@article{65e69e1f46b64d85bdf5f3399e8d7ec1,
title = "Structure of human NADK2 reveals atypical assembly and regulation of NAD kinases from animal mitochondria",
abstract = "All kingdoms of life produce essential nicotinamide dinucleotide NADP(H) using NAD kinases (NADKs). A panel of published NADK structures from bacteria, eukaryotic cytosol, and yeast mitochondria revealed similar tetrameric enzymes. Here, we present the 2.8-{\AA} structure of the human mitochondrial kinase NADK2 with a bound substrate, which is an exception to this uniformity, diverging both structurally and biochemically from NADKs. We show that NADK2 harbors a unique tetramer disruptor/dimerization element, which is conserved in mitochondrial kinases of animals (EMKA) and absent from other NADKs. EMKA stabilizes the NADK2 dimer but prevents further NADK2 oligomerization by blocking the tetramerization interface. This structural change bears functional consequences and alters the activation mechanism of the enzyme. Whereas tetrameric NADKs undergo cooperative activation via oligomerization, NADK2 is a constitutively active noncooperative dimer. Thus, our data point to a unique regulation of NADP(H) synthesis in animal mitochondria achieved via structural adaptation of the NADK2 kinase.",
keywords = "NADK, NADK2, cooperative, dimer, structure",
author = "Jin Du and Michael Estrella and Kristina Solorio-Kirpichyan and Jeffrey, {Philip D.} and Alexei Korennykh",
note = "Funding Information: 12. M. E. Dickinson et al.; International Mouse Phenotyping Consortium; Jackson Laboratory; Infrastructure Nationale PHENOMIN, Institut Clinique de la Souris (ICS); Charles River Laboratories; MRC Harwell; Toronto Centre for Phenogenomics; Wellcome Trust Sanger Institute; RIKEN Methods Human NADK2 and NADK proteins were expressed in Escherichia coli and purified to homogeneity by size exclusion fast protein liquid chromatography (FPLC). NAD(H) kinase kinetics assays were conducted at 37 °C using γ-32P-ATP radioactive substrate and gel electrophoresis, followed by gel quantification on a Typhoon phosphorimager. Initial crystallization of NADK2 was achieved using a high-throughput Mosquito robot, in 100-nL hanging drops. Crystals were optimized and grown in 0.5-μL hanging drops. Diffraction data were collected on the National Synchrotron Light Source II (NSLS-II) Frontier Microfocusing Macromolecular Crystallography (FMX) beamline. The NADK2-substrate cocrystal structure was determined using experimental phasing with selenomethionine. Data Availability. X-ray and diffraction data and coordinates have been deposited in the Protein Data Bank (PDB; accession no. 7N29). All other study data are included in the article and/or SI Appendix. ACKNOWLEDGMENTS. This research used the FMX beamline of the NSLS-II, a US Department of Energy (DOE) Office of Science user facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract DE-SC0012704. The Center for BioMolecular Structure is supported by the NIH, National Institute of General Medical Sciences, through a Center Core P30 Grant (P30GM133893) and by the DOE Office of Biological and Environmental Research (Grant KP1605010). This study was funded by NIH Grant 1R01GM110161-01 (to A.K.) and by the Vallee Foundation (to A.K.). Author affiliations: NJ 08544 aDepartment of Molecular Biology, Princeton University, Princeton, BioResource Center, High-throughput discovery of novel developmental phenotypes. Nature 537, 508–514 (2016). 13. F. Tort et al., Lysine restriction and pyridoxal phosphate administration in a NADK2 patient. Pediatrics 138, e20154534 (2016). 14. D. J. Pomerantz et al.; Collaborators of UDN, Clinical heterogeneity of mitochondrial NAD kinase deficiency caused by a NADK2 start loss variant. Am. J. Med. Genet. A. 176, 692–698 (2018). 15. S. M. Houten et al., Mitochondrial NADP(H) deficiency due to a mutation in NADK2 causes dienoyl-CoA reductase deficiency with hyperlysinemia. Hum. Mol. Genet. 23, 5009–5016 (2014). 16. T. Ando et al., Structural determinants of discrimination of NAD+ from NADH in yeast mitochondrial NADH kinase Pos5. J. Biol. Chem. 286, 29984–29992 (2011). 17. R. Zhang, MNADK, a novel liver-enriched mitochondrion-localized NAD kinase. Biol. Open 2, 432–438 (2013). 18. Y. Fukasawa et al., MitoFates: Improved prediction of mitochondrial targeting sequences and their cleavage sites. Mol. Cell. Proteomics 14, 1113–1126 (2015). 19. S. Kawai, S. Mori, T. Mukai, W. Hashimoto, K. Murata, Molecular characterization of Escherichia coli NAD kinase. Eur. J. Biochem. 268, 4359–4365 (2001). 20. J. H. Grose, L. Joss, S. F. Velick, J. R. Roth, Evidence that feedback inhibition of NAD kinase controls responses to oxidative stress. Proc. Natl. Acad. Sci. U.S.A. 103, 7601–7606 (2006). 21. R. C. Edgar, MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004). 22. A. V. Korennykh et al., The unfolded protein response signals through high-order assembly of Ire1. Nature 457, 687–693 (2009). 23. G. Hoxhaj et al., Direct stimulation of NADP+ synthesis through Akt-mediated phosphorylation of NAD kinase. Science 363, 1088–1092 (2019). 24. E. F. Pettersen et al., UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). Funding Information: ACKNOWLEDGMENTS. This research used the FMX beamline of the NSLS-II, a US Department of Energy (DOE) Office of Science user facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract DE-SC0012704. The Center for BioMolecular Structure is supported by the NIH, National Institute of General Medical Sciences, through a Center Core P30 Grant (P30GM133893) and by the DOE Office of Biological and Environmental Research (Grant KP1605010). This study was funded by NIH Grant 1R01GM110161-01 (to A.K.) and by the Vallee Foundation (to A.K.). Publisher Copyright: Copyright {\textcopyright} 2022 the Author(s).",
year = "2022",
month = jun,
day = "28",
doi = "10.1073/pnas.2200923119",
language = "English (US)",
volume = "119",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
publisher = "National Academy of Sciences",
number = "26",
}