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

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 (AtCESA3^CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)–bound forms. AtCESA3^CatD 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 AtCESA3^CatD 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 AtCESA3^CatD 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.

Cellulose, a linear homopolymer of d-glucopyranose linked by β-1,4-glycosidic bonds, is the major structural component of the cell walls of plants, oomycetes, and algae and constitute the most abundant biopolymer on Earth (1). Cellulose is synthesized by cellulose synthases (CESAs) that belongs to the glycosyltransferase GT-2 superfamily (1, 2). In land plants, cellulose is produced at the plasma membrane by six-lobed rosette-shaped CESA complexes (CSCs) where each CESA is thought to synthesize one cellulose chain (3). The precise number of CESAs per CSC is unresolved but estimated to range between 18 and 36 (4–6).Plants contain multiple cesa genes, with 10 found in the Arabidopsis genome (7). Of these, CESA1, CESA3, CESA6, and the CESA6-like CESAs (i.e., CESA2, CESA5, and CESA9) are involved in primary cell wall formation, whereas CESA4, CESA7, and CESA8 participate in secondary cell wall formation (8–12). These two types of CSCs form heterotrimeric complexes with a ratio of 1:1:1 (13, 14). The Arabidopsis CESAs share an overall sequence identity of ∼60% and have seven transmembrane helices (15). In plants, the catalytic domain (CatD) of the CESAs is located between the second and third transmembrane helices and contains a canonical D, D, D, QxxRW motif (1). While there are similarities between the plant CatD and its counterpart in bacterial cellulose synthases, the CatD is flanked by two plant-specific domains, the so-called plant-conserved region (P-CR) and class-specific region (C-SR) (16). These domains are proposed to have important functions in cellulose synthesis and CESA oligomerization (17).The oligomerization of plant CESAs is thought to be important for the final CSC assembly, and multiple oligomeric states of CESAs, including homodimers, have been reported (18, 19). For example, immunoprecipitation assays using CESA7 fused to a dual His/STRP-tag demonstrated that CESA4, CESA7, and CESA8 could form independent homodimers, and it was hypothesized that the CESA homodimerization may contribute to early stages of CSC assembly. These homodimers might then be converted into CSC heterotrimeric configurations (19). This feature poses a marked difference from the bacterial cellulose synthase complex. However, how CESA homodimers are formed and how they function in cellulose synthesis are unknown.To comprehend the mechanisms behind plant cellulose synthesis, it is essential to acquire structural information about plant CESAs. Indeed, the BcsA–BcsB complex structure from Rhodobacter greatly aided our understanding of the cellulose synthesis in bacteria (20). Nevertheless, there are many differences between bacterial and plant CESAs and the corresponding protein complexes. Extensive efforts have been undertaken to acquire plant CESA structural information, including homology modeling and small-angle X-ray scattering analyses (5, 6, 16, 21, 22). While these efforts have been important to form new hypotheses, they did not reveal significant insights into substrate coordination, cellulose chain extrusion, and complex assembly. Recently, a homotrimeric CESA8 structure from Populus tremula × tremuloides was resolved by cryogenic electron microscopy (cryo-EM), which offered significant new molecular understanding of cellulose microfibril biosynthesis and CESA coordination within the CSC (15). Here we report the crystal structures of Arabidopsis CESA3 CatD (AtCESA3^CatD) in apo and uridine diphosphate (UDP)-glucose (UDP-Glc) bound forms and outline how the CatD might contribute to CESA homodimerization and substrate coordination.