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Please use this identifier to cite or link to this item: http://hdl.handle.net/1807/11126

Title: The Role of the N-Terminal Zinc Binding Domain of ClpX in Cofactor and Substrate Recognition
Authors: Thibault, Guillaume
Advisor: Houry, Walid A.
Department: Biochemistry
Issue Date: 28-Jul-2008
Abstract: ClpX is an ATPase that belongs to a unique group of ATP-dependent chaperones supporting targeted protein unfolding and degradation in concert with their respective proteases. ClpX functions alone or in conjunction with a cylindrical serine protease ClpP. ClpX consists of an N-terminal domain and a C-terminal AAA+ ATP-binding domain. The chaperone oligomerizes into a hexamer with the AAA+ domains forming the base of the hexamer and the N-termini extending out of the base. Here we demonstrate that the N-terminal domain of ClpX is a C4-type zinc binding domain (ZBD) which forms a very stable dimer. ZBD is essential for promoting the degradation of some established ClpXP substrates such as lambdaO and MuA. Experiments indicate that ZBD contains a primary binding site for the lambdaO substrate and for the cofactor SspB. Furthermore, analysis of the binding preferences of the ZBD and AAA+ domains revealed that both domains preferentially bind to hydrophobic residues but have different sequence preferences, with the AAA+ domain preferentially recognizing a wider range of specific sequences than ZBD. As part of this analysis, the binding site of SspB on ZBD in ClpX was determined by NMR and mutational analysis. The SspB C-terminus was found to interact with a hydrophobic patch on the surface of ZBD. The affinity of SspB towards ZBD and the geometry of the SspB-ZBD complex were also investigated. Analysis of ClpX conformational changes during its functional cycle indicated that the ZBDs in ClpX undergo a large nucleotide-dependent block movement into the AAA+ ring. Hence, we propose that ClpX switches between a capture and a feeding conformation. Based on all of these results, the ZBD in ClpX clearly plays a major role in substrate binding and cofactor recognition, as well as in substrate translocation into the ClpP chamber.
URI: http://hdl.handle.net/1807/11126
Appears in Collections:Doctoral
Department of Biochemistry - Doctoral theses

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