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. 2020 Jul 27;11:3740. doi: 10.1038/s41467-020-17457-1

Fig. 6. Key roles for R685, N817, and R868 in DNA loading.

Fig. 6

a Steady-state ATPase activities of single Mfd variants. ATP hydrolysis rates were measured using an ATP/NADH-coupled ATPase assay. Data shown are the means ± SD (n = 3). Variants are color-coded by domain as in Fig. 2a (D5 variants, yellow hues; D6 variants, green hues). Data for MfdR685A and MfdN817A are replotted for convenience from Le et al.16. b–c Equilibrium dissociation constants for the Mfd-dsDNA interaction derived from fluorescence anisotropy binding curves. D5 and D6 variants are color-coded by domain as in Fig. 2a. Data represent mean values ± SD (n = 3). Error bars are often smaller than symbols. Arrows indicate data sets that could not be reliably fit to a binding model owing to compromised affinity for DNA, and likely represent an overestimation of the true affinity. Binding curves are shown in Supplementary Fig. 7. d–j Structural comparison between double-stranded nucleic-acid translocases and Escherichia coli (Eco) Mfd (d) using superpositions restricted to the Cα trace of D5. Shown are Sulfolobus solfataricus (Sso) Snf2 (r.m.s.d. of 1.1 Å, e), Saccharomyces cerevisiae (Sce) Snf2 in its nucleotide-free (r.m.s.d of 1.4 Å, f) and ADP•BeFx -bound state (r.m.s.d. of 1.4, g), RIG-1 (r.m.s.d. of 1.0 Å, h) and ssRNA translocases, Drosophila melanogaster (Dme) Vasa (r.m.s.d. of 0.9 Å, i) and HCV NS3 (r.m.s.d. of 1.1 Å, j). Bound Mg2+ is shown as a dark green sphere, analog ADP•AlF3− as an aquamarine CPK model, bound nucleotide as magenta sticks, ADP as a red CPK model, BeFx as a dark red CPK model and nucleic-acid strands are colored red and orange. The two lobes of the ATPase core are in yellow (N-terminal lobe) and green (C-terminal lobe), except for DNA-binding residues, which are highlighted as slate CPK models. Other domains are shown as a gray ribbon with annotated ATPase motifs. Those motifs contacting DNA are color-coded in slate font and some are not visible in these views. Indicated in parenthesis is the degree of conservation between the various motor cores and D5 and D6 of E. coli Mfd. Source data are provided as a Source Data file.