Fig. 3: Investigation of the PAS-PAC/EGTQF hinge of DosP.

a Atomic models of the straight and bent forms of DosP^R97A depicted as grey cartoon or blue cartoon, respectively. Box highlights the PAS-PAC/EGTQF hinge helix (residues 352-382). Middle inset: Atomic models of residues 352-395 of the bent and straight forms of DosP^R97A. NH₂ and COOH termini are labeled with N and C, respectively. Positions of residues interrogated by mutagenesis are labeled yellow on the model of DosP^R97A straight. Top right inset: Cryo-EM map of DosP^R97A straight form. Asterisk denotes the well-resolved hinge helix. Bottom right inset: Cryo-EM map of DosP^R97A bent form. Asterisk denotes the unresolved hinge helix. b Atomic models of the EAL and EGTQF domains of the straight and bent forms of DosP^R97A depicted as grey cartoon or blue cartoon, respectively. Arrows indicate changes of the helix between the EAL and EGTQF domains. c The EGTQF dimer interface, viewed as if the position in b was rotated 90° upwards towards the reader. Top: the straight form of DosP^R97A shown as grey cartoon. The two chains and dimer interface loops are labeled. Bottom: the bent form of DosP^R97A shown as blue cartoon. d Absorption spectra of DosP^Q378P, DosP^E381P, and DosP^Q382P under aerobic conditions (red, magenta, and orange, respectively), compared to anaerobic conditions (dark blue, blue, and cyan, respectively). e Bar graph showing the k_cat values determined from the turnover of 2 µM c-di-GMP at 25°C by 10 nM aerobic: DosP^WT, DosP^Q378P, DosP^E381P, and DosP^Q382P. Value for DosP^WT is taken from Fig. 1g, values for mutants are the average of two independent experiments. In d and e the assay buffer was 20 mM Tris-HCl, 2.5 mM MgCl₂, 2.0 mM DTT, pH 8.0. Source data are provided as a Source Data file. Cartoons depicted in a and b generated using PyMOL. Inset maps in a and cartoons in c generated using ChimeraX. EAL, c-di-GMP phosphodiesterase domain; EGTQF- diguanylate cyclase domain.