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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Protein Data Bank
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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Observation of in vitro cellulose synthesis by bacterial cellulose synthase with time-resolved small angle X-ray scattering
International Journal of Biological Macromolecules (2019)