An Oriented Peptide Array Library (OPAL) Strategy to Study Protein-Protein Interactions

Abstract

One of the major questions in signal transduction is how the specificities of protein-protein interactions determine the assembly of distinct signaling complexes in response to stimuli. Several peptide library methods have been developed and widely used to study proteinprotein interactions. These approaches primarily rely on peptide or DNA sequencing to identify the peptide or consensus motif for binding and may prove too costly or difficult to accommodate high throughput applications. We report here an oriented peptide array library (OPAL) approach that should facilitate high throughput proteomic analysis of protein-protein interactions. OPAL integrates the principles of both the oriented peptide libraries and array technologies. Hundreds of pools of oriented peptide libraries are synthesized as amino acid scan arrays. We demonstrate that these arrays can be used to map the specificities of a variety of interactions, including antibodies, protein domains such Src homology 2 domains, and protein kinases.Signaling proteins are often composed of distinct protein domains or modules. Some of these serve as protein-protein interaction units, whereas others carry out enzymatic reactions (1). The elucidation of the genetic codes of various organisms, including humans, has presented an increasing number of protein domain families with unknown functions.To understand how signaling proteins interact with each other and maintain the fidelity of signal transduction, it is critical to determine the specificities of these protein domains. Protein domains (such as SH2, 1 SH3, and phosphotyrosine binding (PTB) domains) often mediate protein-protein interactions by recognizing short stretches of peptide sequences on target proteins (2, 3). In the past several years, many combinatorial peptide library based approaches have been developed to analyze the binding or substrate specificities of protein domains (reviewed in Ref. 4). These peptide libraries can be classified as either display or pool peptide libraries.The display libraries employ a variety of matrices for individual peptide presentation, including pin (5), phage (6 -8), beads (9), DNA binding proteins (10), or ribosomes (11). Following multiple rounds of affinity enrichment steps, the identities of the selected peptides can be ascertained by DNA or peptide sequencing (4). Display peptide libraries generally require isolation and sequencing of individual peptides to decode a binding motif, which is time-consuming and costly, limiting it to low throughput applications.In contrast, the pool peptide libraries rely on the comparison of binding affinities of different pools of random peptides to obtain binding consensus motifs (12). Thus, the sequences of individual bound peptides do not need to be resolved. A few years ago, we developed an oriented peptide library method to further simplify and facilitate screening (13). In this method, soluble pools of random peptides are oriented via a central "fixed" amino acid, which reduced the degeneracy of the library and prevented po...

Kinase peptide specificity: Improved determination and relevance to protein phosphorylation

“…Second, the template used for positional scanning is an oriented peptide library (OPL, alternatively referred to as an oriented degenerate peptide) (12,20,25), consisting of two anchor residues and many degenerate residues. The two anchor residues (typically R at PϪ3 and F at Pϩ1) provide sufficient favorable interaction of the investigated kinases to all peptide substrates to permit measurable phosphorylation of each peptide pool; the remaining degenerate positions provide diversity, which ensures that the residue preference measured pertains to diverse sequences (rather than to a single peptide, as is the case for most positional scanning studies of peptide specificity).…”

SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity

“…Furthermore, studies examining SH2 domain specificity have generally identified only a small number of critical residues that are required for the interaction between a given SH2 domain and its cognate phosphopeptide ligands (11,12). Although peptide library-based studies reveal limited specificity (10,14,15), anecdotal evidence from physiological interactions suggests that specificity is more complex. Despite these intense studies, the true selectivity of SH2 domains for physiological peptide ligands may not yet be fully appreciated.…”

“…Pioneering studies by Songyang and co-workers (10,14,15,20) and others (19,21) using degenerate peptide libraries and related techniques established broad specificity profiles for a wide range of SH2 domains. These approaches capture general binding motifs through paneling individual positions (for example at ϩ1 or ϩ2 residues C-terminal from the phosphotyrosine) independently of neighboring positions.…”

Targeting a Novel N-terminal Epitope of Death Receptor 5 Triggers Tumor Cell Death

“…OPAL and Permutation Array-The OPAL was described by Rodriguez et al (22). The peptide arrays were generated on cellulose membranes using an Intavis Multipep instrument (Intavis, Germany) and Fmoc (N-(9-fluorenyl)methoxycarbonyl)-based chemistry according to the manufacturer's instructions.…”

“…There are three potential O-glycosylation regions (25). Two of them ( 19 SSPS 22 and 75 TTTRNTVCQCEEGT 88 ) are in the common ECD sequence of mature human DR5 splice variants, and the third is located within the alternatively spliced DR5 region. However, the effect of O-glycosylation modification on the DR5-AD5-10 complex can be ignored in our study because AD5-10 was generated by immunizing mice with recombinant DR5a expressed in Escherichia coli.…”