Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins

Kathrin Nowak,Andreas Plückthun,Ralph Imhof,Birgit Dreier,Deena M Leslie Pedrioli,Michael O Hottiger,Jonas Grossmann,Ann-Gerd Thorsell,Herwig Schüler,Jens Sobek,Florian Rosenthal,Mareike Bütepage,Bernhard Lüscher,Tobias Karlberg

doi:10.1038/s41467-020-18981-w

Abstract

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.

Highlights

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions
The enriched pools were subcloned into the ribosome display vector pRDV which served as template for the round of selection[38,48,49]
400 mM NaCl binding of engineered Af1521 (eAf1521)-I144G remained similar compared to eAf1521 indicating that I144 and the formation of the tunnel did not contribute to the increased binding we observed with eAf1521. These results provide strong evidence that the two mutations K35E and Y145R and, as a consequence, the generated salt bridge solely contribute to the increased binding affinity of eAf1521

Summary

Introduction

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The ADP-ribosyl-acceptor hydrolases and the macro domain-containing enzymes, have diverse target specificity and hydrolytic activities toward proteins modified with ADPr and, as such, function as ADPr modification erasers[1,14,18,19]. To further elucidate the cellular function of ADP-ribosylation, it is of importance to identify the ADP-ribosylome—the modified proteins and their ADPr acceptor amino acids present in the sample under investigation. This is, challenging due to their low abundance and dynamic turnover compared to other PTMs20,21. Protein ADP-ribosylation was first described in the early 1960s (ref. 22), ADP-ribosylation was traditionally studied and identified in vitro via the incorporation of radioactive

Objectives

Methods

Results

Conclusion