Evidence for Plant-Conserved Region Mediated Trimeric CESAs in Plant Cellulose Synthase Complexes.

Area of Science:

• Plant biology
• Molecular and cellular biology
• Biochemistry

Background:

• Higher plants synthesize cellulose using membrane-bound cellulose synthase complexes (CESAs).
• While heteromeric trimers of different CESA isoforms are supported by molecular biology, homomeric trimers have been observed in vitro and in cryo-electron microscopy (cryo-EM) structures of full-length CESAs.
• The plant-conserved region (P-CR) forms the primary contact between catalytic domains in known CESA structures.

Purpose of the Study:

• To investigate the structure and stability of homotrimers of the catalytic domain of Arabidopsis CESA1 (AtCESA1CatD).
• To identify the key interactions stabilizing these homomeric trimers.
• To assess the potential role of homomeric CESA trimers in plant cell wall biosynthesis.

Main Methods:

• In vitro biochemical assays including inter-subunit lysine crosslinking.
• Negative-stain electron microscopy (EM) to determine structural features.
• Computational modeling and molecular dynamics (MD) simulations based on existing cryo-EM structures.

Main Results:

• Inter-subunit crosslinks were identified within the small P-CR of AtCESA1CatD homotrimers.
• Negative-stain EM and modeling data supported the formation of stable homotrimers.
• MD simulations confirmed the stability of AtCESA1CatD trimers, driven by a limited set of residue contacts.

Conclusions:

• Homomeric CESA trimers, specifically of AtCESA1CatD, are structurally stable.
• These findings suggest that homomeric CESA trimers may play a significant role in both primary and secondary cell wall synthesis in plants.
• Key residues involved in trimer stability were identified, providing targets for future mutagenic studies.