INTERACTION BETWEEN THE ENDOGLUCANASE CELA AND THE SCAFFOLDING PROTEIN CIPC OF THE CLOSTRIDIUM-CELLULOLYTICUM CELLULOSOME

The 5' end of the cipC gene, coding for the N-terminal part of CipC, t he scaffolding protein of Clostridium cellulolyticum ATCC 35319, was c loned and sequenced. It encodes a 586-amino-acid peptide, including se veral domains: a cellulose-binding domain, a hydrophilic domain, and t wo hydrophobic domains (cohesin domains). Sequence alignments showed t hat the N terminus of CipC and CbpA of C. cellulovorans ATCC 35296 hav e the same organization. The mini-CipC polypeptide, containing a cellu lose-binding domain, hydrophilic domain 1, and cohesin domain 1, was o verexpressed in Escherichia coli and purified. The interaction between endoglucanase CelA, with (CelA(2)) and without (CelA(3)) the characte ristic clostridial C-terminal domain called the duplicated-segment or dockerin domain, and the mini-CipC polypeptide was monitored by two di fferent methods: the interaction Western blotting (immunoblotting) met hod and binding assays with biotin-labeled protein. Among the various forms of CelA (CelA(2), CelA(3), and an intermediary form containing o nly part of the duplicated segment), only CelA(2) was found to interac t with cohesin domain 1 of CipC. The apparent equilibrium dissociation constant of the CelA2-mini-CipC complex was 7 . 10(-9) M, which indic ates that there exists a high affinity between these two proteins.