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Published July 14, 2015 | Published + Supplemental Material
Journal Article Open

Structural asymmetry in a conserved signaling system that regulates division, replication, and virulence of an intracellular pathogen

Abstract

We have functionally and structurally defined an essential protein phosphorelay that regulates expression of genes required for growth, division, and intracellular survival of the global zoonotic pathogen Brucella abortus. Our study delineates phosphoryl transfer through this molecular pathway, which initiates from the sensor kinase CckA and proceeds through the ChpT phosphotransferase to two regulatory substrates: CtrA and CpdR. Genetic perturbation of this system results in defects in cell growth and division site selection, and a specific viability deficit inside human phagocytic cells. Thus, proper control of B. abortus division site polarity is necessary for survival in the intracellular niche. We further define the structural foundations of signaling from the central phosphotransferase, ChpT, to its response regulator substrate, CtrA, and provide evidence that there are at least two modes of interaction between ChpT and CtrA, only one of which is competent to catalyze phosphoryltransfer. The structure and dynamics of the active site on each side of the ChpT homodimer are distinct, supporting a model in which quaternary structure of the 2:2 ChpT–CtrA complex enforces an asymmetric mechanism of phosphoryl transfer between ChpT and CtrA. Our study provides mechanistic understanding, from the cellular to the atomic scale, of a conserved transcriptional regulatory system that controls the cellular and infection biology of B. abortus. More generally, our results provide insight into the structural basis of two-component signal transduction, which is broadly conserved in bacteria, plants, and fungi.

Additional Information

© 2015 National Academy of Sciences. Edited by Graham C. Walker, Massachusetts Institute of Technology, Cambridge, MA, and approved June 2, 2015 (received for review February 13, 2015). Published ahead of print June 29, 2015. We thank Aretha Fiebig and members of the S.C. laboratory for discussions and guidance during the preparation of this manuscript and Elena Solomaha and Ryan Duggan for technical assistance. This project has been funded in whole or in part with federal funds from NIH–National Institute of Allergy and Infectious Diseases Grants U19 AI107792 and R01 AI107159 (to S.C.). J.W.W. is supported by NIH Ruth Kirschstein Postdoctoral Fellowship F32 GM109661. Funding for LS-CAT Sector 21 was provided by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor Grant 085P1000817. Small angle X-ray scattering at Advanced Photon Source–BioCAT is supported by NIH Grant P41 GM103622. Author contributions: J.W.W. and S.C. designed research; J.W.W., J.H., A.B., and G.R. performed research; J.W.W., J.H., A.B., G.R., and S.C. contributed new reagents/analytic tools; J.W.W., J.H., A.B., G.R., and S.C. analyzed data; and J.W.W. and S.C. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: Crystallography, atomic coordinates, and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4QPK and 4QPJ). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1503118112/-/DCSupplemental.

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Published - PNAS-2015-Willett-E3709-18.pdf

Supplemental Material - pnas.201503118SI.pdf

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