Distinct binding specificity of the multiple PDZ domains of INADL, a humanprotein with homology to INAD from Drosophila melanogaster

PDZ domains are protein-protein interaction modules that typically bind to short peptide sequences at the carboxyl terminus of target proteins. Protei ns containing multiple PDZ domains often bind to different transmembrane an d intracellular proteins, playing a central role as organizers of multimeri c complexes. To characterize the rules underlying the binding specificity o f different PDZ domains, we have assembled a novel repertoire of random pep tides that are displayed at high density at the carboxyl terminus of the ca psid D protein of bacteriophage lambda. We have exploited this combinatoria l library to determine the peptide binding preference of the seven PDZ doma ins of human INADL, a multi-PDZ protein that is homologous to the INAD prot ein of Drosophila melanogaster. This approach has permitted the determinati on 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 involvemen t of specific residues at contact positions in determining the domain bindi ng preference. However, these experiments failed to reveal simple rules tha t 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 simultaneousl y 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 an y given PDZ domain.