Structural Insights into the MMACHC-MMADHC Protein Complex Involved in Vitamin B12 Trafficking.
Conversion of vitamin B12 (cobalamin, Cbl) into the cofactor forms methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl) is required for the function of two crucial enzymes, mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase, respectively. The intracellular proteins MMACHC and MMADHC play important roles in processing and targeting the Cbl cofactor to its destination enzymes, and recent evidence suggests that they may interact while performing these essential trafficking functions. To better understand the molecular basis of this interaction, we have mapped the crucial protein regions required, indicate that Cbl is likely processed by MMACHC prior to interaction with MMADHC, and identify patient mutations on both proteins that interfere with complex formation, via different mechanisms. We further report the crystal structure of the MMADHC C-terminal region at 2.2 Å resolution, revealing a modified nitroreductase fold with surprising homology to MMACHC despite their poor sequence conservation. Because MMADHC demonstrates no known enzymatic activity, we propose it as the first protein known to repurpose the nitroreductase fold solely for protein-protein interaction. Using small angle x-ray scattering, we reveal the MMACHC-MMADHC complex as a 1:1 heterodimer and provide a structural model of this interaction, where the interaction region overlaps with the MMACHC-Cbl binding site. Together, our findings provide novel structural evidence and mechanistic insight into an essential biological process, whereby an intracellular "trafficking chaperone" highly specific for a trace element cofactor functions via protein-protein interaction, which is disrupted by inherited disease mutations.
From the Division of Metabolism and Children's Research Center, University Children's, Hospital, CH-8032 Zurich, Switzerland, radiz - Rare Disease Initiative Zurich, Clinical Research Priority Program for Rare Diseases, University of Zurich, CH-8032 Zurich, Switzerland.
D Sean
DS
Froese
the Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom, and.
Jolanta
J
Kopec
the Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom, and.
Fiona
F
Fitzpatrick
the Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom, and.
Marion
M
Schuller
the Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom, and.
Thomas J
TJ
McCorvie
the Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom, and.
Rod
R
Chalk
From the Division of Metabolism and Children's Research Center, University Children's, Hospital, CH-8032 Zurich, Switzerland.
Tanja
T
Plessl
From the Division of Metabolism and Children's Research Center, University Children's, Hospital, CH-8032 Zurich, Switzerland.
Victoria
V
Fettelschoss
From the Division of Metabolism and Children's Research Center, University Children's, Hospital, CH-8032 Zurich, Switzerland.
Brian
B
Fowler
From the Division of Metabolism and Children's Research Center, University Children's, Hospital, CH-8032 Zurich, Switzerland, radiz - Rare Disease Initiative Zurich, Clinical Research Priority Program for Rare Diseases, University of Zurich, CH-8032 Zurich, Switzerland, the Zurich Center for Integrative Human Physiology, University of Zurich, 8057 Zurich, Switzerland matthias.baumgartner@kispi.uzh.ch.
Matthias R
MR
Baumgartner
the Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom, and wyatt.yue@sgc.ox.ac.uk.
Wyatt W
WW
Yue
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008659:Metabolic Diseases / Q000378:metabolism; D051379:Mice; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009601:Nitroreductases / Q000737:chemistry; D010088:Oxidoreductases; D010641:Phenotype; D011485:Protein Binding; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014805:Vitamin B 12 / Q000737:chemistry*
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C527938:MMADHC protein, human; D033681:Mitochondrial Membrane Transport Proteins; C570225:Mmadhc protein, mouse; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C508359:MMACHC protein, human; D010088:Oxidoreductases; D009601:Nitroreductases; D014805:Vitamin B 12
crystal structure; metabolic disease; nitroreductase fold; protein-protein interaction; site-directed mutagenesis; small-angle x-ray scattering (SAXS); vitamin B12