Structure of bacterial cellulose synthase subunit D octamer with four inner passageways

Abstract

The cellulose synthesizing terminal complex consisting of subunits A, B, C, and D in Acetobacter xylinum spans the outer and inner cell membranes to synthesize and extrude glucan chains, which are assembled into subelementary fibrils and further into a ribbon. We determined the structures of subunit D (AxCeSD/AxBcsD) with both N- and C-terminal His(6) tags, and in complex with cellopentaose. The structure of AxCeSD shows an exquisite cylinder shape (height: ∼65 Å, outer diameter: ∼90 Å, and inner diameter: ∼25 Å) with a right-hand twisted dimer interface on the cylinder wall, formed by octamer as a functional unit. All N termini of the octamer are positioned inside the AxCeSD cylinder and create four passageways. The location of cellopentaoses in the complex structure suggests that four glucan chains are extruded individually through their own passageway along the dimer interface in a twisted manner. The complex structure also shows that the N-terminal loop, especially residue Lys6, seems to be important for cellulose production, as confirmed by in vivo assay using mutant cells with axcesD gene disruption and N-terminus truncation. Taking all results together, a model of the bacterial terminal complex is discussed.

Fig. 1.

Crystal structure of AxCeSD. (A) Ribbon representation of the dimeric structure of AxCeSD. The two monomers are shown in blue and red, respectively. The helices and sheets are labeled, where the prime refers to the second monomer. (B) Overall structure of the AxCeSD octamer. The octamer structure is viewed along the 4-fold axis (top view) and the dyad axis (side view), with each monomer (A–H) shown in a different color. The N and C termini of all copies that are positioned in the center and outside of the cylinder are indicated by the circled N and C (same as in A), respectively. (C) A schematic diagram of the octamer assembly based on the side view in B. The octamer is represented by a cylinder, and monomers (A, C, E, G) and (B, D, F, H) are distributed in the top and bottom layers, respectively. The colors of each molecule correspond with those in B. The dimer–dimer interfaces are depicted with sloping rectangles, and indicated by arrows. A and B were prepared with the program PyMOL (DeLano Scientific LLC, http://pymol.sourceforge.net/).

Fig. 2.

The structure of AxCeSD complexed with CPT. (A) The CPT and its omitted electron density map. The map along the dimer–dimer interface, shown in cyan chickenwire contoured at 2.0 σ, was calculated in the absence of CPT with coefficients Fo-Fc (Left). The inner view of the CPT passageway is shown by the protein surface of four monomers (Right). (B) Ribbon representation of AxCeSD octamer in complex with CPT (Left), and the relocation of four CPTs in the AxCeSD octamer (Right). A magnified view of the pore is shown in the rectangular box. The CPTs and the AxCeSD residues involved in contact with CPT are shown as stick models. Oxygen and carbon atoms in CPT are colored red and yellow, and oxygen, nitrogen, and carbon atoms in AxCeSD are colored red, blue, and gray, respectively.

Fig. 3.

Effects of axcesD gene deletion and mutations on the cellulose production. AxCeSDs with N termini truncated in various lengths were expressed in DBCD of A. xylinum ATCC23769. All measurements of cellulose production were carried out under the similar expression level of TC. (A) The relative yields of cellulose produced by wild-type (column WT) and following mutant cells: axcesD gene deletion mutant with a control vector (column DBCD), full-length (columns DBCD+D), deletion of the four N-terminal residues (column DBCD+DΔN4), deletion of the five N-terminal residues (column DBCD+DΔN5), and deletion of the six N-terminal residues (column DBCD+DΔN6). The measurements were done five times for each sample. (B) Molecular surfaces of C_AxCeSD (WT; the three N-terminal residues were disordered), DΔN5 (deletion of the five N-terminal residues), and DΔN6 (deletion of the six N-terminal residues) octamer. The colors correspond to eight monomers in the same way as Fig. 1B, and the CPTs are shown as in Fig. 2.