Cohesin‐dockerin microarray: Diverse specificities between two complementary families of interacting protein modules

Abstract

The cellulosome is an intricate multienzyme complex, designed for efficient degradation of plant cell wall polysaccharides, notably cellulose. The supramolecular cellulosome architecture in different bacteria is the consequence of the types and specificities of the interacting cohesin and dockerin modules, borne by the different cellulosomal subunits. In this study, we describe a microarray system for determining cohesin-dockerin specificity, which allows global comparison among the interactions between various members of these two complementary families of interacting protein modules. Matching recombinant fusion proteins were prepared that contained one of the interacting modules: cohesins were joined to an appropriate cellulose-binding module (CBM) and the dockerins were fused to a thermostable xylanase that served to enhance expression and proper folding. The CBM-fused cohesins were immobilized on cellulose-coated glass slides, to which xylanase-fused dockerin samples were applied. Knowledge of the specificity characteristics of native and mutated members of the cohesin and dockerin families provides insight into the architecture of the parent cellulosome and allows selection of suitable cohesin-dockein pairs for biotechnological and nanotechnological application. Using this approach, extensive cross-species interaction among type-II cohesins and dockerins is shown for the first time. Selective intraspecies binding of an archaeal dockerin to two complementary cohesins is also demonstrated.

Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis

“…The recombinant protein was purified as described above except all buffers were supplemented with 5 mm β-mercaptoethanol. The production of recombinant Cel48S and the CBM3a-Coh construct (CBM3a fused to the type I cohesin3 from CipA, the primary scaffoldin of C. thermocellum) were described previously (9,31,32).…”

Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode

“…The CohT construct was cloned as described previously (43), and the CohB and CohF constructs were constructed as reported recently (18). For the construction of the ScafBT plasmid (where ScafBT is a chimeric scaffoldin contain- , the third B. cellulosolvens cohesin from the scaffoldin B subunit was amplified with 5Ј-GCAACCATGGCGGGGAAAAGTTCACCAG-3Ј (NcoI site is in boldface) and 5Ј-GTAGGGTACCTTAGTTACAGTAATGCTTCC-3Ј (KpnI site is in boldface) and ligated into NcoI-KpnI-linearized pET9d C-T scaffoldin (Scaf2 [16]), whereby the B. cellulosolvens cohesin replaced the Clostridium cellulolyticum cohesin.…”

Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors

“…1). These two different type I subclasses do not cross-react (19,28). To identify the residues that modulate type I Coh-Doc specificity in A. cellulolyticus, a mutagenesis study, based on the crystal structure of DocScaB and the alignment of this dockerin with the primary consensus sequence of type I dockerins of A. cellulolyticus cellulosomal enzymes (ϳ100 primary sequences of A. cellulolyticus enzyme dockerins were used), was initiated (Fig.…”