Faculty Scholarship 2019 Crystal Structure of the Mitochondrial Protein mitoNEET Bound to a Benze-sulfonide Ligand Werner J. Geldenhuys Timothy E. long Pushkar Saralkar Toshio Iwasaki Rasia A. A. Nuñez See next page for additional authors Follow this and additional works at: https://researchrepository.wvu.edu/faculty_publications Part of the Pharmacy and Pharmaceutical Sciences Commons Authors Werner J. Geldenhuys, Timothy E. long, Pushkar Saralkar, Toshio Iwasaki, Rasia A. A. Nuñez, Rajesh R. Nair, Mary E. Konkle, Michael A. Menze, Mark V. Pinti, John M. Hollander, Lori A. Hazlehurst, and Aaron R. Robart ARTICLE https://doi.org/10.1038/s42004-019-0172-x OPEN Crystal structure of the mitochondrial protein mitoNEET bound to a benze-sulfonide ligand Werner J. Geldenhuys1,10, Timothy E. Long2,10, Pushkar Saralkar1,10, Toshio Iwasaki 3, Raisa A.A. Nuñez4, Rajesh R. Nair5, Mary E. Konkle 6, Michael A. Menze7, Mark V. Pinti8, John M. Hollander8, Lori A. Hazlehurst1,9 & Aaron R. Robart 4 1234567890():,; MitoNEET (gene cisd1) is a mitochondrial outer membrane [2Fe-2S] protein and is a potential drug target in several metabolic diseases. Previous studies have demonstrated that mitoNEET functions as a redox-active and pH-sensing protein that regulates mitochondrial metabolism, although the structural basis of the potential drug binding site(s) remains elusive. Here we report the crystal structure of the soluble domain of human mitoNEET with a sulfonamide ligand, furosemide. Exploration of the high-resolution crystal structure is used to design mitoNEET binding molecules in a pilot study of molecular probes for use in future develop- ment of mitochondrial targeted therapies for a wide variety of metabolic diseases, including obesity, diabetes and neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. 1 Department of Pharmaceutical Sciences, School of Pharmacy West Virginia University, Morgantown, WV 26506, USA. 2 Department of Pharmaceutical Sciences and Research, School of Pharmacy, Marshall University, Huntington, WV 113-8602, USA. 3 Department of Biochemistry and Molecular Biology, Nippon Medical School, Sendagi, Tokyo 113-8602, Japan. 4 Department of Biochemistry, School of Medicine West Virginia University, Morgantown, WV 26506, USA. 5 Department of Microbiology, School of Medicine West Virginia University, Morgantown, WV 26506, USA. 6 Department of Chemistry, Ball State University, Muncie, IN 47306, USA. 7 Department of Biology, University of Louisville, Louisville, KY 40292, USA. 8 Department of Physiology, School of Medicine West Virginia University, Morgantown, WV 26506, USA. 9 Modulation Therapeutics, Morgantown, WV 26506, USA. 10These authors contributed equally: Werner J. Geldenhuys, Timothy E. Long, Pushkar Saralkar. Correspondence and requests for materials should be addressed to W.J.G. (email: werner. [email protected]) or to A.R.R. (email: [email protected]) COMMUNICATIONS CHEMISTRY | (2019) 2:77 | https://doi.org/10.1038/s42004-019-0172-x | www.nature.com/commschem 1 ARTICLE COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-019-0172-x fi fi fi 16 itoNEET (gene cisd1)wasidentied in 2004 as the rst moderate af nity (IC50 ~40 µM) , using a recombinant mitoNEET Mexample of a mitochondrial outer-membrane iron–sulfur with a mitoNEET-His protein, as opposed to the His-SUMO protein protein,withtheclusterbindingdomainfacingthecyto- we used here for crystallography. We focused on furosemide in this solic side1.Thisproteinwasinitiallydiscoveredastheunintended study due to its appropriateness for the X-ray diffraction studies, due target for the anti-diabetic peroxisome proliferator-activated receptor to its favorable solubility profile, as opposed to the glitazones where gamma (PPAR-γ) agonist pioglitazone, as many of the beneficial solubility in the crystallization mother liquor is limiting. In addition, effects of pioglitazone, a thiazolidinedione (TZD), could not be we did not observe any aggregate formation with furosemide in explained by PPAR-γ activity alone1. MitoNEET assembles as a solution using dynamic light scattering (DLS), which could interfere homodimer, with each subunit containing a rather unusual [2Fe–2S]- with the crystallography studies (Supplementary Fig. 1). type iron–sulfur cluster coordinated by His87 and three cysteine The crystal structure of furosemide bound to mitoNEET is residues2. The amino acids coordinating the [2Fe–2S] cluster form a shown in Fig. 1, with the crystal data (6DE9.pdb) given in signature CDGSH domain common to all members of the CISD- Table 1. In the refined structure, furosemide is bound on the face gene family. Bioinformatics analysis has shown that the CISD-gene close to the [2Fe–2S] cluster in each monomer, which are critical family is highly conserved across a variety of species, including for the biological function of mitoNEET (Fig. 1a). The bound mammals, Caenorhabditis elegans,plantsaswellasthermophilic ligand was supported by strong density after MR-SAD phasing archaea and bacteria, with a high degree of structural homology and refinement, and was further confirmed by simulated (particularly around the cluster binding domain) among the different annealing OMIT map analysis (Fig. 1b, c). The furosemide phyla3. To date three CISD proteins have been crystalized, including carboxylic groups are in reach of the [2Fe–2S] coordinating human mitoNEET (gene cisd1), miner1/NAF-1 (gene cisd2), and His87 side chain for possible hydrogen bonding with His87Nε.In miner2/miNT (gene cisd3)4,5. addition, the furan ring of furosemide can be modeled such that MitoNEET plays an important role in mitochondrial function and the oxygen is pointed toward Sγ of the Cys83 ligand, although the metabolism6–10. Overexpression of mitoNEET in the adipose tissue limited resolution of the refined electron density map cannot of ob/ob mice led to a significantreductionininflammation and discriminate the precise orientation of the furosemide furan ring oxidative stress as compared to the control mice7. In addition, in the present study. The furosemide-binding site observed in the overexpression of mitoNEET in cardiomyocytes was protective crystal structure corresponds to previously hypothesized binding against oxidative stress as induced by hydrogen peroxide11,12.In pocket, from molecular docking studies performed with the contrast, when mitoNEET was knocked out of mice the resulting anticancer drug MAD-28 corroborating our finding17.In phenotype was characterized by a loss of dopamine neurotransmitter addition, the pocket identified by furosemide is suggested to levels from the striatum and Parkinson’s disease type motor defi- correspond to a possible binding site for pioglitazone, for which cits13. Although the detailed mechanistic aspects underlying these nuclear magnetic resonance studies indicated causes perturba- physiological functions remain elusive, current understanding of tions in the overall structure and to be close to a Trp or Phe mitoNEET correlates to the crucial role of the redox-active [2Fe–2S] residues, although the exact residues still need to be determined2. clusters, possibly serving as an outer-membrane redox-sensor and pH In accordance, the Phe82 side chain is within a 5 Å distance from sensor for mitochondrial function and/or a potential source of the the furosemide furan ring in the refined structure, which could iron–sulfur cluster transfer to cytosol in response to the redox states suggest that this is the same binding site that pioglitazone in the cells8,9. occupies. We previously identified other possible binding pockets Despite the fact that mitoNEET was described relatively from docking studies, which was located near the α-helix of recently, numerous crystal structures have been reported for both mitoNEET, and predicted this to be the primary site for the wild-type and mutants of the soluble portions (PDB codes resveratrol-3-sulfate and pioglitazone binding15,18. Considering 2QH7, 2QDO, 2R13, 3EW0, 3REE, 3LPQ, 4EZF, 4F1E, 4F28, and also the previous report that pioglitazone inhibits cluster release 4F2C). However, none of these crystal structures includes a from mitoNEET under oxidative stress19, it seems likely that the bound ligand that has drug-like activity. Here, we describe the compounds binding in the furosemide pocket would stabilize the crystal structure of a mitoNEET–ligand complex with furosemide. ligation environment of the [2Fe–2S] cluster. The observation This structure can be used to gain structural insights into mito- that mutation of HIS87 to CYS has a cluster stabilizing effect and NEET binding to ligands, as well as be used in structure-based caused a significant shift in the reduction potential is also drug discovery (SBDD) studies. consistent with this model20. Regardless of whether the main function of mitoNEET in the cell is as a redox sensor, iron chaperone, or as an electron-transport protein, binding of a Results ligand so near to the cluster is likely to have a significant impact Co-crystallization study. Based on the published literature, mito- on cellular energetics. NEET represents a potential drug target for the development of compounds to treat a variety of metabolic diseases6,7,14.Unfortu- nately, no crystal structure has been solved with a ligand bound to the Ligand development. As proof of principle, we developed a small protein for use in SBDD studies. Here, we present the first crystal pilot set of compounds to validate that the interaction between structure at 1.95 Å resolution of human mitoNEET with a bound furosemide