What’s the most abundant biopolymer on the planet?
If you answered cellulose, you nailed it.
And there’s exciting research to share.

Cellulose is the most abundant biopolymer on the planet, produced at a rate of approximately 85 billion tons per year(1). This polysaccharide comprises hundreds to thousands of β(1->4) linked glucose molecules, and is the primary component of plant cell walls, as well as a component of some bacterial biofilms. Cellulose is used in a number of commercial and manufacturing applications, and cellulose derived biofuels such as cellulosic ethanol is a promising renewable energy source(2).
 
In this month’s newsletter, we’re highlighting some of the structural biology work elucidating how cellulose is synthesized and transported in both plants and bacteria. This work has been pioneered by the lab of Jochen Zimmer at the University of Virginia.

Last month, in the August 28th issue of Science, the Zimmer Lab published the Cryo-EM structure of the plant cellulose synthase CesA isoform 8, which is implicated in secondary cell wall formation(3). This homotrimeric protein complex performs two main functions, synthesizing three cellulose polymers and secreting them through a transmembrane channel formed by each subunit. This work provides evidence for how these cellulose synthase trimeric complexes assemble into supermolecular complexes termed rosettes leading to the formation of micro- and macrofibrils that form the load-bearing component of plant cell walls.

This work continues the Zimmer Lab’s research on cellulose synthase enzymes. In 2013, the lab published the crystal structure of the bacterial BcsA-BcsB cellulose synthase complex which also captured a translocating polysaccharide(4). The results provided the first structural snapshot of how cellulose synthase both synthesizes and translocates a growing cellulose polysaccharide. In 2014, this work continued with additional crystal structures of the cyclic-di-GMP activated BcsA-BcsB complex showing how this signaling molecule modulates the function of the bacterial enzyme(5). A 2016 paper took this work one step further by utilizing in crystallo enzymology to reveal the complete cellulose biosynthesis cycle(6). Lastly, in 2019, they determined the structure of the bacterial cellulose synthase outer membrane channel, BcsC, which is responsible for the translocation of the cellulose polysaccharide across the outer bacterial membrane(7).


Anatrace Detergents:

To determine the Cryo-EM structure of CesA, membranes were solubilized using LMNG:CHS, and purified using GDN. Protein was blotted onto C-flat 400 mesh 1.2/1.3 holey carbon grids for data collection. For the first crystal structure of BcsA-BcsB, membranes were solubilized using Triton X-100, and the protein was purified using LDAO. Later crystal structures used a combination of LDAO and LysoFos Choline Ether 12 for purification and reconstituted the complex into DMPC:POPE:CHAPS bicelles for crystallization. Lastly, for BcsC, membranes were solubilized in LDAO and DDM, and the protein purified using DDM. BcsC was then exchanged into C8E4 for crystallization. Initial crystals of BcsC were obtained in the MemGold Screen from Molecular Dimensions.

 
References:
  1. Duchesne, L. C. & Larson, D. W. Cellulose and the Evolution of Plant Life. BioScience 39, 238–241 (1989).
  2. Liu, C.-G. et al. Cellulosic ethanol production: Progress, challenges and strategies for solutions. Biotechnology Advances 37, 491–504 (2019).
  3. Purushotham, P., Ho, R. & Zimmer, J. Architecture of a catalytically active homotrimeric plant cellulose synthase complex. Science eabb2978 (2020) doi:10.1126/science.abb2978.
  4. Morgan, J. L. W., Strumillo, J. & Zimmer, J. Crystallographic snapshot of cellulose synthesis and membrane translocation. Nature 493, 181–186 (2013).
  5. Morgan, J. L. W., McNamara, J. T. & Zimmer, J. Mechanism of activation of bacterial cellulose synthase by cyclic di-GMP. Nat Struct Mol Biol 21, 489–496 (2014).
  6. Morgan, J. L. W. et al. Observing cellulose biosynthesis and membrane translocation in crystallo. Nature 531, 329–334 (2016).
  7. Acheson, J. F., Derewenda, Z. S. & Zimmer, J. Architecture of the Cellulose Synthase Outer Membrane Channel and Its Association with the Periplasmic TPR Domain. Structure 27, 1855-1861.e3 (2019).