A molecular toolbox for ADP-ribosyl binding proteins

Abstract

Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or viral infections, making them attractive drug targets. We present a robust and accessible assay applicable to both hydrolyzing or non-hydrolyzing binders of mono- and poly-ADP-ribosyl groups. This technology relies on a C-terminal tag based on a G_i protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups or analogs. By fusing the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we generate robust FRET partners and confirm the interaction with 22 known ADP-ribosyl binders. The applicability for high-throughput screening of inhibitors is demonstrated with the SARS-CoV-2 nsp3 macrodomain, for which we identify suramin as a moderate-affinity yet non-specific inhibitor. High-affinity ADP-ribosyl binders fused to nanoluciferase complement this technology, enabling simple blot-based detection of ADP-ribosylated proteins. All these tools can be produced in Escherichia coli and will help in ADP-ribosylation research and drug discovery.

Keywords: ADP-ribosylation; SARS-CoV-2; binding assay; high-throughput screening; inhibitors; macrodomain; post-translational modification; protein labeling.

Graphical abstract

Figure 1

A molecular toolbox for in vitro interaction studies and assay development of ADP-ribosyl binding proteins (A) Site-specific ADP-ribosylation of a C-terminal Gα_i-based 10-mer peptide (GAP-tag) by pertussis toxin subunit S1 (PtxS1) allows for generation of single S-glycosidically linked mono-ADP-ribosyl (MAR) groups. (B) The MAR group of the GAP-tag can be extended to a poly-ADP-ribosyl (PAR) group by PARP2. This system can be used to measure binding of proteins interacting with mono- or poly-ADP-ribosyl groups by FRET or other binding technologies. (C) The GAP-tag can be used for site-specific labeling with NAD⁺ analogs. (D) High-affinity ADP-ribosyl binders fused to nanoluciferase (Nluc) can be used as luminescent probes for fast, sensitive, and selective detection of mono- and poly-ADP-ribosylated proteins in blot-based methods.

Figure 2

Initial development and preparation of toolbox components (A) Testing ADP-ribosylation by PtxS1 with different Gα_i constructs. Unlabeled or YFP-fused full-length Gα_i constructs and GAP-tagged YFP were tested as cysteine-ADP-ribose acceptors when treated with 50 nM (+) or 250 nM (++) PtxS1. As controls, buffer or YFP-GAP in which the acceptor cysteine was mutated to alanine were used. Reactions were blotted on a nitrocellulose membrane, and detection was done using Nluc-eAf1521. (B) The mono-ADP-ribosyl (MAR) group in the GAP-tag can be extended to poly-ADP-ribose by PARP2. YFP-GAP or YFP-GAP(MAR) (10 μM) were mixed with 1 mM NAD and 400 nM (+) or 4 μM (++) PARP2 or buffer as control. The reactions were run on SDS-PAGE and visualized using Coomassie blue or by western blot and detection using Nluc-eAf1521 or Nluc-ALC1. (C) Detection of MAR and PAR by Nluc-eAf1521. (D) Selective detection of PAR by Nluc-ALC1. Dilution series of YFP-GAP(±MAR) or TNKS1(±PAR) were blotted on nitrocellulose membranes. YFP-GAP: 1 fmol = 31 pg; TNKS1: 1 fmol = 75 pg.

Figure 3

The GAP-tag can be used to introduce site-specific modifications with NAD⁺ analogs (A) Site-specific biotinylation of the GAP-tag. GAP-tagged YFP was mixed with NAD⁺ or 6-biotin-17-NAD⁺ in absence or presence of PtxS1. The reactions were blotted on a nitrocellulose membrane, and detection of biotin was done with streptavidin-HRP. (B) YFP-GAP was MARylated with PtxS1 using NAD⁺ or 6-propargyladenine-NAD⁺ containing an alkyne group. The resulting proteins YFP-GAP(MAR) or YFP-GAP(MAR-alkyne) or buffer were mixed with Cy3-azide or Cy5-azide, and the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction was performed by addition of 5 mM sodium ascorbate, 300 μM CuSO₄, and 600 μM L-histidine. The samples were incubated for 3 h at room temperature and blotted on nitrocellulose membranes, and visible-light images were taken. Unreacted Cy3-azide or Cy5-azide was removed by washing of the membranes in Tris-buffered saline/Tween-20. Visible-light and fluorescent images were taken.

Figure 4

Testing interactions of reported and potential readers and erasers with YFP-GAP (A) Interactions of CFP-fused potential and confirmed ADP-ribosyl binders with MARylated YFP-GAP. CFP-fusion proteins (1 μM) were mixed with 5 μM YFP-GAP or with 5 μM YFP-GAP(MAR) in absence or presence of 200 μM ADP-ribose (ADPr.). The ratiometric FRET signals were measured. P9, P14, and P15 denote PARP9, PARP14, and PARP15. (B) Test of ADP-ribosyl removal from GAP-tag. YFP-GAP(MAR) (10 μM) was prepared in absence (−) or presence (+) of 1 μM CFP-fused proteins or 0.01 μM–1 μM snake venom phosphodiesterase I (SVP). Samples were incubated for 24 h at room temperature and blotted on a nitrocellulose membrane. Detection was done with Nluc-eAf1521. (C) Interactions of poly-ADP-ribosyl binders with PARylated YFP-GAP. CFP-fusion proteins (250 nM) were mixed with 500 nM YFP or 500 nM YFP-GAP(PAR) in absence or presence of 100 μM ADP-ribose or 2.5 μM automodified PARP2. The ratiometric FRET signals were measured. (D) Representative dose-response curve of 1 μM CFP-SARS-CoV nsp3 and 5 μM YFP-GAP(MAR) upon competition with ADP-ribose. The control containing no ADP-ribose was set one logarithmic unit below the lowest concentration. Dose-response curves with ADP-ribose for all CFP-fusion proteins are shown in Figure S4. (E) Representative dose-response curve of 250 nM CFP-ALC1 and 500 nM YFP-GAP(PAR) upon competition with PARylated PARP2. The control containing no PARP2(PAR) was set one logarithmic unit below the lowest concentration, while the control using YFP-GAP(MAR) instead of YFP-GAP(PAR) was set one logarithmic unit above the highest PARP2(PAR) concentration. Data shown for FRET assays are mean ± standard deviation with n = 4 replicates.

Figure 5

Various assay technologies can be utilized to detect binding to the MARylated Gα_i (A) Measurement of interaction by FRET. Fluorescence emission spectra of CFP-MDO2 and YFP-GAP(MAR) in absence (control) or presence of 200 μM ADP-ribose (ADPr.). (B) Measurement of interaction by BRET. Luminescence emission spectra of Nluc-MDO2 and YFP-GAP(MAR) in absence (control) or presence of 200 μM ADP-ribose. (C) Measurement of interaction by AlphaScreen. Biotinylated MDO2 and His-tagged MARylated Gα_i were mixed with streptavidin donor beads and chelate acceptor beads in absence (control) or presence of 10 μM ADP-ribose. The luminescence signal was detected upon excitation of donor beads. Data shown are mean ± standard deviation with n = 4 replicates. (D) Measurement of interaction by biolayer interferometry. His-tagged YFP-GAP(MAR) was bound to the optical sensor surface, and the change of signal after association (0 s) or dissociation (120 s, dotted line) of unlabeled MDO2 protein was determined in absence or presence of 3.16 μM ADP-ribose.

Figure 6

Development of a screening assay for the SARS-CoV-2 nsp3 macrodomain (A) Signal validation for a screening assay with CFP-SARS-CoV-2. SARS-CoV-2 (1 μM) was mixed with 5 μM YFP-GAP(MAR) in absence (negative control) or presence (positive control) of 200 μM ADP-ribose, and a Z′ factor of 0.7 was calculated. (B) Screen of ENZO FDA-approved drug library comprising 640 compounds. Only the compound suramin showed inhibition above 30% and was taken to further validation. (C) Structure of the hit compound suramin. (D) Dose-response curve with suramin shows an IC₅₀ of 8.7 μM for the SARS-CoV-2 nsp3 macrodomain in the FRET-based assay. The control containing no compound was set one logarithmic unit below the lowest concentration, while the control containing 200 μM ADP-ribose was set one logarithmic unit above the highest suramin concentration. (E) Suramin shows stabilization of SARS-CoV-2 nsp3 macrodomain by DSF. (F) Inhibition profile of suramin against human and viral ADP-ribosyl binders used in this study. The inhibition was calculated based on the ratiometric FRET signals of the CFP-fused binders mixed with YFP-GAP(MAR) or YFP-GAP(PAR). Data shown for (D) and (F) are mean ± standard deviation with n = 4 replicates.