Uncover New Reactivity of Genetically Encoded Alkyl Bromide Non-Canonical Amino Acids.

Xin Shu,Sana Asghar,Haifan Wu,Shang-Tong Li,Fan Yang,Bing Yang

doi:10.3389/fchem.2022.815991

Abstract

Genetically encoded non-canonical amino acids (ncAAs) with electrophilic moieties are excellent tools to investigate protein-protein interactions (PPIs) both in vitro and in vivo. These ncAAs, including a series of alkyl bromide-based ncAAs, mainly target cysteine residues to form protein-protein cross-links. Although some reactivities towards lysine and tyrosine residues have been reported, a comprehensive understanding of their reactivity towards a broad range of nucleophilic amino acids is lacking. Here we used a recently developed OpenUaa search engine to perform an in-depth analysis of mass spec data generated for Thioredoxin and its direct binding proteins cross-linked with an alkyl bromide-based ncAA, BprY. The analysis showed that, besides cysteine residues, BprY also targeted a broad range of nucleophilic amino acids. We validated this broad reactivity of BprY with Affibody/Z protein complex. We then successfully applied BprY to map a binding interface between SUMO2 and SUMO-interacting motifs (SIMs). BprY was further applied to probe SUMO2 interaction partners. We identified 264 SUMO2 binders, including several validated SUMO2 binders and many new binders. Our data demonstrated that BprY can be effectively used to probe protein-protein interaction interfaces even without cysteine residues, which will greatly expand the power of BprY in studying PPIs.

Highlights

Protein-protein interactions (PPIs) are essential for virtually all cellular processes in all living organisms
Affinity purification mass spectrometry (AP-MS) have been successfully applied to map proteinprotein interactomes in many organisms (Ho et al, 2002; Butland et al, 2005; Krogan et al, 2006; Gordon et al, 2020; Richards et al, 2021), these methods cannot distinguish between direct binders and indirect binders of a protein of interest (POI) (Morris et al, 2014)
Using a recently developed searching algorism OpenUaa (Liu C. et al, 2021), which allowed deeper mining of the interactome data, we observed a broad reactivity of BprY to multiple nucleophilic amino acids—Cys, Asp, Glu, His, Lys, Ser, Thr and Tyr, supported by high-quality MS/MS spectra (Figures 1B; Supplementary Figure S1)

Summary

Introduction

Protein-protein interactions (PPIs) are essential for virtually all cellular processes in all living organisms. There is a significant effort in mapping protein-protein interaction networks to understand relevant biological processes in detail, and many techniques have been developed for this purpose (Low et al, 2021). Affinity purification mass spectrometry (AP-MS) have been successfully applied to map proteinprotein interactomes in many organisms (Ho et al, 2002; Butland et al, 2005; Krogan et al, 2006; Gordon et al, 2020; Richards et al, 2021), these methods cannot distinguish between direct binders and indirect binders of a protein of interest (POI) (Morris et al, 2014). Limited information is available to map the interaction interfaces to gain further structural understanding of these interactions

Methods

Results

Conclusion