Supplementary Figure 5: Cellulose synthesis activity of BcsA mutants and c-di-GMP binding.
From: Mechanism of activation of bacterial cellulose synthase by cyclic di-GMP
(a) Cellulose synthesis assays were performed in inverted membrane vesicles and proteoliposomes as described in the Experimental Procedures. The amount of 3H-glucose-labeled cellulose produced by each mutant is quantified and graphed. 1 μl of IMV's were used for each mutant. For PL assays, the protein concentrations were matched based on UV absorbance and SDS-PAGE followed by Coomassie staining. The apparent lower activity of the R580A mutant may be due to differences in the relative orientation of the enzyme in the PLs or its overall stability. +/-: Experiments performed in the presence and absence of 30 μM c-di-GMP. All data represent the means ± SD for 3 technical replicates. (b) Binding of c-di-GMP to the BcsA-R580A mutant. The ability of the R580A mutant to bind c-di-GMP was assessed using ITC. Left panel, titrating c-di-GMP into wild type (WT) BcsA-B in 1 mM LFCE14 results in an exothermic curve with a Kd of 0.4 μM and a c-di-GMP to BcsA-B stoichiometry of ~2:1 as expected based on the crystal structure. Right panel, BcsA-R580A under the same conditions exhibits a Kd of 1.6 μM and a stoichiometry of ~0.5. The observation that only 1/2-1/4 of the BcsA-R580A population (depending on whether the mutant binds a c-di-GMP monomer or dimer) interacts with c-di-GMP suggests that a fraction of it is structurally compromised, consistent with the results obtained from functional assays (a).
