Structure of Arabidopsis CESA3 catalytic domain with its substrate UDP-glucose provides insight into the mechanism of cellulose synthesis

Abstract

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)–bound forms. AtCESA3CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants.

Point mutations in the catalytic domain disrupt cellulose synthase (CESA6) vesicle trafficking and protein dynamics

“…A recent crystal structure of the catalytic domain of the primary cell wall AtCESA3 revealed that the catalytic core is capable of forming a homodimer at an interface involving β-strand 6 (β6) and a-helix 9 (a9) and that homodimerization may play a role in preventing premature CSC assembly (Qiao et al, 2021). A similar mechanism was proposed in an earlier study of secondary cell wall CSC assembly (Atanassov et al, 2009).…”

“…The DDG, DXD, TED and QXXRW motifs in the catalytic domain are conserved among plant and bacteria and they participate in donor and acceptor UDP-glucose binding, glucan chain polymerization and elongation (Morgan et al, 2013; Morgan et al, 2016; Purushotham et al, 2020). A recent crystal structure of the AtCESA3 catalytic domain reveals the ability to form homodimers through contacts between β-strand 6 (β6) and α-helix 9 (a9) of adjacent subunits and bimolecular fluorescence complementation confirms that homodimers form in the Golgi apparatus (Qiao et al, 2021). This could represent a catalytically-inactive structure of CESA subunits and perhaps is an important form of regulation for CSC assembly, as proposed previously (Atanassov et al, 2009).…”

Updating Insights into the Catalytic Domain Properties of Plant Cellulose synthase (CesA) and Cellulose synthase-like (Csl) Proteins

“…Furthermore, the homotrimer of cotton CesA7 was stabilized by the transmembrane helix 7 (TM7) and the PCR domains, whereas the CSR is located at the periphery of the catalytic domain, suggesting an involvement in the interaction with other rosette units or non-CesA auxiliary proteins [ 48 ]. Additionally, the crystal structure of Arabidopsis CesA3 catalytic domain with its substrate UDP-glucose provided new insights into the mechanism of cellulose synthesis [ 49 ]. This work revealed the structural basis of how the substrate UDP-Glucose and a metal ion Mn 2+ which is required for cellulose synthesis, are coordinated in plant CesA complexes.…”

“…In agreement with previous reports, the crystal structure of Arabidopsis CesA3 catalytic domain was divided into the core domain, the PCR and the CSR domain. The core catalytic domain, including the catalytic sites, was composed of a β -sheet with eight strands, flanked by two α -helices on one side and two α -helices with three β -strands on the other side [ 49 ]. An insertion of a cellulose binding helix towards the catalytic region was proposed without forming a strong interaction with the rest of the catalytic domain, proposing that it is flexible in the absence of cellulose and transmembrane helices [ 49 ].…”

“…The core catalytic domain, including the catalytic sites, was composed of a β -sheet with eight strands, flanked by two α -helices on one side and two α -helices with three β -strands on the other side [ 49 ]. An insertion of a cellulose binding helix towards the catalytic region was proposed without forming a strong interaction with the rest of the catalytic domain, proposing that it is flexible in the absence of cellulose and transmembrane helices [ 49 ]. The plant-specific domains, namely PCR and CSR, were clearly resolved, forming a sandwich with the core catalytic domain as previously described.…”