Structure of the Plant-Conserved Region of Cellulose Synthase and Its Interactions with the Catalytic Core
Creators: name: Rushton, Phillip S
Publisher: name: Purdue University Graduate School
Year: 2020
Subjects: Animal physiology - biophysics
Human biophysics
Plant biology not elsewhere classified
Plant cell and molecular biology
Animal cell and molecular biology
Structural biology (incl. macromolecular modelling)

description: The processive plant cellulose synthase (CESA) synthesizes (1→4)-β-D-glucans. CESAs assemble into a six-fold symmetrical cellulose synthase complex (CSC), with an unknown symmetry and number of CESA isomers. The CSC synthesizes a cellulose microfibril as the fundamental scaffolding unit of the plant cell wall. CESAs are approximately 110 kDa glycosyltransferases with an N-terminal RING-type zinc finger domain (ZnF), seven transmembrane α-helices (TMHs) and a cytoplasmic catalytic domain (CatD). In the CatD, the uridine diphosphate glucose (UDP-Glc) substrate is synthesized into (1→4)-β-D-glucans. The ZnF is likely to facilitate dimers in the CSC. Recombinant class-specific region (CSR), a plant specific insertion to the C-terminal end of the CatD is also known to form dimers<i> in vitro</i>. The CSR sequence is the primary source of distinction between CESA isoforms and class structure. Also within the CESA CatD is a 125-amino acid insertion known as the plant-conserved region (P-CR), whose molecular structure was unknown. The function of the P-CR is still unclear, especially in the context of complete CESA and CSC structures. Thus, one major knowledge gap is understanding how multimeric CSCs synthesize multiple chains of (1→4)-β-D-glucans that coalesce to form microfibrils. The specific number of CESAs in a CSC and how interactions of individual CESA isoforms contribute to the CSC are not known. Elucidating the structure-function relationships of the P-CR domain, and with the consideration of the ability of CSR and ZnF domains to dimerize, it is possible to more completely model the structure of the CSC. Recombinantly expressed rice (<i>Oryza sativa</i>) secondary cell wall OsCESA8 P-CR domain purifies as a monomer and shows distinct α-helical secondary structure by circular dichroism analysis. A molecular envelope of the P-CR was derived by small angle X-ray scattering (SAXS). The P-CR was crystallized and structure solved to 2.4 Å resolution revealing an anti-parallel coiled-coiled domain. Connecting the coiled-coil α-helices is an ordered loop that bends back towards the coiled-coils. The P-CR crystal structure fits the molecular envelope derived by SAXS, which in turn fits into the CatD molecular envelope. The best fit places the P-CR between the membrane and substrate entry portal. In depth analysis of structural similarity to other proteins, and 3D-surface structure of the P-CR, leads to hypotheses that it could function in protein-protein interactions as a dimer, trimer or tetramer in the CSC, that it could form protein-protein interactions with CESA-interacting proteins, and/or modulate substrate entry through its N- and/or C-terminus. From modeling, hypothetically important residues within the P-CR or related to the P-CR through potential protein contacts were mutated in <i>Arabidopsis thaliana</i> <i>AtCESA1</i> constructs. These constructs were expressed in the temperature-sensitive <i>radial swelling</i> (<i>rsw</i>)<i> rsw1-1</i> mutant of <i>AtCESA1 </i>to test for complementation of growth phenotypes at restrictive temperatures. Preliminary experiments indicate that some mutated CESA1 sequences fail to complement the <i>rsw1-1</i> phenotype, suggesting that specific functions of individual amino can be tested using this system.