Alteration of a single tryptophan residue of the cellulose-binding domain blocks secretion of the Erwinia chrysanthemi Cel5 cellulase (ex-EGZ) via the type II system

Ce15 (formerly known as endoglucanase Z) of Erwinia chrysanthemi is secrete d by the Out type II pathway. Previous studies have shown that the catalyti c domain (CD), linker region (LR) and cellulose-binding domain (CBD) each c ontain information needed for secretion. The aim of this work was to furthe r investigate the secretion-related information present in the CBDCe15. Fir stly, deleting a surface-exposed flexible loop had no effect on secretion. This indicated that some structural freedom is tolerated by the type II sys tem. Secondly, mutation of a single tryptophan residue, previously shown to be important for binding to cellulose, i.e. Trp43, was found also to impai r secretion. This indicated that the flat cellulose-binding surface of CBDC e15 contains secretion-related information. Thirdly, CBDCe15 was substitute d by the CBDEGG of Alteromonas haloplanctis endoglucanase G, yielding a hyb rid protein CDCe15-LRCe15-CBDEGG that exhibited 90 % identity with Ce15, in cluding the Trp43 residue. The hybrid protein was not secreted. This indica ted that the Trp43 residue is necessary but not sufficient for secretion. H ere we propose a model in which the secretion of Ce15 involves a transient intramolecular interaction between the cellulose-binding surface of CBDCe15 and a region close to the entry into the active site in CDCe15. Once secre ted, the protein may then open out to allow the cellulose-binding surface o f CBDCe15 to interact with the surface of the cellulose substrate. An impli cation of this model is that protein molecules fold to a specific secretion -competent conformation prior to secretion that is different from the foldi ng state of the secreted species. (C) 2000 Academic Press.