Dockerins

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. ... 

After catalytic domains, the next most common domains are CBDs which are usually j oined to the catalytic domain by a short tinker peptide. Cellulases that are present in cellulosomes contain short domains called dockerins that bind to specific sites on a scaffoldin protein to form a cellulosome [30]. A number of cellulases contain fibronectin-like domains, but the function of these domains is not known [6]. There are several other domains with unknown functions [6]. 

A key component of cellulosomes is a 200,000 MW protein, scaffoldin, that contains at least nine copies of a cohesin domain, a family Ilia cellulose-binding domain, and a dockerin-like domain that appears to bind scaffoldin to the cell surface. The cohesin domains are the binding sites for the dockerin domains that are present at the C-terminus of each cellulase molecule present in the cellulosome. At this time, it appears that all the cohesin domains of a given species bind with nearly equal affinity to the dockerin domains present on the cellulo-somal enzymes of that species [92]. This result suggests that cellulosomes are a... 

CE Family 1 is very large and contains members which do not act on carbohydrate-derived substrates. The crystal structure of a CE 1 domain of XynlOB modular enzyme from Clostridium thermocellum has been solved. " The CE 1 domain is a feruloyl esterase which hydrolyses the feruloyl groups attached to some arabinofuranosyl 05 groups in native xylan. (The Xyn lOB protein as a whole consists of two CBM 22 domains, a dockerin domain, and a GH 20 xylanase domain, and forms part of a cellulosome - see Section 5.10.) The enzyme has the common a/p hydrolase fold. Studies of ferulic acid complexes of the inactive alanine mutant of the active site serine revealed the classic catalytic triad, and two main-chain peptide NH bonds are in place to form an oxyanion hole . A remarkable feature is that the enzyme as repeatedly isolated was esterilied on the active site serine by phosphate or sulfate. 

As an alternative to these three approaches, two other sequence-based methods have been developed to predict interaction events. The mirror tree method is based on extracting information from the possible coevolution of interacting proteins. Consequently the phylogenetic trees of coevolving proteins are expected to have a significant similarity, as detected in examples such as insulin and its receptors [53], and dockerins and cohexins [54]. Current methods are based on the direct comparison of the distance matrices of pairs of protein families [55] and its extension to large data sets [56], Improvements have been described to predict interaction specificity [57],... 

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. 

Effect of Rational Mutagenesis of Selected Cohesin Residues on the High-Affinity Cohesin-Dockerin... 

The crystal structures of three cohesins, two from C. thermocellum and one from C. cellulolyticum, have been reported (7-9). The cohesin domains form a nine-stranded P sandwich with a jelly-roll topology. The P sandwich results from the association of a four-stranded antiparallel P sheet, and a five-stranded mixed P sheet, stabilized by a hydrophobic core. The two P sheets are composed of strands 8,3,6,5 and strands 9,1,2,7,4 respectively. In addition, the three-dimensional structure of one dockerin (from the family-48 C. thermocellum cellulosomal enzyme, CelS) was solved by NMR spectroscopy (70). The dockerin structure consists of two Ca -binding loop-helix motifs connected by a linker. 

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. 

In the present work, we have systematically mutated the latter residues and have selected additional residues on the cohesin surface as targets for site-directed mutagenesis. Using an approach similar to that described above for the mutagenesis of the dockerin domain, we have tried to convert the specificity of the cohesin of one sf ies to match that of the other (i.e., from C. thermocellum... 

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. 

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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