Extended Data Fig. 4: Characterization of the D-2HG-HPHD-1 interaction.
From: A feedback loop engaging propionate catabolism intermediates controls mitochondrial morphology
a, Predication of D-2HG binding to HPHD-1. HPHD-1 structure is predicted using alpha-Fold (https://alphafold.ebi.ac.uk/). D-2HG (in green and orange) docking on HPHD-1 (in blue) is predicted using the Molegro Virtual Docker (http://molexus.io/molegro-virtual-docker/). Amino acid residues involved in binding with D-2HG are labeled in orange. b, Left: Graphic depiction of HPHD-1 truncations (top) and gel images showing recombinant HPHD-1 proteins (bottom). Right: Binding curves of D-2HG with HPHD-1 proteins using MST assays. Data (mean) are from two independent experiments. c, Top: Schematic depiction of the forward reaction of HPHD-1 dehydrogenase using isopropanol and NAD+ as substrates. Bottom: HPHD-1 dehydrogenase activity without or with D-2HG was measured at the reaction times 0.5 h (left) and 1.5 h (right) by quantifying the production of NADH as in Fig. 4e. n = 3 independent experiments. d, Top: Schematic depiction of the reverse reaction catalyzed by HPHD-1 using 2-propanone and NADH as substrates. HPHD-1 activity without or with D-2HG at the indicated reaction time points was measured by quantifying the reduction of NADH. The B. licheniformis NADH Oxidase (B.l. Oxidase) was used as positive control. n = 3 independent experiments. For statistical analyses, P values were determined using one-way ANOVA (c and d). Individual data points (mean ± s.e.m. and exact P value) are shown. ****P < 0.0001; ***P < 0.001; ns, P > 0.05. Source numerical data and unprocessed gels are available in source data.
