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Biochemical and structural characterization of CpxP and CpxA, key components of an envelope stress response in Escherichia coli Open Access


Other title
envelope stress
two-component system
Gram-negative bacteria
Type of item
Degree grantor
University of Alberta
Author or creator
Thede, Gina L.
Supervisor and department
Glover, Mark (Biochemistry)
Young, Howard (Biochemistry)
Examining committee member and department
Glover, Mark (Biochemistry)
Raivio, Tracy (Biological Sciences)
MacMillan, Andrew (Biochemistry)
Cygler, Mirek (University of Saskatchewan, Biochemistry)
Young, Howard (Biochemistry)
Department of Biochemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
The Cpx two‐component signal transduction pathway of Escherichia coli consists of an inner membrane‐localized sensor histidine kinase, CpxA, the response regulator, CpxR, and the novel periplasmic accessory protein, CpxP. These proteins mediate a bacterial stress response, sensing envelope perturbations including damage caused by misfolded periplasmic or inner membrane proteins, and regulating the expression of a host of factors that contribute to preservation of the envelope. While structural information exists for a number of bacterial two‐component systems, there is limited data to describe the constituents of the Cpx system. Since CpxP has no homologues of known function, we initially focused on its biophysical and structural characterization. Biochemical studies demonstrated that CpxP maintains a dimeric state, but may undergo a slight structural adjustment in response to the Cpx pathway inducing cue, alkaline pH. The crystal structure of CpxP, determined to 2.85 Å resolution, revealed an antiparallel dimer of intertwined α‐helices with a highly basic concave surface. We identified that the core fold adopted by CpxP is also found in a number of other periplasmic stress response proteins. Additionally, we proposed that the conserved LTXXQ motifs that define a family of proteins have a structural role in the formation of diverging turns. Finally, we identified several sites, including two solvent‐exposed residues and the charged surfaces of CpxP, which are likely involved in intermolecular interactions. In an effort to understand the molecular mechanism(s) by which CpxA detects specific inducing signals, we set out to describe the uncharacterized periplasmic sensor domain of CpxA biochemically and structurally. We demonstrated that the isolated sensor domain likely exists in a unique tetramer‐dimer equilibrium. Further, we suggested that the periplasmic domain of CpxA forms a PAS‐like PDC fold, and identified regions that could possibly be involved in a structural rearrangement upon stimulus perception. Lastly, we established preliminary crystallization conditions that will be optimized for high‐resolution structure determination. Ultimately, these studies will provide insight into the molecular characteristics of CpxA and CpxP that contribute to the regulation of the response to envelope stress in pathogenic bacteria such as E. coli.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Thede, G. L., D. C. Arthur, R. A. Edwards, D. R. Buelow, J. L. Wong, T. L. Raivio, and J. N. M. Glover. 2011. Structure of the periplasmic stress response protein CpxP. J Bacteriol 193:2149‐57.Chaulk, S. G., G. L. Thede, O. A. Kent, Z. Xu, E. M. Gesner, R. A. Veldhoen, S. K. Khanna, I. S. Goping, A. M. MacMillan, J. T. Mendell, H. S. Young, R. P. Fahlman, and J. N. M. Glover. 2011. Role of pri‐miRNA tertiary structure in miR‐17~92 miRNA biogenesis. RNA Biol 8:1105‐14.

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