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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 : LCB-UPR9043 2 BIP-UPR9036 IBSM-CNRS 31 Chemin Joseph Aiguier. 13402, Marseille cedex 20 France 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 ¯exible 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 ¯at cellulose binding surface of CBD Cel5 contains secretion-related information. Thirdly, CBD Cel5 was substituted by the CBD EGG of Alteromonas haloplanc-tis 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 suf®cient 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 speci®c secretion-competent confor-mation 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 <>
Submitted on : Wednesday, July 12, 2017 - 10:26:24 AM
Last modification on : Wednesday, March 13, 2019 - 4:36:05 PM

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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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