Abstract
ADP-ribosylation is a reversible and site-specific post-translational modification that regulates a wide array of cellular signaling pathways. Regulation of ADP-ribosylation is vital for maintaining genomic integrity, and uncontrolled accumulation of poly(ADP-ribosyl)ation triggers a poly(ADP-ribose) (PAR)–dependent release of apoptosis-inducing factor from mitochondria, leading to cell death. ADP-ribosyl-acceptor hydrolase 3 (ARH3) cleaves PAR and mono(ADP-ribosyl)ation at serine following DNA damage. ARH3 is also a metalloenzyme with strong metal selectivity. While coordination of two magnesium ions (MgA and MgB) significantly enhances its catalytic efficiency, calcium binding suppresses its function. However, how the coordination of different metal ions affects its catalysis has not been defined. Here, we report a new crystal structure of ARH3 complexed with its product ADP-ribose and calcium. This structure shows that calcium coordination significantly distorts the binuclear metal center of ARH3, which results in decreased binding affinity to ADP-ribose, and suboptimal substrate alignment, leading to impaired hydrolysis of PAR and mono(ADP-ribosyl)ated serines. Furthermore, combined structural and mutational analysis of the metal-coordinating acidic residues revealed that MgA is crucial for optimal substrate positioning for catalysis, whereas MgB plays a key role in substrate binding. Our collective data provide novel insights into the different roles of these metal ions and the basis of metal selectivity of ARH3 and contribute to understanding the dynamic regulation of cellular ADP-ribosylations during the DNA damage response.
Highlights
Rapid and effective responses to extracellular and intracellular signals are crucial for the maintenance of genomic integrity and determination of cell fate [1]
It has been shown that serine ADP-ribosylation, which is synthesized by PARP1/ histone PARylation factor 1 (HPF1) and PARP2/histone PARylation factor 2 complexes, is the major cellular post-translational modification (PTM) following DNA damage [27,28,29]
Our new crystal structure of acceptor hydrolase 3 (ARH3)–ADPR–Ca2+ complex reveals that Ca2+ coordination significantly distorts the structure of the dimetallic catalytic center and interferes with optimal positioning of the 100-OH group of the terminal ribose of ADPR, corresponding to the 100-O-linkage in substrates, which results in impaired hydrolysis of PAR and serine mono(ADPribosyl)ated substrates
Summary
Yasin Pourfarjam , Zhijun Ma1, Igor Kurinov , Joel Moss, and In-Kwon Kim1,* From the 1Department of Chemistry, University of Cincinnati, Cincinnati, Ohio, USA; 2Department of Chemistry and Chemical Biology, NE-CAT APS, Cornell University, Argonne, Illinois, USA; 3Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA
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