Alteration of a single tryptophan residue of the cellulose-binding domain blocks secretion of the Erwinia chrysanthemiCel5 cellulase (ex-EGZ) via the type II system

Cel5 (formerly known as endoglucanase Z) of Erwinia chrysanthemi is secreted by the Out type II pathway. Previous studies have shown that the catalytic domain (CD), linker region (LR) and cellulose-binding domain (CBD) each contain information needed for secretion. The aim of this work was to further investigate the secretion-related information present in the CBDCel5. Firstly,deleting a surface-exposed flexible loop had no effect on secretion. This indicated that some structural freedom is tolerated by the type II system. Secondly, mutation of a single tryptophan residue, previously shown to be important for binding to cellulose, i.e. Trp43, was found also to impair secretion. This indicated that the flat cellulose-binding surface of CBDCel5contains secretion-related information. Thirdly, CBDCel5 was substituted by the CBDEGG of Alteromonas haloplanctis endoglucanase G, yielding a hybrid protein CDCel5-LRCel5-CBDEGG that exhibited 90 % identity with Cel5, including the Trp43 residue. The hybrid protein was not secreted. This indicated that the Trp43 residue is necessary but not sufficient for secretion. Here we propose a model in which the secretion of Cel5 involves a transient intramolecular interaction between the cellulose-binding surface of CBDCel5 and a region close to the entry into the active site in CDCel5. Once secreted, the protein may then open out to allow the cellulose-binding surface of CBDCel5to interact with the surface of the cellulose substrate. An implication of this model is that protein molecules fold to a specific secretion-competent conformation prior to secretion that is different from the folding state of the secreted species.