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.

Effects of the linker region on the structure and function of modular GH5 cellulases

“…This observation is quite relevant in processive cellulases, in which the inter-domain region directly interacts with the modules31. Our findings provide evidence that the inter-modular distance is also important in non-processive GH5 endoglucanases.…”

Crystal Structures of the C‐Terminally Truncated Endoglucanase Cel9Q from Clostridium thermocellum Complexed with Cellodextrins and Tris

“…The lengths of the disulfide and hydrogen bonds in the dimerization interface of oligosaccharide‐free Ct Cel9QΔc are summarized in Table . This dimerization interface, with an area of 634 Å 2 , of the CBM has not been observed in the similar structures of Tf Cel9A, Cc Cel9G, and Ct Cel9I . In addition, the structures of Tf Cel9A, Cc Cel9G, and Ct Cel9I, which contain type 3c CBMs, have been solved.…”

“…These two domains are separated by a 17‐residue linker. The linker region, which is essential for the endoglucanase activity, has been shown to play a role in modulating the function of the GH9 catalytic domain and CBM . A disulfide bond is formed between the two Cys535 residues of the protein monomers in the asymmetric unit, resulting in the formation of a stable Ct Cel9QΔc homodimer.…”

“…The residues His145, Trp267, Trp325 and Tyr431 are highly conserved in family 9 cellulases. The aromatic residue Trp209 (PDB ID: 1JS4) is located in subsite −3 of the processive cellulase Tf Cel9A, similar to Trp209 (PDB ID: 2XFG) in Ct Cel9I, Trp230 (PDB ID: 4DOD) in Caldicellulosiruptor bescii Cb CelA, and His220 in Ct Cel9Q (this study), whereas the corresponding residue Thr207 (PDB ID: 1IA6) in the nonprocessive cellulase Cc Cel9M is located toward subsite −4, similar to Thr356 (PDB ID: 1CLC) in Ct Cel9D, Asp302 (PDB ID: 3EZ8) in Aa Cel9A, and Ser276 (PDB ID: 2YIK) in Ct CelT (Figure ). This key residue has been proposed to distinguish processive cellulases—if the residue forms a stacking interaction with the substrate—from nonprocessive cellulases .…”

“…The second Ca 2+ ion, located at the CBM, is coordinated to one water molecule, Asp514 O, Glu517 O ϵ 1 , Glu517 O ϵ 2 , Asp592 O, Asn595 O δ1 , and Asp596 O δ1 in a pentagonal bipyramidal geometry (Figure B). Again, this calcium‐binding site is also highly conserved in type 3 CBM structures . However, in the type 3b CBM structure (PDB ID: 2XBT), calcium ions were not observed in this conserved calcium‐binding site .…”