Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder

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 120 min. However, with the addition of OHCbl, MMACHC-wt becomes significantly stabilized (ΔTm max = 8 °C, half-maximal effective ligand concentration, AC50 = 3 μM). 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 50 μM 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 1–3 μM, which compares well with the Kd of 6 μM 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 (ΔTm max = 4 °C). The dose–response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC50 of 7 μM. 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

An approach to quality management in structural biology: Biophysical selection of proteins for successful crystallization

Aggregation, incorrect folding and low stability are common obstacles for protein structure determination, and are often discovered at a very late state of protein production. In many cases, however, the reasons for failure to obtain diffracting crystals remain entirely unknown. We report on the contribution of systematic biophysical characterization to the success in structural determination of human proteins of unknown fold. Routine analysis using dynamic light scattering (DLS), differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) was employed to evaluate fold and stability of 263 purified protein samples (98 different human proteins). We found that FTIR-monitored temperature scanning may be used to detect incorrect folding and discovered a positive correlation between unfolding enthalpy measured with DSC and the size of small, globular proteins that may be used to estimate the quality of protein preparations. Furthermore, our work establishes that the risk of aggregation during concentration of proteins may be reduced through DLS monitoring. In summary, our study demonstrates that biophysical characterization provides an ideal tool to facilitate quality management for structural biology and many other areas of biological research

X ray structure of fumarylacetoacetate hydrolase family member Homo sapiens FLJ36880

The human protein FLJ36880 belongs to the fumarylacetoacetate hydrolase family. The Xray structure of FLJ36880 has been determined to 2.2 a resolution employing the semi automated hight hroughput structural genomics approach of the Protein Structure Factory. FLJ36880 adopts a mixed [beta]sandwich roll fold and forms homodimers in crystals as well as in solution. One Mg[2 ] ion is bound to each subunit of the dimeric protein by coordination to three carboxylate oxygens and three water molecules. These metal binding sites are accessible from the same surface of the dimer, partly due to the disorder of the undecapeptide stretch D29 to L39. The overall structure and metal binding site of FLJ36880 bear clear similarities to the Cterminal domain of the bifunctional enzyme HpcE from Escherichia coli C, fumarylacetoacetate hydrolase from Mus musculus and to YcgM Apc5008 from E. coli 1262. These similarities provide a framework for suggesting biochemical functions and evolutionary relationships of FLJ36880. It appears highly probable that the metal binding sites are involved in an enzymatic activity related to the catabolism of aromatic amino acids. Two point mutations in the activesite of FAH, responsible for the metabolic disease hereditary tyrosinemia type I HTI in humans, affect residues that are structurally conserved in FLJ36880 and located in the putative catalytic sit

Crystal structure of Homo sapiens PTD012 reveals a zinc containing hydrolase fold

The human protein PTD012 is the longer product of an alternatively spliced gene and was described to be localized in the nucleus. The X ray structure analysis at 1.7 resolution of PTD012 through SAD phasing reveals a monomeric protein and a novel fold. The shorter splice form was also studied and appears to be unfolded and non functional. The structure of PTD012 displays an amp; 945; amp; 946; amp; 946; amp; 945; four layer topology. A metal ion residing between the central amp; 946; sheets is partially coordinated by three histidine residues. X ray absorption near edge structure XANES analysis identifies the PTD012 bound ion as Zn2 . Tetrahedral coordination of the ion is completed by the carboxylate oxygen atom of an acetate molecule taken up from the crystallization buffer. The binding of Zn2 to PTD012 is reminiscent of zinc containing enzymes such as carboxypeptidase, carbonic anhydrase, and amp; 946; lactamase. Biochemical assays failed to demonstrate any of these enzyme activities in PTD012. However, PTD012 exhibits ester hydrolase activity on the substrate p nitrophenyl acetat