Fig. 6: Structural details of DosPR97A with bound c-di-GMP.
a Turnover of 100 µM c-di-GMP at 25 °C by 10 nM aerobic DosPWT or DosPR97A. The buffer for the assays was 20 mM Tris-HCl, 2.5 mM MgCl2, 2.0 mM DTT, pH 8.0. Values are the mean of two independent experiments. b Cryo-EM map of substrate-bound DosPFLAG-R97A in complex with c-di-GMP (colored magenta) with the two protein chains colored green and lavender. c Atomic model depicted as cartoon and overlaid with a map that was gaussian filtered to level 1.5. d Density and atomic model for the hPAS domains with their bound heme. e Density and atomic model for one of the EAL domains with its bound c-di-GMP and magnesium ions. f Atomic models of substrate-bound DosPFLAG-WT and DosPFLAG-R97A aligned by their hPAS domains and colored green and magenta, respectively. Domains are labeled with brackets. Arrows indicate differences in conformation. g The hPAS domains of substrate-bound DosPFLAG-WT and DosPFLAG-R97A aligned and colored as in f. One monomer is set partially transparent. Arrows label differences in conformation. h The hPAS dimer of DosPFLAG-WT depicted as green cartoon. Heme depicted as sticks. Residues 93-95 and 114−114 of one monomer are highlighted as yellow cartoon and the side chain atoms of M30 from the other monomer are depicted with spheres. i The hPAS dimer of DosPFLAG-R97A depicted as magenta cartoon. Heme depicted as sticks. Residues 93-95 of one monomer are highlighted as yellow cartoon and the side chain atoms of M30 from the other monomer are depicted with spheres. j A hydrogen bond between R131 and E136 links the hPAS domain to the helix leading to the PAS-PAC domain. DosPFLAG-WT and DosPFLAG-R97A are colored green and magenta, respectively. k Changes in the position of Me1 and Me2 between DosPFLAG-WT and DosPFLAG-R97A. The c-di-GMP and DosP are depicted as in f. The magnesium ions shown as green or pink spheres correspond to DosPFLAG-WT and DosPFLAG-R97A, respectively. Source data are provided as a Source Data file. Panels b-k Generated using ChimeraX and PyMOL. hPAS, heme-binding Per-Arnt-Sim; EAL, c-di-GMP phosphodiesterase domain; EGTQF- diguanylate cyclase domain.
