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Engineering Af1521 macro domain increases ADP-ribose binding affinity and leads to the identification of more ADP-ribosylated proteins

ABSTRACT: Protein ADP-ribosylation is a covalent, reversible post-translational modification that has important functions in several cellular mechanisms. The identification of modified proteins in cells has proven challenging and, due to the low abundance of protein ADP-ribosylation, has mainly been possible via enrichment methodologies using ADP-ribose binding domains. Here, using random mutagenesis and in vitro selection with an ADP-ribosylated histone peptide, we engineered the archaeal Af1521 macro domain to generate an engineered isoform containing nine mutations that displays significantly increased affinity towards ADP-ribose. Comparison of the wild-type and the engineered Af1521 macro domains using our proteomic ADP-ribosylome enrichment workflow demonstrated an increased identification rate of ADP-ribosylated proteins with the engineered Af1521.

INSTRUMENT(S): Orbitrap Fusion

TISSUE(S): Epithelial Cell

SUBMITTER: Kathrin Nowak

LAB HEAD: Michael O. Hottiger

PROVIDER: PXD016686 | Pride | 2020-10-20

REPOSITORIES: Pride

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

Nowak Kathrin K Rosenthal Florian F Karlberg Tobias T Bütepage Mareike M Thorsell Ann-Gerd AG Dreier Birgit B Grossmann Jonas J Sobek Jens J Imhof Ralph R Lüscher Bernhard B Schüler Herwig H Plückthun Andreas A Leslie Pedrioli Deena M DM Hottiger Michael O MO

Nature communications 20201015 1

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 A ...[more]

PMID: 33060572

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Project description:UNLABELLED:ADP-ribosylation is a posttranslational protein modification in which ADP-ribose is transferred from NAD(+) to specific acceptors to regulate a wide variety of cellular processes. The macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1, and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their macro domains. Here, we report that the macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase for mono-ADP-ribose (MAR) and poly(ADP-ribose) (PAR) chain removal (de-MARylation and de-PARylation, respectively) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of an HEV subgenomic replicon. The de-MARylation activity is present in all three pathogenic positive-sense, single-stranded RNA [(+)ssRNA] virus families which carry a macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus and human coronavirus 229E), Togaviridae (Venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant. IMPORTANCE:Protein ADP-ribosylation is a covalent posttranslational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae, possess macro domains embedded in their polyproteins. Here, we show that viral macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various poly(ADP-ribose) polymerases which are notorious guardians of cellular integrity are demodified by macro domains from members of these virus families. In the case of hepatitis E virus, the adjacent viral helicase domain dramatically increases the binding of the macro domain to PAR and simulates the demodification activity.

			Action	DRS
	10mg_eAf_1_EThcD.csv	Csv
	10mg_eAf_1_HCD.csv	Csv
	10mg_eAf_2_EThcD.csv	Csv
	10mg_eAf_2_HCD.csv	Csv
	10mg_eAf_3_EThcD.csv	Csv