<p><a>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</a>
(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.</p>
<p>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.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/12678833 |
| Date | 29 July 2020 |
| Creators | Phillip S Rushton (9143657) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/Structure_of_the_Plant-Conserved_Region_of_Cellulose_Synthase_and_Its_Interactions_with_the_Catalytic_Core/12678833 |
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