Crystallographic snapshot of cellulose synthesis and membrane translocation

Abstract

Cellulose, the most abundant biological macromolecule, is an extracellular, linear polymer of glucose molecules. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose production frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB from Rhodobacter sphaeroides containing a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose molecule at a time.

Figure 1. Architecture of the BcsA-B complex

a, BcsA and BcsB form an elongated complex with large cytosolic and periplasmic domains. BcsA’s TM-helices are colored green, the glycosyltransferase (GT) domain sand and the C-terminal domain red. BcsB is shown in light and dark blue for its periplasmic and membrane associated regions, respectively. The N- and C-termini of both subunits are indicated and the translocating glucan and UDP are shown as cyan and violet spheres. Horizontal bars indicate the membrane boundaries. IF: Amphipathic interface helices of BcsA. b, Unbiased FoFc-difference Fourier electron density (pink mesh, contoured at 4.5σ) calculated before modeling the glucan and UDP molecules. The continuous density runs from the intracellular catalytic site to the periplasmic BcsA-B interface and accommodates 18 glucose molecules (cyan sticks). The UDP density was contoured at 3σ (right panel).

Figure 2. Organization of BcsA’s catalytic site and PilZ domain

a, Conserved residues of BcsA coordinate UDP and the terminal disaccharide of the glucan. Side chains represented in sticks belong to the sequence motifs shown in single letter code. Conserved residues are highlighted in bold and the depicted residues are underlined. All side chains shown could be positioned unambiguously in the electron density map. No density was observed for the side chains of Lys226 and Arg382 (shown as spheres for their Cα atoms). The likely position of the donor Glc is indicated by a dashed ellipsoid. b, Surface representation of BcsA with its C-terminal domain shown as cartoon in red. The linker connecting TM8 with the β-barrel forms a 2-stranded β-sheet (shown in cyan) with the IF3/TM7-loop. TM7 is colored dark gray and IF3 orange. The Cα atoms of residues likely involved in cd-GMP binding are shown as spheres and are labeled. The horizontal bar indicates the cytoplasmic membrane boundary.

Figure 3. The membrane-integrated domain of BcsA-B

a, The TM-region includes nine TM-helices and three cytoplasmic and two periplasmic interface helices (IF1-5). BcsA and BcsB are shown as green and blue ribbons. TM- and IF-helices are shown as cartoons and colored in shades of green and orange for BcsA’s TM1-8 and IF1-3, respectively, and shades of blue for BcsB’s IF4 and -5 and its C-terminal TM-anchor. TM3-8 of BcsA form a narrow channel. Its cytoplasmic entrance is formed by IF1-3 as well as the N-terminal half of TM5 and its periplasmic exit is between the periplasmic 5/6- and 7/8-loops of BcsA. Trp383 of the “Q(Q/R)xRW” motif is shown in sticks. b, Conserved residues of BcsA interact with the translocating glucan. BcsA and BcsB are shown as ribbons and side chains of BcsA contacting the glucan (cyan surface) are shown as sticks. Residues of BcsB contacting the polymer are not conserved and not shown.

Figure 4. Organization of the periplasmic domain

a, The periplasmic region of the cellulose synthase is primarily formed by BcsB. It consists of two periplasmic carbohydrate binding domains (CBD-1 and -2) that are connected to two α/β-domains (FD1 and FD2). The CBDs are covalently attached via a disulfide bond between the conserved Cys163 and Cys430 (yellow spheres). BcsB’s TM-anchor and IF-helices 4 and 5 are colored in dark blue. The N- and C-termini of BcsB are indicated. b, Surface representation of BcsB colored according to sequence conservation from red (variable) to deep blue (invariant). The tips of CBD-1 and CBD-2 form a patch of conserved, primarily hydrophobic residues above the periplasmic exit of the TM-channel. BcsA is shown as a grey surface and the glucan as purple spheres.

Figure 5. Proposed model for cellulose synthesis and translocation

Following glycosyl transfer, the newly added Glc could rotate around the acetal linkage into the plane of the polymer. The rotation direction would be determined by steric interactions and formation of the β-1,4 glucan characteristic intramolecular O3-H••O5 hydrogen bond. The glucan might translocate into the channel during this relaxation. This process would be repeated with a second UDP-Glc but the rotation direction after glycosyl transfer would be in the opposite direction due to steric constraints. Alternatively, the glucan might not translocate into the channel until UDP is replaced by UDP-Glc. Trp383 and Cys318 mark the entrance to the TM-channel (only shown in the right panel).