Characterization of a novel protein‐binding module — the WW domain

Abstract

We have identified, characterized and cloned human, mouse and chicken cDNA of a novel protein that binds to the Src homology domain 3 (SH3) of the Yes proto-oncogene product. We subsequently named it YAP for Yes-associated protein. Analysis of the YAP sequence revealed a protein module that was found in various structural, regulatory and signaling molecules. Because one of the prominent features of this sequence motif is the presence of two conserved tryptophans (W), we named it the WW domain. Using a functional screen of a cDNA expression library, we have identified two putative ligands of the WW domain of YAP which we named WBP-1 and WBP-2. Peptide sequence comparison between the two partial clones revealed a homologous proline-rich region. Binding assays and site-specific mutagenesis have shown that the proline-rich motif binds with relatively high affinity and specificity to the WW domain of YAP, with a preliminary consensus that is different from the SH3-binding PXXP motif. This suggests that the WW domain has a role in mediating protein-protein interactions via proline-rich regions, similar but distinct from Src homology 3 (SH3) domains. Based on this finding, we hypothesize that additional protein modules exist and that they could be isolated using proline-rich peptides as functional probes.Key words: Protein protein interaction; Protein module; Polyproline Background and rationale -Retrospective lookWhat started as a pilot project ended up being the main focus of an entire lab. Our general aim has been to decipher molecular steps of signaling by the Yes proto-oncogene product which represents a non-receptor type protein-tyrosine kinase of the Src family [1]. The specific goal was to isolate substrates and regulators of the Yes kinase in order to understand at least some aspects of the molecular role it plays in normal and the viral-Yes oncogene transformed cells [2]. Initially, our experimental approaches were descriptive of nature [3,4]. We argued that by characterizing the pattern of Yes expression in various tissues and cells, we would have been able to find a common denominator that would provide a clue regarding the physiological function of the Yes protein [5]. Localization of the Yes proto-oncogene transcript and protein in cerebellar Purkinje cells was an exciting finding which gave us hope for functional clues [6]. Unfortunately, that was immediately followed by the frustration of trying to study the Yes kinase in difficult experimental systems of isolated Purkinje neurons or cerebellar slices. The direction of our research activities shifted swiftly when Hirai and Varmus proposed that the amino termini of Src family kinases form complexes with cellular proteins and that these apparently transient and dynamic complexes constitute a part of the mechanism by which Src, Yes and other kinases signal [7]. The SH2 domain, residing at the amino terminal half of the Src kinases had already been delineated at that time and was a primary candidate for a signaling domain [8]. The proposal of Hirai...

A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function

“…We used deep mutational scanning to measure the ability of 47,000 unique variants of the hYAP65 WW domain to bind to their polyproline peptide ligand (2,4). WW domains mediate protein-protein interactions, have a well-defined structure, and fold through a two-state mechanism, simplifying subsequent measurements of thermodynamic stability (11)(12)(13). We displayed a library of variants of the hYAP65 WW domain on the surface of T7 bacteriophage.…”

Activation of the E3 ubiquitin ligase Itch through a phosphorylation-induced conformational change

“…The C2 domain binds Ca 2ϩ and phospholipids and mediates protein recruitment to intracellular membranes (12). WW domains comprise a ␤-sheet structure that contains two conserved Trp residues, and mediates protein-protein interactions (13). Targets for WW motifs are usually Pro-rich regions (PRR) (14).…”

A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: Implications for filovirus budding

“…For example, the PY motifs within the RSV Gag, VSV M, and rabies virus M proteins were shown to interact with WW-domains of specific cellular proteins (22,27). WW-domains are modular, hydrophobic domains found in a wide variety of cellular proteins, and these domains are classified as types I-IV, depending on the core sequence of the ligand (e.g., PPxY) with which they interact (36)(37)(38)(39)(40)(41)(42). The main function of WW-domains is to facilitate protein-protein interactions in the cell (36).…”