Abstract
Methylmalonic aciduria and homocystinuria, cblC type, is the most common inborn error of cellular vitamin B12 metabolism. We previously showed that the protein carrying the mutation responsible for late-onset cblC (MMACHC-R161Q), treatable with high dose OHCbl, is able to bind OHCbl with wild-type affinity, leaving undetermined the disease mechanism involved [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. To assess whether the mutation renders the protein unstable, we investigated the thermostability of the wild-type and mutant MMACHC proteins, either unbound or bound to different cobalamins (Cbl), using differential scanning fluorimetry. We found that MMACHC-wt and MMACHC-R161Q are both very thermolabile proteins in their apo forms, with melting temperatures (Tm) of 39.3±1.0 and 37.1±0.7°C, respectively; a difference confirmed by unfolding of MMACHC-R161Q but not MMACHC-wt by isothermal denaturation at 35°C over 120min. However, with the addition of OHCbl, MMACHC-wt becomes significantly stabilized (ΔTmmax=8°C, half-maximal effective ligand concentration, AC50=3μM). We surveyed the effect of different cobalamins on the stabilization of the wild-type protein and found that AdoCbl was the most stabilizing, exerting a maximum increase in Tm of ∼16°C, followed by MeCbl at ∼13°C, each evaluated at 50μM cofactor. The other cobalamins stabilized in the order (CN)2Cbi>OHCbl>CNCbl. Interestingly, the AC50’s for AdoCbl, MeCbl, (CN)2Cbi and OHCbl were similar and ranged from 1–3μM, which compares well with the Kd of 6μM for OHCbl [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. Unlike MMACHC-wt, the mutant protein MMACHC-R161Q is only moderately stabilized by OHCbl (ΔTmmax=4°C). The dose–response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC50 of 7μM. MMACHC-R161Q showed the same order of stabilization as MMACHC-wt, but each cobalamin stabilized this mutant protein less than its wild-type counterpart. Additionally, MMACHC-R161Q had a higher AC50 for each cobalamin form compared to MMACHC-wt. Finally, we show that MMACHC-R161Q is able to support the base-off transition for AdoCbl and CNCbl, indicating this mutant is not blocked in that respect. Taken together, our results suggest that protein stability, as well as propensity for ligand-induced stabilization, contributes to the disease mechanism in late-onset cblC disorder. Our results underscore the importance of cofactor stabilization of MMACHC and suggest that even small increases in the concentration of cobalamin complexed with MMACHC may have therapeutic benefit in children with the late-onset, vitamin responsive cblC disease.
Highlights
Vitamin B12 is a complex organometallic molecule whose structure was determined by Hodgkin et al [1] in1096-7192 Ó 2010 Elsevier Inc
Using differential scanning fluorimetry (DSF), we demonstrate that MMACHC-wt is an exceedingly labile protein, with a melting temperature near physiologic temperature, which, is strongly stabilized by binding to cobalamins
In order to investigate the thermostability of MMACHC we used DSF, a method that measures protein stability and ligand-induced changes in stability [20,21]
Summary
Vitamin B12 (cobalamin, Cbl) is a complex organometallic molecule whose structure was determined by Hodgkin et al [1] in. 1956 after being first isolated by Smith [2] and Rickes et al [3]. The cobalamin core consists of a cobalt atom caged in a corrin ring (Fig. 1). Extending beneath the corrin ring is the 5,6-dimethylbenzimidazole (DMB) base which may be coordinated to the cobalt atom to form the lower or a-axial ligand. The structure is considered ‘‘base-on”; when unattached, ‘‘base-off”. If the DMB moiety is not present, the structure is called cobinamide (Cbi).
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