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. 2015 Oct 19;290(49):29167–29177. doi: 10.1074/jbc.M115.683268

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© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Author's Choice—Final version free via Creative Commons CC-BY license.

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FIGURE 1. — Interaction study of MMACHC and MMADHC. A, domain diagram showing recombinant human MMACHC and MMADHC constructs described in this figure. For reference, mouse construct MmMMADHC_Δ128 used to generate the crystal structure is also shown. B, interaction of human MMACHC full-length and MMADHC truncated proteins by BN-PAGE. Lanes 1–8: single protein controls including MMACHC_FL alone (lane 1) and with MeCbl and GSH (lane 2); and MMADHC truncations including MMADHC_Δ61 (lane 3), MMADHC_Δ123 (lane 4), MMADHC_Δ153 (lane 5), MMADHC_Δ157 (lane 6), MMADHC_Δ167 (lane 7), and MMADHC_Δ172 (lane 8). Lanes 9–14: combined protein incubation of MMACHC_FL in presence of MeCbl and GSH with MMADHC_Δ61 (lane 9), MMADHC_Δ123 (lane 10), MMADHC_Δ153 (lane 11), MMADHC_Δ157 (lane 12), MMADHC_Δ167 (lane 13), and MMADHC_Δ172 (lane 14). C, interaction of MMACHC mutants and MMADHC_Δ123 by BN-PAGE, in the presence of MeCbl and GSH. Lanes 1–6: MMACHC_ΔC alone (lane 1) and with MMADHC_Δ123 (lane 2); MMACHC_R161Q alone (lane 3) and with MMADHC_Δ123 (lane 4); and MMACHC_ΔPNR alone (lane 5) and with MMADHC_Δ123 (lane 6). For B and C, white dots indicate MMACHC homodimer, and white asterisks indicate MMACHC-MMADHC heterodimer. D, analysis of complex formation by size-exclusion chromatography including chromatographs of MmMMADHC_Δ123 (gray line), HsMMACHC_ΔC + MeCbl + GSH (black line), and MmMMADHC_Δ123 + HsMMACHC_ΔC + MeCbl + GSH (black dotted line). x axis: elution volume; left y axis: absorbance (milliabsorbance units (mAU)) for HsMMACHC_ΔC + MeCbl + GSH; right y axis: absorbance (mAU) for MmMMADHC_Δ123 and MmMMADHC_Δ123 + HsMMACHC_ΔC + MeCbl + GSH. Molecular weights of each peak are calculated from a calibration curve using molecular weight protein standards (Sigma-Aldrich). E, sequence alignment of MMADHC from H. sapiens (HS, NP_056517.1, human) and predicted MMADHC protein (Y76A2B.5) from C. elegans (Ce) using the BLAST server (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The boxed region represents the smallest interacting construct of HsMMADHC for HsMMACHC. F, BN-PAGE of HsMMACHC_FL and HsMMADHC_Δ123 under various conditions. White asterisks indicate MMACHC-MMADHC heterodimer. G, native mass spectrometry of HsMMACHC_FL and HsMMADHC_Δ123 incubated with MeCbl and GSH. Expected sizes: HsMMACHC_FL, 31818.6 Da; HsMMADHC_Δ123, 19727.7 Da; MeCbl, 1343.6 Da; and GSH, 307.3 Da.