The Development and Application of a Quantitative Peptide Microarray Based Approach to Protein Interaction Domain Specificity Space

Abstract

Protein interaction domain (PID) linear peptide motif interactions direct diverse cellular processes in a specific and coordinated fashion. PID specificity, or the interaction selectivity derived from affinity preferences between possible PID-peptide pairs is the basis of this ability. Here, we develop an integrated experimental and computational cellulose peptide conjugate microarray (CPCMA) based approach for the high throughput analysis of PID specificity that provides unprecedented quantitative resolution and reproducibility. As a test system, we quantify the specificity preferences of four Src Homology 2 domains and 124 physiological phosphopeptides to produce a novel quantitative interactome. The quantitative data set covers a broad affinity range, is highly precise, and agrees well with orthogonal biophysical validation, in vivo interactions, and peptide library trained algorithm predictions. In contrast to preceding approaches, the CPCMAs proved capable of confidently assigning interactions into affinity categories, resolving the subtle affinity contributions of residue correlations, and yielded predictive peptide motif affinity matrices. Unique CPCMA enabled modes of systems level analysis reveal a physiological interactome with expected node degree value decreasing as a function of affinity, resulting in minimal high affinity binding overlap between domains; uncover that Src Homology 2 domains bind ligands with a similar average affinity yet strikingly different levels of promiscuity and binding dynamic range; and parse with unprecedented quantitative resolution contextual factors directing specificity. The CPCMA platform promises broad application within the fields of PID specificity, synthetic biology, specificity focused drug design, and network biology. Molecular & Cellular Proteomics 13: 10.1074/mcp.O114.038695, 3647-3662, 2014. Protein interaction domains (PIDs)1 often compete for the same linear motif binding sites across a range of affinities, resulting in many potential interactions that may enable the rapid assembly and disassembly of signaling proteins in response to external and internal cues (1, 2). PID-peptide interactions have small binding interfaces, resulting in moderate affinity interactions mediated primarily by a few amino acid "hot-spots" within motifs specific for a particular PID family (3-5). The power of individual residues to direct interactions, the absence of structural constraint for linear motifs, and the modularity of PIDs has enabled the rapid evolution of these networks resulting in many large multimember PID families in higher eukaryotes (6 -9). For these large families dedicated to the recognition of similar ligands, PID specificity-or the interaction selectivity derived from affinity preferences between possible PID-peptide pairs-underpins the effective conveyance of specific cell signals. High throughput interaction mapping efforts are used to decipher how this PID "specificity space" is populated, thereby providing insight into protein function and the principles ...

Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads

“…However, these methods typically identify only strong binders, cannot return negative information about residues that ablate binding critical for downstream target prediction in vivo (Gfeller et al, 2011), and do not directly probe effects of PTMs. Array-based methods (Fodor et al, 1991) allow quantification of binding of labeled proteins to 10s-100s (SPOT arrays) (Frank et al, 1990), tens of thousands (Atwater et al, 2018), or millions (ultra-high density arrays) (Buus et al, 2012; Forsström et al, 2014; Price et al, 2012; Carmona et al, 2015) of peptides chemically synthesized in situ , permitting direct incorporation of PTMs or unnatural amino acids at specific positions (Engelmann et al, 2014; Tinti et al, 2013; Filippakopoulos et al, 2012). However, it is difficult to evaluate the yield and purity of peptides in each spot, contributing to false positives and negatives (Tinti et al, 2013; Blikstad and Ivarsson, 2015).…”

High-throughput methods for identification of protein-protein interactions involving short linear motifs

“…A growing number of domains have been found to engage in peptide-mediated interactions. Today, there are about 200 known peptide binding domain families [ 11 ] with well studied examples being the PDZ (postsynaptic density protein 95/discs large/zona occludens 1) domains that typically bind to C-terminal peptides of target proteins [ 12 – 14 ], the poly proline binding WW domains [ 15 ] and SH3 (Src Homology 3) domains [ 16 , 17 ], and the phosphotyrosine binding SH2 (Src Homology 2) domains [ 18 – 22 ] (Table 1 ). Manually curated databases such as the eukaryotic linear motif (ELM) resource [ 23 ] and the Linear Motif mediated Protein interaction Database (LMPID) [ 24 ] contain over 2,000 annotated instances of domain-motif interactions, most of which have been discovered by low-throughput experiments such as pulldowns, co-immunoprecipitation (co-IPs), mutational analysis and detailed structural studies of domain-peptide complexes.…”

“…This allow for direct and controlled mapping of interactions regulated by PTMs, such as phosphorylation [ 21 ] and acetylation [ 36 ]. For example, the tyrosine phosphopeptide binding of SH2 domains have been elucidated using a quantitative peptide microarray based approach [ 18 ] and by the use of a high-density peptide chip technology [ 21 ]. Similarly, Filippakopolous and co-workers created SPOT arrays that covered all possible sites for ε-N-acetylation of lysine residues of human histones [ 36 ].…”

A Methodological Assessment and Characterization of Genetically-Driven Variation in Three Human Phosphoproteomes

“…Binding of short linear peptides and phosphopeptides by PRPs directs specific catalysis and the formation of transient protein-protein interactions during signal transduction 1 , 60 . Motif defining amino acids are typically found within +/− 5 residues of the phosphorylation site 61 – 65 , with other residues extending beyond the motif also playing an important role to ensure interaction specificity 66 . While we were not able to obtain a large enough sampling of variants that disrupt annotated motifs (here only 5) to perform enrichment analysis, we did observe increased phosphopeptide variability as the distance between the phosphorylated site and the closest variant (in cis ) decreased (R = −0.11; p = 4.37 × 10 −5 , Supplemental Fig.…”