Cohesin-Dockerin Interactions within and between Clostridium josui and Clostridium thermocellum: BINDING SELECTIVITY BETWEEN COGNATE DOCKERIN AND COHESIN DOMAINS AND SPECIES SPECIFICITY

Sadanari Jindou,Akane Soda,Shuichi Karita,Tsutomu Kajino,Pierre Béguin,J.H David Wu,Minoru Inagaki,Tetsuya Kimura,Kazuo Sakka,Kunio Ohmiya

doi:10.1074/jbc.m308673200

Sadanari Jindou, Akane Soda + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m308673200

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Abstract

The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component. We prepared five recombinant cohesin polypeptides of the Clostridium thermocellum scaffolding protein CipA, two dockerin polypeptides of C. thermocellum Xyn11A and Xyn10C, four cohesin polypeptides of Clostridium josui CipA, and two dockerin polypeptides of C. josui Aga27A and Cel8A, and qualitatively and quantitatively examined the cohesin-dockerin interactions within C. thermocellum and C. josui, respectively, and the species specificity of the cohesin-dockerin interactions between these two bacteria. Surface plasmon resonance (SPR) analysis indicated that there was a certain selectivity, with a maximal 34-fold difference in the K(D) values, in the cohesin-dockerin interactions within a combination of C. josui, although this was not detected by qualitative analysis. Affinity blotting analysis suggested that there was at least one exception to the species specificity in the cohesin-dockerin interactions, although species specificity was generally conserved among the cohesin and dockerin polypeptides from C. thermocellum and C. josui, i.e. the dockerin polypeptides of C. thermocellum Xyn11A exceptionally bound to the cohesin polypeptides from C. josui CipA. SPR analysis confirmed this exceptional binding. We discuss the relationship between the species specificity of the cohesin-dockerin binding and the conserved amino acid residues in the dockerin domains.

Highlights

The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component
This was demonstrated by swapping these residues between the dockerin domains of C. thermocellum Cel48A and C. cellulolyticum Cel5A: each of the mutated dockerins acquired the ability to bind to its noncognate cohesin domain but did not lose the affinity for its cognate cohesin domain, suggesting that the conserved 11th and 12th residues do play a role in biorecognition, whereas additional residues may contribute to the specificity of the interaction [10]
Production and Purification of Various Cohesin and Dockerin Polypeptides—C. thermocellum CipA consists of nine cohesin domains, a family 3 carbohydrate-binding module (CBM), and a type II dockerin domain (Fig. 1A)

Summary

Introduction

The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component. The second and third cohesin domains of C. thermocellum CipA interacted with all the catalytic subunits, except for the largest catalytic subunit known as Cel9D-Cel44A (formerly CelJ) [14], of the cellulosome in an affinity blotting analysis [14], suggesting that generally there is no selectivity in the interactions among cohesin and dockerin domains within the same species, but that there may be at least one exception.

Results

Conclusion