains of human INADL, a multi-PDZ protein that is homologous to the INAD protein of Drosophila melanogaster. This approach has permitted the determination of the consensus ligand for each PDZ domain and the assignment to class I, class II, and to a new specificity class, class IV, characterized by the presence of an acidic residue at the carboxyl-terminal position. Homology modeling and site-directed mutagenesis experiments confirmed the involvement of specific residues at contact positions in determining the domain binding preference. However, these experiments failed to reveal simple rules that would permit the association of the chemical characteristics of any given residue in the peptide binding pocket to the preference for specific amino acid sequences in the ligand peptide. Rather, they suggested that to infer the binding preference of any PDZ domain, it is necessary to simultaneously take into account all contact positions by using computational procedures. For this purpose we extended the SPOT algorithm, originally developed for SH3 domains, to evaluate the probability that any peptide would bind to any given PDZ domain.A large number of interactions in the cell are mediated by families of protein binding modules that are found repeatedly and in different combinations in several proteins. Typically these modules mediate protein-protein interactions through recognition of short peptides in the target protein (1). Several approaches, based upon the screening of repertoires of combinatorial peptides, have been developed to investigate the recognition specificity of these domain families. Phage display of small peptides of random sequence has been successfully used for the characterization of binding domains such as SH2, SH3, WW, EH, etc. (reviewed in Ref. 2). PDZ domains (identified as conserved elements in postsynaptic density protein PSD-95, Disc-large tumor suppressor Dlg, Zonula occludens protein ZO-1) differ from the remaining domains since they bind to specific carboxyl-terminal sequences of target proteins and/or dimerize with other PDZ domains (reviewed in Ref. 3). This peculiarity has limited the possibility of using "classical" peptide repertoires displayed by fusion to M13 coat proteins, since these display systems present random peptides by fusing them to the amino terminus of pIII or pVIII coat proteins. As a consequence, PDZ specificity ha...

Potassium channel Kir4.1 macromolecular complex in retinal glial cells

“…This idea is lent credence by the findings that PDZ domains can discriminate interaction partners based on single amino acid substitutions (Songyang et al, 1997;Gee et al, 2000;Vaccaro et al, 2001). Therefore, the differences in the PDZ domain-binding C-termini of Kir4.1 (-R-I-S-N-V) and AQP4 (-V-L-S-S-V) may facilitate the preferential binding of AQP4 to a particular syntrophin isoform and Kir4.1 to another.…”

PDZ domains: troubles in classification

“…Several experimental data con¢rm this statement: we have shown that the substitution of the histidine at the crucial position KB1 of hINADL-7 (a class I binding PDZ) is not su¤cient to change its binding speci¢city and to confer a clear ligand preference [1]. Furthermore, the ¢rst PDZ of MUPP1 is a member of the most abundant group (G,H); therefore it should bind to class I motifs.…”

“…On the other hand, target peptides may be classi¢ed according to the (few) speci¢c residues that constitute the binding motif, through analysis of molecular repertoires (libraries of synthetic peptides; phage display; two hybrid, etc.) that allow identifying collections of di¡erent ligands.We have recently pointed out that, in order to characterize PDZ domains and to infer their binding speci¢city, it is necessary to exploit computational procedures, which simultaneously take into account all the contact positions of the domain binding pocket and the corresponding residues of the ligand [1]. PDZ domains are protein interaction modules that recognize and bind the C-terminal four residues of their target.…”

Fly cryptochrome and the visual system

“…Although a homologous complex of the fly Signalplex has not been described in ipRGCs, several components of this multiprotein complex seem to be conserved (18). Specifically, a protein homolog of dINAD, INAD-like (INADL), bearing seven PDZ domains, has been identified in humans (46). A search for a functional protein interaction network, performed with the STRING database (Fig.…”