@article{4a9b498c6c154e969d49aeb95937a8e2,
title = "Crystal structure of human NADK2 reveals a dimeric organization and active site occlusion by lysine acetylation",
abstract = "NAD+ kinases (NADKs) are metabolite kinases that phosphorylate NAD+ molecules to make NADP+, a limiting substrate for the generation of reducing power NADPH. NADK2 sustains mitochondrial NADPH production that enables proline biosynthesis and antioxidant defense. However, its molecular architecture and mechanistic regulation remain undescribed. Here, we report the crystal structure of human NADK2, revealing a substrate-driven mode of activation. We find that NADK2 presents an unexpected dimeric organization instead of the typical tetrameric assemblage observed for other NADKs. A specific extended segment (aa 325–365) is crucial for NADK2 dimerization and activity. Moreover, we characterize numerous acetylation events, including those on Lys76 and Lys304, which reside near the active site and inhibit NADK2 activity without disrupting dimerization, thereby reducing mitochondrial NADP(H) production, proline synthesis, and cell growth. These findings reveal important molecular insight into the structure and regulation of a vital enzyme in mitochondrial NADPH and proline metabolism.",
keywords = "crystal structure, mitochondrial metabolism, NAD kinases, NADK2, NADPH metabolism, post-translational modifications, proline metabolism",
author = "Charline Mary and Soflaee, {Mona Hoseini} and Rushendhiran Kesavan and Muriel Gelin and Harrison Brown and G. Zacharias and Mathews, {Thomas P.} and Andrew Lemoff and Corinne Lionne and Gilles Labesse and Gerta Hoxhaj",
note = "Funding Information: We thank Amin Sagar for recording the SAXS data and the staff at the BioSAXS BM29 ESRF (Grenoble, France) for their help. We also acknowledge the staff of the ID30B beamline at the ESRF for the X-ray diffraction experiments. We also thank Chad Brautigam for his assistance with mass photometry, Feng Cai and Hieu Vu for assistance with MS analysis, and the UT Southwestern Proteomics Core for the help with the PTM analysis. The authors also acknowledge the UT Southwestern Quantitative Light Microscopy Core, a Shared Resource of the Harold C. Simmons Cancer Center, which is supported in part by an NCI Cancer Center Support grant (1P30 CA142543-01). Some data in this study were acquired with a mass photometer that was supported by award S10OD030312-01 from the National Institutes of Health. This research was supported by grants from the NIH: R01GM143236 (G.H.), a Welch foundation award (I-2067-20210327 [G.H.]), and a TS Alliance Research Grants Program award (885252 [G.H.]). C.M. G.L. and C.L. were supported from grants from the Agence Nationale de la Recherche NADKiller contract (ANR-17-CE18-0011-01) and Cov-H2L contract (ANR-20-CE18-25). G.H. is a recipient of a CPRIT Scholar (CPRIT; RR190087) and a V Scholar (V2021-019) award. G.L. and G.H. conceptualized, directed the study, and wrote the manuscript. M.H.S. and R.K. performed and analyzed the biochemical and cell biological work. H.B. assisted with immunoblotting, and A.L. assisted with PTM data analysis. L.G.Z. and T.P.M. performed LC-MS/MS analyses and assisted with mass-spectrometry-related methods. C.M. cloned and purified NADK2 for enzymology, biophysical characterization, and crystallogenesis. M.G. performed X-ray crystallography and assisted with the in silico ligand docking. C.L. performed the in vitro enzymatic assays. C.M. M.G. C.L. and G.L. analyzed biophysical data. The authors declare no competing interests. Funding Information: We thank Amin Sagar for recording the SAXS data and the staff at the BioSAXS BM29 ESRF (Grenoble, France) for their help. We also acknowledge the staff of the ID30B beamline at the ESRF for the X-ray diffraction experiments. We also thank Chad Brautigam for his assistance with mass photometry, Feng Cai and Hieu Vu for assistance with MS analysis, and the UT Southwestern Proteomics Core for the help with the PTM analysis. The authors also acknowledge the UT Southwestern Quantitative Light Microscopy Core, a Shared Resource of the Harold C. Simmons Cancer Center, which is supported in part by an NCI Cancer Center Support grant ( 1P30 CA142543-01 ). Some data in this study were acquired with a mass photometer that was supported by award S10OD030312-01 from the National Institutes of Health . This research was supported by grants from the NIH: R01GM143236 (G.H.), a Welch foundation award ( I-2067-20210327 [G.H.]), and a TS Alliance Research Grants Program award ( 885252 [G.H.]). C.M., G.L., and C.L. were supported from grants from the Agence Nationale de la Recherche NADKiller contract ( ANR-17-CE18-0011-01 ) and Cov-H2L contract ( ANR-20-CE18-25 ). G.H. is a recipient of a CPRIT Scholar ( CPRIT ; RR190087 ) and a V Scholar (V2021-019) award. Publisher Copyright: {\textcopyright} 2022 Elsevier Inc.",
year = "2022",
month = sep,
day = "1",
doi = "10.1016/j.molcel.2022.06.026",
language = "English (US)",
volume = "82",
pages = "3299--3311.e8",
journal = "Molecular Cell",
issn = "1097-2765",
publisher = "Cell Press",
number = "17",
}