Target-assisted iterative screening applied to random peptide libraries unveiled a novel and atypical recognition consensus shared by CIN85/SETA/Ruk SH3 domains, PX(P/A)XXR. Confirmed by mutagenesis and in vitro binding experiments, the novel consensus allowed for the accurate mapping of CIN85 SH3 binding sites within known CIN85 interactors, c-Cbl, BLNK, Cbl-b, AIP1/Alix, SB1, and CD2 proteins, as well as the prediction of CIN85 novel-interacting partners in protein databases. Synaptojanin 1, PAK2, ZO-2, and TAF II 70, which contain CIN85 SH3 recognition consensus sites, were selectively precipitated from mouse brain lysates by CIN85 SH3 domains in glutathione S-transferase pulldown experiments. A direct interaction of synaptojanin 1 and PAK2 with CIN85 SH3 domains was confirmed by Far Western blotting.One of the initial approaches to study a novel gene product is the identification of its interacting partners. As increasingly more proteins become functionally characterized it is more and more probable that the identification of interacting partners of a novel protein will suggest a possible function for this protein by associative reasoning and, thus, provide a framework for its characterization. Using this approach, a novel CIN85/CMS adaptor protein family was functionally implicated in a remarkably wide spectrum of different cellular processes such as the down-regulation of receptor tyrosine kinases and endocytosis (through the association of CIN85 with Cbl/Cbl-b and endophilins) (1, 2), apoptosis (through the association of CIN85 with p85-phosphatidylinositol 3-kinase and AIP1/Alix) (3, 4), and adhesion phenomena (through the association of CMS with p130Cas and the interactions of CD2AP/CMS with CD2 and cortactin) (5-7). However, the mapping of CIN85/CMS SH3 domains binding sites within the characterized CIN85/CMS interactors has remained an elusive goal, giving rise to discordant conclusions in the literature. The absence of information on the recognition properties of CIN85 SH3 domains hampers the further characterization of its interactions and the understanding of the biological role of this protein, which has emerged as a highly connected node linking functionally diverse protein interaction sub-networks. Intriguingly, it has been suggested that CIN85/CMS may have unique function(s) in higher eukaryotes, as CIN85 orthologues of the human and rat proteins were not found in Caenorhabditis elegans, Drosophila melanogaster, or yeast (8).At least five discrete protein interaction modules can be inferred from the analysis of the CIN85 amino acid sequence. These are three consecutive SH3 domains at the N terminus followed by a central proline-rich region and a coiled-coil domain situated at the C terminus (Fig. 1). CIN85 was shown to homodimerize through its coiled-coil domains, and a number of proteins interacting with the CIN85 proline-rich region were identified including Grb2, p130Cas , endophilins, p85-phosphatidylinositol 3-kinase, and cortactin (1,3,7,9,10). The SH3 domains of CIN85 bound c-Cbl, Cbl-b,...

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The present work is intended to demonstrate that most of the paradoxes, controversies, and contradictions accumulated in molecular and cell biology over many years of research can be readily resolved if the cell and living systems in general are re-interpreted within an alternative paradigm of biological organization that is based on the concepts and empirical laws of nonequilibrium thermodynamics. In addition to resolving paradoxes and controversies, the proposed re-conceptualization of the cell and biological organization reveals hitherto unappreciated connections among many seemingly disparate phenomena and observations, and provides new and powerful insights into the universal principles governing the emergence and organizational dynamics of living systems on each and every scale of biological organizational hierarchy, from proteins and cells to economies and ecologies.

Syntrophins, a family of intracellular peripheral membrane proteins of the dystrophin-associated protein complex (DAPC), each contain a single PDZ domain. Syntrophin PDZ domains bind C-terminal peptide sequences with the consensus R/K-E-S/T-X-V-COOH, an interaction that mediates association of skeletal muscle sodium channels with the DAPC. Here, we have isolated cyclic peptide ligands for syntrophin PDZ domains from a library of combinatorial peptides displayed at the N terminus of protein III of bacteriophage M13. Affinity selection from a library of X 10 C peptides yielded ligands with the consensus X-(R/K)-E-T-C-L/M-A-G-X-⌿-C, where ⌿ represents any hydrophobic amino acid. These peptides contain residues (underlined) similar to the C-terminal consensus sequence for binding to syntrophin PDZ domains and bind to the same site on syntrophin PDZ domains as C-terminal peptides, but do not bind to other closely related PDZ domains. PDZ binding is dependent on the formation of an intramolecular disulfide bond in the peptides, since treatment with dithiothreitol, or substitution of either of the two cysteines with alanines, eliminated this activity. Furthermore, amino acid replacements revealed that most residues in the phage-selected peptides are required for binding. Our results define a new mode of binding to PDZ domains and suggest that proteins containing similar conformationally constrained sequences may be ligands for PDZ domains. PDZ1 domains are 80 -90-amino acid modules present in numerous eukaryotic proteins. They were first described as a series of three internal, repeated elements within the postsynaptic density (PSD)-95 protein (1). In fact, the name PDZ is derived from three proteins first recognized to contain repeats of this domain: PSD-95; the Drosophila discs-large tumor suppressor protein, Dlg; and the mammalian tight-junction protein zona occludens-1, ZO-1 (2-5). PDZ domains have since been identified in a large number of multifunctional proteins, many of which are associated with specialized regions of cell to cell contact such as tight junctions, septate junctions, and synaptic junctions (6). The PDZ domain may be an evolutionarily old domain, as it has been detected in mammalian, nematode, yeast, plant, and bacterial genomic sequences by computer analysis (7).PDZ domains mediate protein-protein interactions by at least two distinct mechanisms. Certain PDZ domains bind directly to specific recognition sequences at the C terminus of transmembrane proteins. For example, the second PDZ domain of PSD-95 interacts with an S/T-X-V-COOH motif in N-methyl-D-aspartate receptor 2B subunits (8, 9) and in Shaker-type potassium channels (10). PDZ domains can also form heterotypic dimers with other PDZ domains. For instance, the Nterminal region of nNOS, which itself contains a PDZ domain, binds directly to PDZ domains in both PSD-95 and ␣ 1 -syntrophin, a component of the dystrophin-associated protein complex (DAPC) (11). A third possible mode of interaction, in which the consensus binding sequence is locat...