title: Reassembly and co-crystallization of a family 9 processive endoglucanase from its component parts: structural and functional significance of the intermodular linker.
abstract: Non-cellulosomal processive endoglucanase 9I (Cel9I) from Clostridium thermocellum is a modular protein, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b), separated by linker regions. GH9 does not show cellulase activity when expressed without CBM3c and CBM3b and the presence of the CBM3c was previously shown to be essential for endoglucanase activity. Physical reassociation of independently expressed GH9 and CBM3c modules (containing linker sequences) restored 60-70% of the intact Cel9I endocellulase activity. However, the mechanism responsible for recovery of activity remained unclear. In this work we independently expressed recombinant GH9 and CBM3c with and without their interconnecting linker in Escherichia coli. We crystallized and determined the molecular structure of the GH9/linker-CBM3c heterodimer at a resolution of 1.68 Å to understand the functional and structural importance of the mutual spatial orientation of the modules and the role of the interconnecting linker during their re-association. Enzyme activity assays and isothermal titration calorimetry were performed to study and compare the effect of the linker on the re-association. The results indicated that reassembly of the modules could also occur without the linker, albeit with only very low recovery of endoglucanase activity. We propose that the linker regions in the GH9/CBM3c endoglucanases are important for spatial organization and fixation of the modules into functional enzymes.
authors.affiliation: Department of Molecular Microbiology and Biotechnology, The Daniella Rich Institute for Structural Biology, Tel Aviv University , Ramat Aviv , Israel.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Petkun
authors.affiliation: Department of Molecular Microbiology and Biotechnology, The Daniella Rich Institute for Structural Biology, Tel Aviv University , Ramat Aviv , Israel.
authors.forename: Inna
authors.initials: I
authors.lastname: Rozman Grinberg
authors.affiliation: Department of Molecular Microbiology and Biotechnology, The Daniella Rich Institute for Structural Biology, Tel Aviv University , Ramat Aviv , Israel.
authors.forename: Raphael
authors.initials: R
authors.lastname: Lamed
authors.affiliation: Department of Life Sciences, Meijo University , Nagoya , Japan.
authors.forename: Sadanari
authors.initials: S
authors.lastname: Jindou
authors.affiliation: Department of Molecular Microbiology and Biotechnology, The Daniella Rich Institute for Structural Biology, Tel Aviv University , Ramat Aviv , Israel.
authors.forename: Tal
authors.initials: T
authors.lastname: Burstein
authors.affiliation: Department of Molecular Microbiology and Biotechnology, The Daniella Rich Institute for Structural Biology, Tel Aviv University , Ramat Aviv , Israel.
authors.forename: Oren
authors.initials: O
authors.lastname: Yaniv
authors.affiliation: Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology , Haifa , Israel.
authors.forename: Yuval
authors.initials: Y
authors.lastname: Shoham
authors.affiliation: Department of Chemical Research Support, The Weizmann Institute of Science , Rehovot , Israel.
authors.forename: Linda J W
authors.initials: LJ
authors.lastname: Shimon
authors.affiliation: Department of Biological Chemistry, The Weizmann Institute of Science , Rehovot , Israel.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Bayer
authors.affiliation: Department of Molecular Microbiology and Biotechnology, The Daniella Rich Institute for Structural Biology, Tel Aviv University , Ramat Aviv , Israel.
authors.forename: Felix
authors.initials: F
authors.lastname: Frolow
keywords: Carbohydrate-binding module (CBM); Clostridium thermocellum; Family-9 glycoside hydrolase; Protein–protein interaction; X-ray structure