Cellulosome from Clostridium cellulolyticum: Molecular study of the dockerin/cohesin interaction

Clostridium cellulolyticum produces cellulolytic complexes (cellulosomes) m ade of 10-13 cell wall degrading enzymes tightly bound to a scaffolding pro tein (CipC) by means of their dockerin domain. It has previously been shown that the receptor domains in CipC are the cohesin domains and that the coh esin/dockerin interaction is calcium-dependent. In the present study, surfa ce plasmon resonance was used to demonstrate that the free cohesin 1 from C ipC and dockerin from CelA have the same K-D (2.5 x 10(-10) M) as that of t he entire CelA and a larger fragment of CipC, the latter of which contains, in addition to cohesin 1, a cellulose binding domain and a hydrophilic dom ain of unknown function. This demonstrates that neither the catalytic domai n of CelA nor the noncohesin domains of CipC have any influence on the inte raction. Dockerin domains are composed of two conserved segments of 22 resi dues: removal of the second segment abolishes the affinity for cohesin 1, w hereas modified dockerins having twice the first segment, twice the second, or both segments but in a reverse order have K-D values for cohesin 1 in t he same range as that observed for wild-type dockerin. These data indicate that if two segments ate required for the complexation with the cohesin, se gments 1 and 2 are similar enough to replace each other. Calcium overlay ex periments revealed that the dockerin domain has one calcium binding site pe r conserved segment. Circular dichroism performed on wild-type and mutant d ockerins indicates that this domain is well structured and that removal of calcium only weakly affects the secondary structure, which remains 40-45% h elical.