Abstract
ADP-ribosylation is a post-translational modification that occurs on chemically diverse amino acids, including aspartate, glutamate, lysine, arginine, serine and cysteine on proteins and is mediated by ADP-ribosyltransferases, including a subset commonly known as poly(ADP-ribose) polymerases. ADP-ribose can be conjugated to proteins singly as a monomer or in polymeric chains as poly(ADP-ribose). While ADP-ribosylation can be reversed by ADP-ribosylhydrolases, this protein modification can also be processed to phosphoribosylation by enzymes possessing phosphodiesterase activity, such as snake venom phosphodiesterase, mammalian ectonucleotide pyrophosphatase/phosphodiesterase 1, Escherichia coli RppH, Legionella pneumophila Sde and Homo sapiens NudT16 (HsNudT16). Our studies here sought to utilize X-ray crystallographic structures of HsNudT16 in complex with monomeric and dimeric ADP-ribose in identifying the active site for binding and processing free and protein-conjugated ADP-ribose into phosphoribose forms. These structural data guide rational design of mutants that widen the active site to better accommodate protein-conjugated ADP-ribose. We identified that several HsNudT16 mutants (Δ17, F36A, and F61S) have reduced activity for free ADP-ribose, similar processing ability against protein-conjugated mono(ADP-ribose), but improved catalytic efficiency for protein-conjugated poly(ADP-ribose). These HsNudT16 variants may, therefore, provide a novel tool to investigate different forms of ADP-ribose.
Highlights
ADP-ribosylation is a post-translational modification in which ADP-ribose (ADPr) is added onto glutamate, aspartate, cysteine, serine, lysine, and arginine residues of proteins using NAD+ as their substrate[1,2,3,4,5]
A starter culture of LB supplemented with 50 μg/mL kanamycin and 34 μg/mL chloramphenicol was inoculated using a glycerol stock of CodonPlus RIPL E. coli cells (Agilent) that had been transformed with the pNIC28-BSA4-NudT16 plasmid and left to grow at 37 °C overnight. 10 mL of starter culture was used to inoculate each four 1 L cultures containing TB supplemented with 50 μg/mL kanamycin and 34 μg/mL chloramphenicol
Crystal structures of HsNudT16 in complex with ADPr further guided our mutagenesis of the enzyme to improve its ability to process PARylated proteins to phosphoribosylated proteins
Summary
ADP-ribosylation is a post-translational modification in which ADP-ribose (ADPr) is added onto glutamate, aspartate, cysteine, serine, lysine, and arginine residues of proteins using NAD+ as their substrate[1,2,3,4,5]. ADP-ribosylation is involved in diverse cellular functions, including DNA repair, transcription, chromosome segregation, cell cycle, cell metabolism, cell death and RNA metabolism[3,9,10,11] This protein modification often acts as a scaffolding mechanism of other proteins[2] in DNA damage repair[3,12,13,14], mitotic spindle[15] and www.nature.com/scientificreports/. One possible solution is to treat MARylated and PARylated proteins/peptides with SVP, ENPP1 or NUDIX enzymes EcRppH and HsNudT1623,30–34, resulting in a 212.0086 Da phosphoribose tag at the otherwise modified residues. This phosphoribose tag allows for enrichment by phosphoproteomics approaches and site identification by mass spectrometry. Compared with the wild-type, HsNudT16 mutants Δ17, F36A, and F61S have reduced hydrolysis activity towards free ADPr, comparable processing ability for MARylated proteins, and improved catalytic efficiency for PARylated proteins
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