Mechanism of activation of bacterial cellulose synthase by cyclic di-GMP

Abstract

The bacterial signaling molecule cyclic di-GMP (c-di-GMP) stimulates the synthesis of bacterial cellulose, which is frequently found in biofilms. Bacterial cellulose is synthesized and translocated across the inner membrane by a complex of cellulose synthase BcsA and BcsB subunits. Here we present crystal structures of the c-di-GMP-activated BcsA-BcsB complex. The structures reveal that c-di-GMP releases an autoinhibited state of the enzyme by breaking a salt bridge that otherwise tethers a conserved gating loop that controls access to and substrate coordination at the active site. Disrupting the salt bridge by mutagenesis generates a constitutively active cellulose synthase. Additionally, the c-di-GMP-activated BcsA-BcsB complex contains a nascent cellulose polymer whose terminal glucose unit rests at a new location above BcsA's active site and is positioned for catalysis. Our mechanistic insights indicate how c-di-GMP allosterically modulates enzymatic functions.

Figure 1

Structure of the c-di-GMP-activated BcsA–B complex. (a) Cartoon representation of the BcsA–B structure in the presence of a c-di-GMP homo-dimer. BcsA is colored brown, green and red for its GT domain, TM region, and C terminus, respectively. The 6-stranded β-barrel within BcsA’s C terminus forms a c-di-GMP binding PilZ domain. BcsB is shown in blue. The c-di-GMP dimer and translocating cellulose polymer are shown in spheres. BcsA’s finger helix and gating loop are colored yellow and steel blue, respectively. IF: Amphipathic interface helices that surround the cytosolic entrance to BcsA’s TM channel. Horizontal bars indicate the putative membrane boundaries. (b) Comparison of BcsA’s PilZ positions in the presence and absence of c-di-GMP. BcsA is shown as a pale gray surface, and BcsA’s C terminus is shown as a red cartoon. The position of the β-barrel in the c-di-GMP-free state (pdb 4HG6) is shown as a gray cartoon and c-di-GMP is shown as spheres. (c) Interactions of the “RxxxR” and “DxSxxG” motifs with the c-di-GMP dimer. Residues of each motif are shown as yellow sticks and c-di-GMP is shown in sticks and spheres.

Figure 2

Conformational changes of BcsA’s gating loop. (a) Gating loop positions in the absence and the presence of c-di-GMP (shown in cyan and steel blue, respectively). Phe503 and Val505 of the “FxVTxK” motif are shown as sticks and the gating loop’s pivots, Arg499 and Glu514, are shown as spheres. The C terminus is colored as in Fig. 1. Inset: residues involved in stabilizing the gating loop in the “open” position are shown as sticks. (b) Accessible volume at the active site entrance (dark blue mesh) in the absence (left) and the presence (right) of c-di-GMP, calculated with a 3.5 Å probe sphere. UDP in the resting (pdb 4HG6) and c-di-GMP in the open BcsA–B structure are shown as spheres.

Figure 3

Stabilization of the gating loop by Arg580. A comparison of the Arg580 position in the absence and presence of c-di-GMP. Arg580 is shown as dark gray and red in the absence and presence of c-di-GMP, respectively. The gating loop is shown in cyan and steel blue representing the “resting” and “open” states, respectively. Glu371 is shown in sticks, and putative interactions are indicated. BcsA’s PilZ domain is colored red and the TM8-β-barrel linker in the resting state is shown as a dark gray cartoon.

Figure 4

Insertion of the gating loop into the catalytic pocket. (a) A comparison of the gating loop positions of c-di-GMP-bound BcsA–B in the absence and presence of UDP (shown in steel blue and green, respectively). The inserted gating loop is colored green, IF2 is shown as a gray cartoon helix, and UDP as well as the translocating cellulose polymer are shown as sticks. Trp383 of the “QxxRW” motif at the entrance to the TM channel is shown as gray sticks. (b) Coordination of UDP at the active site by the gating loop’s “FxVTxK” motif. The gating loop is colored green, representing the “inserted” state. UDP, the conserved residues of the gating loop as well as Gln379 and Arg382 of the “QxxRW” motif are shown as sticks. The terminal glucose of the cellulose polymer and the putative catalytic base (Asp343) are shown as cyan and gray sticks, respectively.

Figure 5

Comparison of BcsA-catalyzed in vitro cellulose synthesis in the absence and the presence of c-di-GMP. (a and b) Inverted membrane vesicles and proteoliposomes (PL), respectively, containing BcsA–B with the indicated mutations in BcsA were used for cellulose synthesis assays. (WT: wild type). The activity in the absence of c-di-GMP is quantified relative to the activity in the presence of 30 μM c-di-GMP. Insets: Western analysis of IMVs against the C-terminal poly-histidine tag on BcsA (a) and Coomassie-stained SDS-PAGE of the purified BcsA–B complexes (b). (c) Catalytic rates of the indicated PL-reconstituted BcsA–B mutants in the presence and absence of 30 μM c-di-GMP as measured by quantifying the formation of UDP. (d) Activity of the PL-reconstituted BcsA R580A mutant at increasing c-di-GMP concentrations. No activity is observed in the presence of 30 μM c-di-GMP when magnesium is depleted with 25 mM EDTA (–Mg²⁺). DPM: Disintegrations per minute. (All data represent the means ± SD for 3 technical replicates).

Figure 6

Movement of the finger helix, cellulose translocation, and the acceptor position. (a) Comparison of the positions of BcsA’s finger helix and translocating glucan in the resting (colored gray, pdb 4HG6) and c-di-GMP-bound states. An unbiased SigmaA-weighted mFo–DFc difference electron density of the translocating cellulose polymer in the UDP-free, c-di-GMP-bound state is contoured at 3.5σ and shown as a magenta mesh. The positions of the glucan as observed in the resting state and the c-di-GMP-bound structure are shown as gray and cyan sticks, respectively. BcsA’s finger helix and the preceding small loop are colored yellow. (b) Cut-away view of BcsA’s TM channel with the position of the finger helix in the c-di-GMP-bound and resting states shown as yellow and gray solid cylinders, respectively. The translocating glucan is shown as cyan and red spheres and Asp343 is shown as sticks. Membrane boundaries are indicated by horizontal black lines. (c) Conserved residues involved in stabilizing the finger helix in the “up” position are shown as sticks. (d) Residues coordinating the polymer’s terminal glucose are shown as sticks. (e) Stabilization of a single galactose molecule by the sodium-dependent sugar transporter vSGLT. Residues coordinating galactose are shown as sticks (pdb 3DH4).