Observing cellulose biosynthesis and membrane translocation in crystallo

Abstract

Many biopolymers, including polysaccharides, must be translocated across at least one membrane to reach their site of biological function. Cellulose is a linear glucose polymer synthesized and secreted by a membrane-integrated cellulose synthase. Here, in crystallo enzymology with the catalytically active bacterial cellulose synthase BcsA–BcsB complex reveals structural snapshots of a complete cellulose biosynthesis cycle, from substrate binding to polymer translocation. Substrate- and product-bound structures of BcsA provide the basis for substrate recognition and demonstrate the stepwise elongation of cellulose. Furthermore, the structural snapshots show that BcsA translocates cellulose via a ratcheting mechanism involving a ‘finger helix’ that contacts the polymer’s terminal glucose. Cooperating with BcsA’s gating loop, the finger helix moves ‘up’ and ‘down’ in response to substrate binding and polymer elongation, respectively, thereby pushing the elongated polymer into BcsA’s transmembrane channel. This mechanism is validated experimentally by tethering BcsA’s finger helix, which inhibits polymer translocation but not elongation.

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Figure 1: In crystallo cellulose biosynthesis.
Figure 2: Movement of BcsA’s finger helix is essential for cellulose translocation.
Figure 3: The product-bound state.
Figure 4: The substrate-bound state.
Figure 5: Model of cellulose biosynthesis.

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Acknowledgements

We thank T. Rapoport for critical comments on the manuscript and J. Acheson for advice on reducing BcsA–BcsB complexes in crystallo. Diffraction data were collected at the Argonne National Laboratory’s Advanced Photon Source (APS) beam lines 23-ID-D (GM/CA-), 22-ID (SER-) and 24-ID-C (NE-CAT). GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). The NE-CAT beam lines are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6M detector on 24-ID-C beam line is funded by a NIH-ORIP HEI grant (S10 RR029205). Data for this research was also in part collected at the APS SER-CAT beam line, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by ANL under Contract No. DE-AC02-06CH11357. J.L.W.M. is supported by a National Science Foundation Graduate Research Fellowship, Grant No. DGE-1315231. M.F. thanks the Austrian Science Fund (FWF) (J3293-B21) for an Erwin Schrödinger postdoctoral fellowship. This research was primarily supported by the National Institutes of Health, Grant 1R01GM101001, awarded to J.Z.; S.G.W. thanks the Natural Sciences and Engineering Research Council of Canada for financial support.

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Contributions

J.T.M. and J.L.W.M. purified and crystallized BcsA–BcsB and performed all crystal soaking experiments. J.T.M. cloned and analysed all BcsA cysteine mutants. J.T.M. and J.L.W.M. collected and processed diffraction data and built and refined the BcsA–BcsB models. M.F. synthesized the fluorinated and phosphonate UDP-Glc analogues and J.R. and H.-M.C. synthesized the UDP-thio-galactose analogues. J.T.M., J.L.W.M. and J.Z. analysed the data. J.Z. and J.L.W.M. wrote the paper and all authors edited the text.

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Correspondence to Jochen Zimmer.

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The authors declare no competing financial interests.

Additional information

Structure factors and coordinates have been deposited at the Protein Data Bank under entry codes 5EJ1, 5EIY and 5EJZ.

Extended data figures and tables

Extended Data Figure 1 Conformational flexibility of the gating loop after cellulose extension.

Unbiased Sigma-A weighted Fo − Fc difference electron density of the gating loop in the pre-translocation state contoured at 2σ. The ordered part of the gating loop is shown as a thick green ribbon and two alternative backbone positions are indicated by a black dashed line. The position of the gating loop in the inserted state in the presence of a UDP molecule as observed in PDB entry 4P00 is shown as a cartoon representation coloured blue.

Extended Data Figure 2 In crystallo translocation of a 6-thio-galactose-containing cellulose polymer.

The position of the 6-thio-galactose group at the polymer’s non-reducing end was determined after polymer extension (upper panel) and upon subsequent incubation with UDP/Mg2+ (lower panel) in an anomalous difference Fourier electron density (DANO) map. DANO peaks detected at a wavelength of 1.74 Å are shown as a red mesh contoured at 3.5σ. Unbiased Sigma-A weighted Fo − Fc difference electron density for the cellulose polymer is shown as a green mesh contoured at 4σ. The cellulose polymer was extended and translocated as described in Fig. 1 with the exception that UDP-6-thio-galactose was used as substrate and Mg2+ was included during the initial soaking step. The extended DANO peak around Cys318 in the post-translocation state might arise from overlapping peaks originating from Cys318 and the thio-Gal unit in an opposite orientation. All Cys and Met residues close to BcsA’s active site are shown as sticks. UDP is shown as sticks in violet for its carbon atoms.

Extended Data Figure 3 Position of the disulfide-tethered finger helix.

The BcsA-2C–BcsB complex was crystallized as described for wild-type BcsA–BcsB. a, Unbiased Sigma-A weighted Fo − Fc difference electron density of BcsA’s finger helix contoured at 4σ (magenta mesh). Cellulose and BcsA’s Trp383 at the entrance to the transmembrane channel are shown as sticks in cyan and grey for their carbon atoms, respectively. The finger helix and IF2 are shown as cartoon helices coloured yellow and grey, respectively. b, The finger helix-tethered BcsA–BcsB complex was refined in a resolution range from 34 to 3.2Å to a final R/Rfree of 19.9/23.9% in Phenix_refine34 with Ala residues at positions 338 and 394 of BcsA. A strong difference electron density peak indicates the position of the omitted disulfide bond in a Sigma-A weighted Fo − Fc difference electron density map, (green mesh, contoured at 2σ).

Extended Data Figure 4 Comparison of the UDP conformation in the substrate and UDP-bound states of BcsA.

The substrate-bound BcsA structure was superimposed with PDB entry 4P00 by secondary structure matching in Coot. The substrate is shown as ‘balls and sticks’ in violet for the carbon atoms and the UDP molecule from PDB entry 4P00 is shown as grey sticks. BcsA’s finger helix is shown as a yellow cartoon and the cellulose polymer is shown as cyan and red ‘balls and sticks’ as observed in PDB entry 4P00. Magnesium is shown as a green sphere.

Extended Data Figure 5 UDP-Glc induced polymer translocation.

The nascent cellulose polymer was extended with a chain-terminating galactose residue upon soaking BcsA–BcsB crystals with UDP-Gal. Following dilution of the substrate as described in Fig. 1, crystals were incubated for 150 min either in the absence of a nucleotide or in the presence of UDP/Mg2+ or UDP-Glc/Mg2+, respectively. The unbiased SigmaA-weighted Fo − Fc difference electron density of the nascent polymer (green mesh) is shown at three different contour levels, indicating that UDP-Glc also induces polymer translocation.

Extended Data Figure 6 Stabilization of BcsA’s finger helix by conserved residues.

Top panel: stick representation of BcsA’s finger helix and nascent cellulose polymer shown in yellow and cyan for their carbon atoms. The finger helix is shown as a poly-glycine helix except for the labelled residues. Bottom panel: The finger helix’s ‘TEDxxT’ motif is conserved among pro- and eukaryotic cellulose synthases. Finger helix sequences are aligned for Micrasterias denticulata CesA, Physcomitrella patens CesA5, Arabidopsis thaliana CesA8, Rhodobacter sphaeroides and Escherichia coli BcsA, and Ciona savignyi CesA. The conserved threonine following the TED motif is indicated with a red box. Of note, the threonine residue is absent from the Ciona CesA sequence; however, this protein contains a serine residue at the following position, which could perform a similar function.

Extended Data Table 1 Crystallographic data collection and refinement statistics

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Morgan, J., McNamara, J., Fischer, M. et al. Observing cellulose biosynthesis and membrane translocation in crystallo. Nature 531, 329–334 (2016). https://doi.org/10.1038/nature16966

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