NudT16

Introduction

NudT16 is an archetypical member of the Nudix superfamily of hydrolases. Nudix hydrolase enzymes have in common a stretch of 23 amino acids called the Nudix signature sequence which is the binding site for the metal involved in diphosphate hydrolysis. Nudix hydrolases prefer substrates of the form NUcleoside, DIphosphate and variable motifX, hence their name NUDIX. Examples of such substrates are ATP where X is PPI, ADP-ribose where X is phospho-ribose or m7GTP-RNA where X is phospho-RNA. Their catalytic activity is to hydrolyze a phosphorus-oxygen bond resulting in a nucleoside monophosphate (NMP) and a phosphate linked to moiety X. While NudT16 was initially described as a nuclear RNA and cytoplasmic mRNA decapping enzyme, further studies have shown that it also effectively hydrolyzes inosine diphosphate (IDP) and its hazardous deoxyribose cognate (dIDP) into inosine monophosphate (IMP) and deoxy inosine monophosphate (dIMP), respectively ^[1]. NudT16 has also been shown to hydrolyze ADP-ribose (ADPr) and Poly-ADP-ribose (polyADPr) ^[2]. Physiologically, NudT16 has been shown to regulate levels of 53BP1, an adaptor protein that recruits other proteins to the site of a DNA breakage, through hydrolytic removal of ADPr from Poly-ADP-ribosylated 53BP1 ^[3].

Structure

NudT16 is a homodimer. The ribbon diagram shows Nudt16’s structure with A structure on the right shows the enzyme (one monomer in cyan and the other in purple) in complex with diADPr. This dimerization allows for each subunit to have a deeper ADPr binding pocket. Each monomer consists of a two beta-sheets surrounded by alpha-helices, as per the canonical canonical NNudix fold, an a/b fold of two . One beta-sheets sandwiched by a-helices. is composed entirely of antiparallel strands, while the other has two central parallel strands sandwiched between two outer anti-parallel strands. The catalytically relevant Nudix signature characteristic of Nudix hydrolases consists of 23 highly conserved residues (G1Z2-6E7Z8-14R15E16U17Z18E19E20Z21G22U23, where Z is any amino acid and U is an aliphatic and hydrophobic residue) in a loop-helix-loop region. The residues of the Nudix signature sequence in NudT16 (GARRLELGEALALGSGWRHVCHA) are shown in light pink and dark blue. The Glu residues in the Nudix box play a role in metal ion chelation essential to substrate binding and in the deprotonation of a water molecule to form a hydroxide nucleophile. ^[4] In contrast to the negatively charged pockets where metal ligands chelate, the adenosine binding pocket is positively charged. The mouth of the binding site is about 9 Å in width. Contrary to Nudix ADPRases,^{[5] [6]} Homo sapiens NudT16 (HsNudT16) binds adenosine of ADPr and buries it deep in the core, while leaving the non-adenosine ribose exposed to the surface. This orientation allows the exposed ribose to conjugate another protein. Many residues in the mouth of this binding pocket are also involved in hydrogen bonding, the binding of metal ligands, and serve to delimit the binding site.

Biological Function

Although initially it was postulated that HsNudT16 biological function was to decapp mRNA, the work of Gong and co-workers shows that HsNudT16 removes the posttranslational modification ADP-ribosylation of 53BP1 and so it regulates 53BP1 levels^[7].

Interestingly, Thirawatananond et. al. investigated whether the widening of the ADPr binding site would allow for increased hydrolysis activity by NudT16. Nudt16 mutants F36A, F61S, and a double mutant with both F36A and F61S have NudT16 hydrolysis activity decreased in free ADPr, and it remained comparably efficient in mono(ADP-ribosylated) proteins, and increased in poly(ADP-ribosylated) proteins. ^[8]

Iyama et. al. determined that NudT16 is a (Deoxy)inosine diphosphatase and that the loss of this protein is followed by increased accumulation of single-strand breaks in DNA, reduced proliferation, and increased cell arrest. They also found increased levels of inosine in RNA, which informed the conclusion that HsNudT16 functions in the nucleus to protect the cell from ITP and its detrimental effects ^[9] .

References

1. ↑ Tresaugues L, Lundback T, Welin M, Flodin S, Nyman T, Silvander C, Graslund S, Nordlund P. Structural Basis for the Specificity of Human NUDT16 and Its Regulation by Inosine Monophosphate. PLoS One. 2015 Jun 29;10(6):e0131507. doi: 10.1371/journal.pone.0131507., eCollection 2015. PMID:26121039 doi:http://dx.doi.org/10.1371/journal.pone.0131507
2. ↑ Thirawatananond P, McPherson RL, Malhi J, Nathan S, Lambrecht MJ, Brichacek M, Hergenrother PJ, Leung AKL, Gabelli SB. Structural analyses of NudT16-ADP-ribose complexes direct rational design of mutants with improved processing of poly(ADP-ribosyl)ated proteins. Sci Rep. 2019 Apr 11;9(1):5940. doi: 10.1038/s41598-019-39491-w. PMID:30976021 doi:http://dx.doi.org/10.1038/s41598-019-39491-w
3. ↑ Zhang F, Lou L, Peng B, Song X, Reizes O, Almasan A, Gong Z. Nudix Hydrolase NUDT16 Regulates 53BP1 Protein by Reversing 53BP1 ADP-Ribosylation. Cancer Res. 2020 Mar 1;80(5):999-1010. doi: 10.1158/0008-5472.CAN-19-2205. Epub, 2020 Jan 7. PMID:31911551 doi:http://dx.doi.org/10.1158/0008-5472.CAN-19-2205
4. ↑ Tresaugues L, Lundback T, Welin M, Flodin S, Nyman T, Silvander C, Graslund S, Nordlund P. Structural Basis for the Specificity of Human NUDT16 and Its Regulation by Inosine Monophosphate. PLoS One. 2015 Jun 29;10(6):e0131507. doi: 10.1371/journal.pone.0131507., eCollection 2015. PMID:26121039 doi:http://dx.doi.org/10.1371/journal.pone.0131507
5. ↑ https://doi.org/10.1021/bi0259296
6. ↑ Gabelli SB, Bianchet MA, Bessman MJ, Amzel LM. The structure of ADP-ribose pyrophosphatase reveals the structural basis for the versatility of the Nudix family. Nat Struct Biol. 2001 May;8(5):467-72. PMID:11323725 doi:10.1038/87647
7. ↑ Zhang F, Lou L, Peng B, Song X, Reizes O, Almasan A, Gong Z. Nudix Hydrolase NUDT16 Regulates 53BP1 Protein by Reversing 53BP1 ADP-Ribosylation. Cancer Res. 2020 Mar 1;80(5):999-1010. doi: 10.1158/0008-5472.CAN-19-2205. Epub, 2020 Jan 7. PMID:31911551 doi:http://dx.doi.org/10.1158/0008-5472.CAN-19-2205
8. ↑ Thirawatananond P, McPherson RL, Malhi J, Nathan S, Lambrecht MJ, Brichacek M, Hergenrother PJ, Leung AKL, Gabelli SB. Structural analyses of NudT16-ADP-ribose complexes direct rational design of mutants with improved processing of poly(ADP-ribosyl)ated proteins. Sci Rep. 2019 Apr 11;9(1):5940. doi: 10.1038/s41598-019-39491-w. PMID:30976021 doi:http://dx.doi.org/10.1038/s41598-019-39491-w
9. ↑ Iyama T, Abolhassani N, Tsuchimoto D, Nonaka M, Nakabeppu Y. NUDT16 is a (deoxy)inosine diphosphatase, and its deficiency induces accumulation of single-strand breaks in nuclear DNA and growth arrest. Nucleic Acids Res. 2010 Aug;38(14):4834-43. doi: 10.1093/nar/gkq249. Epub 2010, Apr 12. PMID:20385596 doi:http://dx.doi.org/10.1093/nar/gkq249