Cohesin-dockerin interaction

Pages, S., A. Belaich, J. R Belaich, E. Morag, R. Lamed, Y. Shoham, and E. A. Bayer. 1997. Species-specificity of the cohesin-dockerin interaction between Clostridium thermocellum and Clostridium cellulolyticum Prediction of specificity determinants of the dockerin domain. Proteins 29 517-27. 

The objective of one direction of ongoing research in our group has been to define the molecular basis behind the highly specific, tenacious cohesin-dockerin interaction (11), During the course of our work, we have employed site-directed mutagenesis in attempts to assess the dockerin residues that may contribute to the observed interspecies specificity of cohesin binding. 

Microtiter plates (MaxiSorp-immunoplates, NUNC A/S, Roskilde, Denmark) were coated overnight at 23°C with the cohesin test samples (200 pl/well, 270 nM of miniCipC c, wild-type or mutated Coh2CBD t). The plates were blocked for 2.5 h with blocking solution (300 pl/well 3% (w/v) bovine serum albumin in TrisNC buffer) and washed three times with TrisNC buffer (300 pl/well). The cohesin-dockerin interaction was initiated upon addition of dockerin samples (200 pl/well, 94 nM of XynDocA c or XynDocS t), and the plates were incubated for 2.5 h. After five washes, the bound dockerins were detected by means of the fused-xylanase activity substrate solution (240 pl/well 2.9 mM / -nitrophenyl p-D-cellobioside) was added followed by incubation at 60 C. Optical density was detOTnined at 420 nm on a VERSAmax microplate reader (Molecular Devices Corp., Sunnyvale CA). 

Microtiter plates were coated overnight with wild-type C. thermocellum cohesin samples (200 pl/well, 270 nM of Coh2CBD t). Plates were blocked for 2.5 h with the above-described blocking solution, and washed three times with TrisNC buffer. The cohesin-dockerin interaction was carried out by the addition of 100 pi of the desired competitor cohesin sample (i.e., wild-type or mutant Coh2CBD t at various concentrations, up to a maximum of 1.3 pM), immediately followed by the addition of dockerin solution (100 pi of XynDocS t to final concentration of 47 nM). Dilutions of the competitor cohesins were carried out in TrisNC buffer containing BSA, to maintain a constant protein concentration. After incubation for 2.5 h, the wells were washed five times, and the amount of dockerin bound to the coating cohesin was detected by means of the fiised-xylanase activity, as described above. 

In previous work, we employed a combined bioinformatics-based approach with site-directed mutagenesis, in order to identify and corroborate the involvement of dockerin residues in the recognition of the cohesin domain. This approach revealed a group of 8 positions on the dockerin domain suspected to be critical to the observed species-specific selectivity of the cohesin-dockerin interaction. In like fashion, we attempted to employ a similar approach for identification of recognition residues on the surface of the molecular counterpart - the cohesin. 

In general, two major types of subunit compose cellulosomes the noncatalytic scaffoldin(s) and the catalyticahy active components. Each of these structures may be quite complex. The assembly of the cehulosome is facilitated by the high-affinity recognition between the scaffoldin cohesin and the enzymes dockerin modules. The scaffoldin often contains multiple cohesin modules, thereby enabling numerous different enzymes to be assembled into the cehulosome complex. In addition, in some species, such as Acetivibrio cellulolyticus, the cellulosomes present multiple scaffoldins with different cohesins [41]. The interaction cohesin-dockerins is type and specie-specific. 

Haimovitz, R Barak, Y Morag, E Voronov-Goldman, M Shoham, Y Lamed, R et al. Cohesin-dockerin microarray Diverse specificities between two complementary families of interacting protein modules. Proteomics, 2008, 8(5), 968 - 79. 

Within the cellulosome complex, type I dockerin domain is responsible for incorporating its associated glycosyl hydrolase in the bacterial cellulosome via interaction with a reception domain, the cohesin domain. The three-dimensional solution structure of the 69-residue dockerin domain from the thermophilic Clostridium thermocellum (Topt = 55-65 °C) was solved by NMR and was found to consist of two Ca " -binding loop-helix motifs connected by a linker. Each Ca " -binding subdomain is stabilized by a cluster of buried hydrophobic sidechains. Recently, the NMR sequence-specific resonance assignment of type II cohesin module from C. thermocellum has been published. ... 

Consequently, in order to determine whether any of the combination of mutations did in fact include binding-site residues, the binding affinities of the mutated cohesins were also evaluated in a quantitative manner. The results are presented in Figure 1. In competitive enzyme-linked interaction assay, cELIA, the native cohesin was used as a standard to coat microtiter plates. The immobilized cohesin was then allowed to interact with an enzyme-linked dockerin solution together with a competitor cohesin (native or mutated) in the solution phase. The measured enzymatic activity, expressed as the percentage of activity detected in the absence of the soluble competitor, reflects die amount of dockerin bound to the immobilized cohesin standard. The IC50, i.e., the... 

Figure L Competitive-EUA of native and mutated cohesinsfrom C. thermocellum interacting with a native dockerin of the same species, A solution containing the native cohesin was used to coat microtiter plates, and the immobilized cohesin was allowed to interact with an enzyme-linked dockerin in the presence of native (O) or mutated competitor cohesin [A, mut 20 (O) B, mut 28 (A) C, mut 30 (V) A mut 33 ( )]. The observed enzymatic activity reflects the amount of dockerin bound to the immobilized cohesin.

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