Extended Data Fig. 2: Biochemical and structural analysis of BsForCE1 and variants.
From: A scaffold for quinone channeling between membrane and soluble bacterial oxidoreductases
a. ForC and ForE form a complex with a 4:4 stoichiometry as shown by SEC-MALS analysis of BsForCE1 WT (blue curve), BsForCE1F128D-R132D (green curve) and BsForCE1ΔHMP (orange curve). Left y-axis, molar mass; right y-axis normalized UV chromatograms, measured molar mass in coloured stars. For BsForCE1 WT and BsForCE1F128D-R132D, complexes have a MW of 512 ± 1.5 and 495 ± 1.5 kDa respectively. For BsForCE1ΔHMP, the complex has a MW of 470 ± 1.9 kDa. In addition, two proteins of 117 ± 1.2 kDa and 111 ± 0.8 kDa could correspond to a monomer of ForC1. b., c. Inhibition of the formate:O2 consumption by respiratory chain inhibitors. WT strain was grown with 12.5 mM formate. O2 consumption was measured using an oxygen electrode after formate spiking, in presence of 1.29 to 27.1 μM HQNO (b) or 0.16 to 7.6 mM KCN (c). 100% corresponds to 153 nmol O2/min/mg. d., e. Michaelis Menten plot of BsForCE1F128-DR132D (d) and BsForCE1ΔHMP (e) coupling formate oxidation to menadione reduction in vitro. Data from n = 3 biological replicates are presented as mean values +/− SD. f. Conservation of hydrophobic and positively charged residues at the respective F128 and R132 positions in the DUF1641 family. Sequences from Interpro IPR012440 (DUF1641) family were clustered and restricted to 644 representatives using MMseqs2 (Many-against-Many searching, https://toolkit.tuebingen.mpg.de/tools/mmseqs2) online software (minimum sequence identity>0.5; minimum alignment coverage>0.8). Then, multiple sequence alignment (MSA) was performed using Clustal Ω (default parameters). Finally, HMM logo was generated using http://skylign.org/ with the previously described MSA (from K118 to Q155, BsForE1 numbering) to consider both helix 7 and 8. Each stack represents one residue position within the MSA. Height of a letter within a stack represents the frequency of the corresponding residue at this position. The sequence of BsForE1 is indicated below the HMM logo and helices 7 and 8 are indicated. g. X-ray structure of BsForCE1ΔHMP superimposes with BsForCE1. BsForCE1ΔHMP is colored according to the b-factor value while BsForCE1 is colored in gray. For clarity, one ForC subunit is shown on both structures.
