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. 2022 Nov 16;144(45):20582-20589.
doi: 10.1021/jacs.2c06249. Epub 2022 Nov 1.

Arginine ADP-Ribosylation: Chemical Synthesis of Post-Translationally Modified Ubiquitin Proteins

Jim Voorneveld  1 , Max S Kloet  2 , Sven Wijngaarden  1 , Robbert Q Kim  2 , Angeliki Moutsiopoulou  2 , Marnix Verdegaal  2 , Mohit Misra  3 , Ivan Đikić  3 , Gijsbert A van der Marel  1 , Herman S Overkleeft  1 , Dmitri V Filippov  1 , Gerbrand J van der Heden van Noort  2
Affiliations

Arginine ADP-Ribosylation: Chemical Synthesis of Post-Translationally Modified Ubiquitin Proteins

Jim Voorneveld et al. J Am Chem Soc. .

Abstract

We describe the development and optimization of a methodology to prepare peptides and proteins modified on the arginine residue with an adenosine-di-phosphate-ribosyl (ADPr) group. Our method comprises reacting an ornithine containing polypeptide on-resin with an α-linked anomeric isothiourea N-riboside, ensuing installment of a phosphomonoester at the 5'-hydroxyl of the ribosyl moiety followed by the conversion into the adenosine diphosphate. We use this method to obtain four regioisomers of ADP-ribosylated ubiquitin (UbADPr), each modified with an ADP-ribosyl residue on a different arginine position within the ubiquitin (Ub) protein (Arg42, Arg54, Arg72, and Arg74) as the first reported examples of fully synthetic arginine-linked ADPr-modified proteins. We show the chemically prepared Arg-linked UbADPr to be accepted and processed by Legionella enzymes and compare the entire suite of four Arg-linked UbADPr regioisomers in a variety of biochemical experiments, allowing us to profile the activity and selectivity of Legionella pneumophila ligase and hydrolase enzymes.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
(A) Advances presented in this study and (B) schematic representation of the pathway L. pneumophila enzymes use to (de)ubiquitinate host cell substrate proteins.
Scheme 1
Scheme 1. Synthetic Scheme toward Arginine-Linked ADPr-Peptides
(A) Solution-phase chemistry toward building blocks 1α and 1β and (B) solid-phase chemistry toward ADPr-peptides 14–17.
Figure 2
Figure 2
(A) Schematical representation of the experimental setup, where DupA cleaves the pyrophosphate linkage in α- or β-configured Arg-ADPr-peptides. (B) DupA-mediated hydrolysis of heptamer 14 followed over time using 1H NMR. The anomeric (α- or β-glycosidic-linked 14) is hydrolyzed into the α- or β-linked phosphoribose variant providing different chemical shifts for each product. The associated protons are annotated and integrated. (C) DupA-mediated hydrolysis of 14–17 as compared to enzymatically produced Arg42UbADPr. The conversion is measured over time and followed with HRMS. 14 is prepared using 1α, and 15 is prepared using 1β.
Figure 3
Figure 3
DupA-mediated hydrolysis of UbADPr into UbPr. (A) DupA-mediated pyrophosphate bond cleavage in UbADPr arginine variants after overnight incubation. (B) Hydrolysis of UbADPr by DupA followed over a time course of 0–90 min. Both graphs are analyzed with HRMS. The measurements in both graphs are normalized for background UbPr present as impurity associated with the synthesis.
Figure 4
Figure 4
SdeA-mediated ligation of UbADPr and fluorescent RTN4b 20-mer fragment 23. (A) Schematic representation of the conducted assay showing SdeA-mediated conjugation of UbADPr and peptide 23 to form a fluorescent product. (B) Arg42UbADPr is recognized and processed by SdeA. SdeA-mediated ligation assay performed for all (synthetic) UbADPr′s and analyzed by SDS-PAGE; top panel: gel stained with Coomassie blue protein stain, bottom panel; fluorescence scan. M: molecular weight marker.
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