Protein painting, an optimized MS-based technique, reveals functionally relevant interfaces of the PD-1/PD-L1 complex and the YAP2/ZO-1 complex

Amanda Haymond,Douglass Dey,Rachel Carter,Angela Dailing,Vaishnavi Nara,Pranavi Nara,Sravani Venkatayogi,Mikell Paige,Lance Liotta,Alessandra Luchini

doi:10.1074/jbc.ra118.007310

Abstract

Protein-protein interactions lie at the heart of many biological processes and therefore represent promising drug targets. Despite this opportunity, identification of protein-protein interfaces remains challenging. We have previously developed a method that relies on coating protein surfaces with small-molecule dyes to discriminate between solvent-accessible protein surfaces and hidden interface regions. Dye-bound, solvent-accessible protein regions resist trypsin digestion, whereas hidden interface regions are revealed by denaturation and sequenced by MS. The small-molecule dyes bind promiscuously and with high affinity, but their binding mechanism is unknown. Here, we report on the optimization of a novel dye probe used in protein painting, Fast Blue B + naphthionic acid, and show that its affinity for proteins strongly depends on hydrophobic moieties that we call here "hydrophobic clamps." We demonstrate the utility of this probe by sequencing the protein-protein interaction regions between the Hippo pathway protein Yes-associated protein 2 (YAP2) and tight junction protein 1 (TJP1 or ZO-1), uncovering interactions via the known binding domain as well as ZO-1's MAGUK domain and YAP's N-terminal proline-rich domain. Additionally, we demonstrate how residues predicted by protein painting are present exclusively in the complex interface and how these residues may guide the development of peptide inhibitors using a case study of programmed cell death protein 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1). Inhibitors designed around the PD-1/PD-L1 interface regions identified via protein painting effectively disrupted complex formation, with the most potent inhibitor having an IC50 of 5 μm.

Highlights

Protein–protein interactions lie at the heart of many biological processes and represent promising drug targets
We identified from a screen of smallmolecule dyes a total of four compounds with suitably low offrates and high protein binding that could be used in protein painting: Acid Orange 50 (AO50; CAS 10214-07-0), Reactive Blue 19 (CAS 2580-78-1), Red 49 (CAS 5517-38-4), and Congo Red (CAS 573-58-0) [5]
We answered remaining questions about how small dyes bind to proteins by comparing structurally similar dyes, which revealed the importance of a hydrophobic clamp region for achieving high numbers of bound dye molecules

Summary

Introduction

Protein–protein interactions lie at the heart of many biological processes and represent promising drug targets. We have previously reported a new technique called protein painting, shown, that identifies protein–protein interfaces via binding of small-molecule dyes, or “paints” [5]. In this technique, preformed protein complexes are pulsed with molecular paints that noncovalently coat the solvent-accessible surface of the complex. Peptides that are identified in an unpainted protein sample, yet are absent in a painted sample, represent sequences to which molecular dyes have bound to prevent trypsinization, and are considered the solvent-accessible peptide fragments. For peptides to be considered absent from the sample, they must be

Methods

Results

Conclusion