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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

Abstract : 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 CBD(Cel5). 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 CBD(Cel5 )contains secretion-related information. Thirdly, CBD(Cel5) was substituted by the CBD(EGG) of Alteromonas haloplanctis endoglucanase G, yielding a hybrid protein CD(Cel5)-LR(Cel5)-CBD(EGG) 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 CBD(Cel5) and a region close to the entry into the active site in CD(Cel5). Once secreted, the protein may then open out to allow the cellulose-binding surface of CBD(Cel5 )to 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.
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https://hal-amu.archives-ouvertes.fr/hal-01560905
Contributor : Virginie Chapon Connect in order to contact the contributor
Submitted on : Wednesday, July 12, 2017 - 10:26:24 AM
Last modification on : Thursday, January 13, 2022 - 10:00:03 AM

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Virginie Chapon, Helen Simpson, Xavier Morelli, Emmanuel Brun, Frederic Barras. 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. Journal of Molecular Biology, Elsevier, 2000, 303, pp.117-123. ⟨10.1006/jmbi.2000.4103⟩. ⟨hal-01560905⟩

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