Screening phage-displayed combinatorial peptide libraries.

Among the many techniques available to investigators interested in mapping protein-protein interactions is phage display. With a modest amount of effort, time, and cost, one can select peptide ligands to a wide array of targets from phage-display combinatorial peptide libraries. In this article, protocols and examples are provided to guide scientists who wish to identify peptide ligands to their favorite proteins.

Characterizing Class I WW domains defines key specificity determinants and generates mutant domains with novel specificities.

INTRODUCTION: WW domains are small protein interaction modules found in a wide range of eukaryotic signaling and structural proteins. Five classes of WW domains have been annotated to date, where each class is largely defined by the type of peptide ligand selected, rather than by similarities within WW domains. Class I WW domains bind Pro-Pro-Xxx-Tyr containing ligands, and it would be of interest to determine residues within the domains that determine this specificity. RESULTS: Fourteen WW domains selected Leu/Pro-Pro-Xxx-Tyr containing peptides ligands via phage display and were thus designated as Class 1 WW domains. These domains include those present in human YAP (hYAP) and WWP3, as well as those found in ubiquitin protein ligases of the Nedd4 family, including mouse Nedd4 (mNedd4), WWP1, WWP2 and Rsp5. Comparing the primary structures of these WW domains highlighted a set of highly conserved residues, in addition to those originally noted to occur within WW domains. Substitutions at two of these conserved positions completely inhibited ligand binding, whereas substitution at a non-conserved position did not. Moreover, mutant WW domains containing substitutions at conserved positions bound novel peptide ligands. CONCLUSIONS: Class I WW domains contain a highly conserved set of residues that are important in selecting Pro-Xxx-Tyr containing peptide ligands. The presence of these residues within an uncharacterized WW domain can be used to predict its ability to bind Pro-Xxx-Tyr containing peptide ligands.

Convergent evolution with combinatorial peptides.

Once the sequence of a genome is in hand, understanding the function of its encoded proteins becomes a task of paramount importance. Much like the biochemists who first outlined different biochemical pathways, many genomic scientists are engaged in determining which proteins interact with which proteins, thereby establishing a protein interaction network. While these interactions have evolved in regard to their specificity, affinity and cellular function over billions of years, it is possible in the laboratory to isolate peptides from combinatorial libraries that bind to the same proteins with similar specificity, affinity and primary structures, which resemble those of the natural interacting proteins. We have termed this phenomenon 'convergent evolution'. In this review, we highlight various examples of convergent evolution that have been uncovered in experiments dissecting protein-protein interactions with combinatorial peptides. Thus, a fruitful approach for mapping protein-protein interactions is to isolate peptide ligands to a target protein and identify candidate interacting proteins in a sequenced genome by computer analysis.

Physical and functional interactions between the transactivation domain of the hematopoietic transcription factor NF-E2 and WW domains.

Tandem binding sites for the hematopoietic transcription factor NF-E2 in the beta-globin locus control region activate high-level beta-globin gene expression in transgenic mice. NF-E2 is a heterodimer consisting of a hematopoietic subunit p45 and a ubiquitous subunit p18. Gavva et al. [Gavva, N. R., Gavva, R., Ermekova, K., Sudol, M., and Shen, J. C. (1997) J. Biol. Chem. 272, 24105-24108] reported that human p45 contains a PPXY motif that binds WW domains. We show that murine NF-E2, which contains two PPXY motifs (PPXY-1 and -2) within its transactivation domain, differentially interacted with nine GST-WW domain fusion proteins. Quantitative analysis revealed high-affinity binding (KD = 5.7 nM) of p45 to a WW domain from a novel human ubiquitin ligase homologue (WWP1) expressed in hematopoietic tissues. The amino-terminal WW domain of WWP1 formed a multimeric complex with DNA-bound NF-E2. A WWP1 ligand peptide, isolated by phage display, and a peptide spanning PPXY-1 inhibited p45 binding, whereas an SH3 domain-interacting peptide and a peptide spanning PPXY-2 did not. Mutation of PPXY-1, but not PPXY-2, inhibited the transactivation function of NF-E2, providing support for the hypothesis that WW domain interactions are important for NF-E2-mediated transactivation.