Structural features of recombinant MMADHC isoforms and their interactions with MMACHC, proteins of mammalian vitamin B12 metabolism

• Published in: Molecular genetics and metabolism 2012-11, Vol.107 (3), p.352-362
• Main Authors: Deme, Justin C., Miousse, Isabelle R., Plesa, Maria, Kim, Jaeseung C., Hancock, Mark A., Mah, Wayne, Rosenblatt, David S., Coulton, James W.
• Format: Article
• Language: English
• Subjects: aciduria; Amino Acid Sequence; Binding Sites; C.D; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; cblC; cblD; Cell Line; Cell Surface Display Techniques; Circular Dichroism; Conformation; Escherichia coli - genetics; Gene Expression; Homocystinuria; Humans; Light scattering; Metabolism; Mitochondrial Membrane Transport Proteins - chemistry; Mitochondrial Membrane Transport Proteins - genetics; Mitochondrial Membrane Transport Proteins - metabolism; MMACHC; MMADHC; Models, Molecular; Molecular Sequence Data; Mutation; Native Polyacrylamide Gel Electrophoresis; Panning; Phages; Protein Binding; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein structure; Protein Structure, Secondary; Protein–protein interaction; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Secondary structure; Vitamin B 12 - chemistry; Vitamin B 12 - metabolism; Vitamin B12; Western blotting
• ISSN: 1096-7192; 1096-7206
• DOI: 10.1016/j.ymgme.2012.07.001

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. ► Endogenous MMACHC and MMADHC detected in human cell lines. ► MMADHC did not bind cobalamin ► Identified MMADHC- and MMACHC- binding regions on MMACHC ► Disordered N-terminus of MMADHC is not involved in binding MMACHC ► Proposed function of MMADHC exerted through interaction with MMACHC