Structural features of recombinant MMADHC isoforms and their interactions with MMACHC, proteins of mammalian vitamin B 12 metabolism Justin C. Deme a , Isabelle R. Miousse b , Maria Plesa a, b , Jaeseung C. Kim b , Mark A. Hancock c , Wayne Mah a, b , David S. Rosenblatt b , James W. Coulton a, ⁎ a Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada b Department of Human Genetics, McGill University, Montreal, Quebec, Canada c Sheldon Biotechnology Centre, McGill University, Montreal, Quebec, Canada abstract article info Article history: Received 28 June 2012 Accepted 1 July 2012 Available online 11 July 2012 Keywords: cblC cblD MMACHC MMADHC Protein–protein interaction Vitamin B 12 The genes MMACHC and MMADHC encode critical proteins involved in the intracellular metabolism of cobalamin. Two clinical features, homocystinuria and methylmalonic aciduria, define inborn errors of these genes. Based on disease phenotypes, MMADHC acts at a branch point for cobalamin delivery, apparently exerting its function through interaction with MMACHC that demonstrates dealkylase and decyanase activities. Here we present biophysical analyses of MMADHC to identify structural features and to further characterize its interaction with MMACHC. Two recombinant tag-less isoforms of MMADHC (MMADHCΔ1-12 and MMADHCΔ1-61) were expressed and purified. Full length MMACHC and full length MMADHC were detected in whole cell lysates of human cells; by Western blotting, their molecular masses corresponded to purified recombinant proteins. By clear-native PAGE and by dynamic light scattering, recombinant MMADHCs were stable and monodisperse. Both species were monomeric, adopting extended conformations in solution. Circular dichroism and secondary structure predictions correlated with significant regions of disorder within the N-terminal domain of MMADHC. We found no evidence that MMADHC binds cobalamin. Phage panning against MMADHC predicted four binding regions on MMACHC, two of which overlap with predicted sites on MMACHC at which it may self-associate. Specific, concentration-dependent responses were observed for MMACHC binding to itself and to both MMADHC constructs. As estimated in the sub-micromolar range, the binding of MMACHC to itself was weaker compared to its interaction with either of the MMADHC isoforms. We propose that the function of MMADHC is exerted through its structured C-terminal domain via interactions with MMACHC. © 2012 Elsevier Inc. All rights reserved. 1. Introduction Vitamin B 12 , or cobalamin (Cbl), is a water soluble vitamin required by all mammals. Because mammals lack the enzymes required for de novo synthesis, Cbl is acquired through dietary intake [1,2]. The physio- logically relevant Cbl forms in mammals are methylcobalamin (MeCbl) and deoxyadenosylcobalamin (AdoCbl), utilized by methionine synthase (MS) and methylmalonyl-CoA mutase (MUT), respectively. MS is a cytoplasmic enzyme that catalyzes methionine regeneration through remethylation of homocysteine, whereas MUT resides in the mitochon- drion and is responsible for the terminal metabolism of odd carbon number fatty acids and the amino acids valine, methionine, isoleucine and threonine, for entry into the tricarboxylic acid cycle. Based on somatic complementation assays, nine inherited defects of intracellular cobalamin metabolism have been discovered, designated cblA to cblJ and mut [3–9]. These defects are characterized by increased production of methylmalonic acid and/or homocysteine, with symp- toms that may include lethargy, hypotonia, developmental delay, seizures, and megaloblastic anemia. Four of these defects (cblC, cblD, cblF, cblJ) can cause combined homocystinuria and methylmalonic aciduria. The cblC class of cobalamin disorders correspond to mutations in the MMACHC ( methyl malonic aciduria type C and homo cystinuria) gene [10] and are the most frequent inborn errors of vitamin B 12 metab- olism with ~500 known cases and over 70 different causal mutations [11–13]. MMACHC encodes a protein of 282 amino acids (31.7 kDa), and based on disease phenotype is predicted to localize to the cyto- plasm. Recent evidence suggests possible localization to the mitochon- drion [14] despite its lacking a predicted mitochondrial leader sequence. MMACHC binds Cbl [15–17] in a base-off conformation [18]. Molecular Genetics and Metabolism 107 (2012) 352–362 Abbreviations: AdoCbl, 5′-deoxyadenosylcobalamin; AUC, analytical ultracentrifuga- tion; Cbl, cobalamin; CD, circular dichroism; CNCbl, cyanocobalamin; CN-PAGE, clear native polyacrylamide gel electrophoresis; DLS, dynamic light scattering; MBP, maltose bind- ing protein; MeCbl, methylcobalamin; MS, methionine synthase; MUT, methylmalonyl-coA mutase; OHCbl, hydroxocobalamin; RU, resonance unit; SEC, size exclusion chromatography; SPR, surface plasmon resonance; TCEP, tris(2-carboxyethyl)phosphine; TEV, tobacco etch virus. ⁎ Corresponding author at: Lyman Duff Medical Building, Room 403, 3775 University Street, Montreal, QC, Canada H3A 2B4. Fax: +1 514 398 7052. E-mail address: james.coulton@mcgill.ca (J.W. Coulton). 1096-7192/$ – see front matter © 2012 Elsevier Inc. 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