Human intracellular vitamin B12 metabolism: Insight into human disease from structural and interaction studies

Abstract

Vitamin B12 (cobalamin, Cbl) in its cofactor forms methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl), is essential for the function of the enzymes methionine synthase (MS) and methylmalonyl-CoA mutase (MUT), respectively. MS catalyzes the remethylation of homocysteine to methionine, which may function as an essential amino acid or be further converted S-adenosylmethionine, an important cellular methyl-donor. MUT catalyzes the conversion of L-methylmalonyl-CoA to succinyl-CoA, an important step in the breakdown of odd-chain fatty acids, branched-chain amino acids and the side-chain of cholesterol, and whose product functions as an anapleurotic substrate for the tricarboxylic acid cycle. Both enzymes are vitally important to the human body, as deficiency of either enzyme, or the production of their cofactor forms, results in inborn errors that vary widely in their clinical presentation but at their most severe state cause coma and death. In order to facilitate the intracellular transport and modification of Cbl to these destination enzymes, a sophisticated pathway of protein transporters, chaperones and modifiers has evolved. This pathway is necessary to ferry free Cbl out of the lysosome and across the cytosol for MS, and into the mitochondria for MUT. Additionally, since Cbl is a scarce and reactive substance inside the cell, this pathway must also ensure Cbl sequestration and timely delivery. Thus far, 7 proteins have been identified to perform these essential functions. The goal of the presented work was to use cellular, biochemical and structural techniques to understand how they achieve these processes, and why autosomally inherited mutations in the genes encoding these proteins results in dysfunction and disease. Following intracellular uptake by receptor-mediated endocytosis, intralysosomal Cbl must be exported across the lysosomal membrane into the cytosol, a process that requires the integral membrane proteins LMBD1 and ABCD4. Although known to be required for lysosomal Cbl export, little was known about the function of these two proteins. We found that ABCD4 requires LMBD1 for targeting to the lysosome and that these proteins specifically and selectively interact. Further, we found that this interaction, and concomitantly cofactor synthesis, is defective when ABCD4 harbors the patient mutations p.Arg432Gln or p.Asn141Lys, or artificial mutations disrupting the ATPase domain, suggesting the requirement of interaction for lysosomal Cbl export. Cytosolically released Cbl is chaperoned and modified by the protein MMACHC, and targeted either towards MS or MUT by the protein MMADHC. Our X-ray crystal structures of MMACHC and MMADHC demonstrated that, despite very different amino acid sequences, both proteins retain a modified nitroreductase fold. MMACHC utilizes this fold to enhance binding and modification of Cbl, while MMADHC takes advantage of the structural similarity to MMACHC to displace the MMACHC homodimer with a MMACHC-MMADHC heterodimer, thereby targeting Cbl-bound MMACHC to the destination enzymes. However, heterodimerization only occurs following MMACHC processing of Cbl, and is blocked by mutations in MMACHC which reduce processing ability, suggesting Cbl must be in a specific form before it can be further transported within the cell. The MMACHC-MMADHC protein complex is also abrogated by mutations in MMADHC in a specific region known to be important for the delivery of Cbl to both MS and MUT, revealing that this interaction is required for synthesis of both Cbl cofactor forms and interaction disruption may result in disease. Once in the mitochondria, Cbl is processed to AdoCbl by the protein MMAB. MMAB then directly transfers the cofactor to MUT, a process which has been shown in homologous bacterial proteins to be gated by MMAA. Determination of the structures of human MUT and MMAA revealed altered homodimeric assembly compared to bacterial homologs, despite strong subunit structural conservation. Correspondingly, human MUT and MMAA have unique requirements for physical interaction and their hetero-protein complex has a novel molar ratio compared to orthologous proteins. By recapitulating known patient missense mutations on recombinant MMAA, we identified first one (p.Gly188Arg) and then fourteen further MMAA mutations that specifically interfere with the functional interaction of these two proteins, while leaving the intrinsic stability and catalytic activity of MMAA intact. Further, we found that these mutations interfere with the ability of MMAA to gate the transfer of AdoCbl from MMAB to MUT, providing a direct relation to loss of MUT function in patients with MMAA mutations. Together, these studies reveal direct protein-protein interaction to be a crucial component of intracellular Cbl processing. Tandem interactions were found in each cellular organelle investigated, and mutation of the proteins involved, in many cases by known patient mutations, causes disruption of these interactions and loss of Cbl cofactor synthesis. Therefore, maintenance or rescue of these interactions may be an important consideration for future treatment options, and investigation into multi-protein complexes is promising area of further research.