title: The c-di-GMP recognition mechanism of the PilZ domain of bacterial cellulose synthase subunit A.
abstract: In some Proteobacteria and Firmicutes such as Pseudomonas aeruginosa, Vibrio cholerae, Xanthomonas campestris, and Clostridium difficile, cyclic dimeric guanosine monophosphate (c-di-GMP) is known to regulate cellular processes, including motility, biofilm formation, and virulence, as a second messenger. Cellulose production in Acetobacter xylinum, a model organism of cellulose biosynthesis, also depends on by cellular c-di-GMP level. In cellulose-synthesizing bacteria, a terminal complex localized in the cell membrane synthesizes cellulose and regulates the production of cellulose sensed by c-di-GMP. Although previous studies indicated that the PilZ domain conserved in cellulose synthase subunit A (CeSA) was part of a receptor for c-di-GMP, the recognition mechanism by PilZ domain of CeSA remains unclear. In the present study, we studied the interaction between c-di-GMP and the PilZ domain of CeSA from a structural viewpoint. First, we solved the crystal structure of the PilZ domain of CeSA from A. xylinum (AxCeSA-PilZ) at 2.1Å resolution. Then, comparison of the sequence and structure of AxCeSA-PilZ to those of known structures of PilZ, such as VCA0042, PP4397, and PA4608, indicated the involvement of Lys573 and Arg643 of AxCeSA-PilZ in the recognition of c-di-GMP besides the RxxxR motif. Finally, the binding characteristics of c-di-GMP to AxCeSA-PilZ and mutants were determined with isothermal titration calorimetry, indicating that the residues corresponding to Lys573 and Arg643 in AxCeSA-PilZ generally contribute to the binding of c-di-GMP to PilZ.
authors.affiliation: Graduate School of Life Science, Hokkaido University, Kita-10, Nishi-8, Kita-ku, Sapporo 060-0810, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Keisuke
authors.initials: K
authors.lastname: Komoda
authors.forename: Naofumi
authors.initials: N
authors.lastname: Sakurai
authors.forename: Kenji
authors.initials: K
authors.lastname: Tajima
authors.forename: Isao
authors.initials: I
authors.lastname: Tanaka
authors.forename: Min
authors.initials: M
authors.lastname: Yao
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics; D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry; D041822:Gluconacetobacter xylinus / Q000201:enzymology*; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics; D008956:Models, Chemical; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: C062025:bis(3',5')-cyclic diguanylic acid; D005964:Glucosyltransferases; C076062:cellulose synthase (cyclic diguanylic acid); D006152:Cyclic GMP; D000409:Alanine