NMR Structural and Biophysical Analysis of the Disease-Linked Inner Mitochondrial Membrane Protein MPV17 |
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| Institution: | 1. Structural Membrane Biochemistry, Bavarian NMR Center (BNMRZ) at the Department of Chemistry, Technical University of Munich, Ernst-Otto-Fischer-Str. 2, 85748 Garching, Germany;2. Institute of Structural Biology, Helmholtz Center Munich, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany;1. Department of Biochemistry and Molecular Biophysics, Box 8231, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, United States;2. Department of Physics, Washington University in St. Louis, St. Louis, MO 63130, United States;1. New York Structural Biology Center, New York, NY, USA;2. Department of Biomedical Sciences, University at Albany, Albany, NY, USA;3. Division of Infectious Diseases, Wadsworth Center, New York State Department of Health, Albany, NY, USA;4. Department of Molecular Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Montebello, Oslo, Norway;5. Department of Biosciences, University of Oslo, Oslo, Norway;1. Centre de Biophysique Moléculaire, CNRS UPR4301, Rue Charles Sadron, 45071 Orléans cedex 2, France;2. ED 549, Santé, Sciences Biologiques & Chimie du Vivant, Université d''Orléans, France;1. Institute of Biochemistry, Graz University of Technology, 8010 Graz, Austria;2. BioTechMed-Graz, 8010 Graz, Austria;1. Department of Biochemistry, University of Bayreuth, 95447 Bayreuth, Germany;2. Biophysical Chemistry, Humboldt-University Berlin, 10115 Berlin, Germany;3. Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Victoria 3800, Australia;4. Biopolymers, University of Bayreuth, 95447 Bayreuth, Germany;5. Bayreuth Center for Biochemistry & Molecular Biology, University of Bayreuth, 95447 Bayreuth, Germany;6. North-Bavarian NMR Center, University of Bayreuth, 95447 Bayreuth, Germany |
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| Abstract: | MPV17 is an integral inner mitochondrial membrane protein, whose loss-of-function is linked to the hepatocerebral form of the mitochondrial-DNA-depletion syndrome, leading to a tissue-specific reduction of mitochondrial DNA and organ failure in infants. Several disease-causing mutations in MPV17 have been identified and earlier studies with reconstituted protein suggest that MPV17 forms a high conductivity channel in the membrane. However, the molecular and structural basis of the MPV17 functionality remain only poorly understood. In order to make MPV17 accessible to high-resolution structural studies, we here present an efficient protocol for its high-level production in E. coli and refolding into detergent micelles. Using biophysical and NMR methods, we show that refolded MPV17 in detergent micelles adopts a compact structure consisting of six membrane-embedded α-helices. Furthermore, we demonstrate that MPV17 forms oligomers in a lipid bilayer that are further stabilized by disulfide-bridges. In line with these findings, MPV17 could only be inserted into lipid nanodiscs of 8–12 nm in diameter if intrinsic cysteines were either removed by mutagenesis or blocked by chemical modification. Using this nanodisc reconstitution approach, we could show that disease-linked mutations in MPV17 abolish its oligomerization properties in the membrane. These data suggest that, induced by oxidative stress, MPV17 can alter its oligomeric state from a properly folded monomer to a disulfide-stabilized oligomeric pore which might be required for the transport of metabolic DNA precursors into the mitochondrial matrix to compensate for the damage caused by reactive oxygen species. |
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| Keywords: | dynamics membrane protein stability mitochondrial diseases nanodiscs reactive oxygen species |
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