Fig. 4: The c-di-GMP-bound synthase macrocomplex.
a Locally refined cryo-EM map and fitted structure of the crownless c-di-GMP-saturated synthase macrocomplex in two different views. IM inner membrane. b Cartoon representation of the same assembly, summary of the BcsA interactions with the cytosolic vestibule partners and stimulatory effects of BcsR overexpression as detected by binding and UV-fluorescence of E. coli macrocolonies grown on Congo Red-supplemented plates. c A zoom-in on the BcsA-BcsR interface with key residues shown as sticks. The R-D-R triad is indicated with a yellow arrowhead. d c-di-GMP coordination, together with its corresponding electron density, and overall core synthase fold showing unstructured gating loop and an accessible active site. e Effects on cellulose secretion upon BcsRNTD mutagenesis using plasmid-based complementation with various BcsR mutants. KDDA D21K-L25D-F29D-L31D, ADDDA D21A-L25D-F29D-L31D-Y36A. CR Congo Red, CF calcofluor. Data representative of three independent experiments with two biological replicates each. f Consensus ColabFold structural models of Type II BcsA-BcsR (based on multiple BcsA homologs encoded by bcsR- and bcsEF-positive enterobacterial bcs clusters), Type III BcsA (derived from bcsK-positive bcs clusters) and Type I and hybrid BcsA-BcsPNTD (derived from bcsPDQ-positive bcs clusters). BcsA is shown as Coulombic electrostatic potential-colored surface, and BcsR (magenta) and BcsPNTD (cyan) are shown in cartoon. The stabilizing pairs of hydrophobic residues in BcsR and BcsPNTD are shown as sticks.
