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Title: Crystal structure of B acillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity: AtxA multimerization, phosphorylation and activity

Abstract

The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (HisAsp) and phosphoablative (HisAla) amino acid changes for activity in B.anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.

Authors:
 [1];  [1];  [1];  [2];  [2];  [1]
  1. Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston, Houston TX USA
  2. Center for Structural Genomics of Infectious Diseases, Argonne National Laboratory, Argonne IL USA; Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne IL USA
Publication Date:
Research Org.:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH). National Institute of Allergy and Infectious Diseases (NIAID); Keck Center for Computational and Structural Biology in Biodefense Training Program
OSTI Identifier:
1339284
DOE Contract Number:  
AC02-06CH11357
Resource Type:
Journal Article
Journal Name:
Molecular Microbiology
Additional Journal Information:
Journal Volume: 95; Journal Issue: 3; Journal ID: ISSN 0950-382X
Publisher:
Wiley
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Hammerstrom, Troy G., Horton, Lori B., Swick, Michelle C., Joachimiak, Andrzej, Osipiuk, Jerzy, and Koehler, Theresa M. Crystal structure of B acillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity: AtxA multimerization, phosphorylation and activity. United States: N. p., 2014. Web. doi:10.1111/mmi.12867.
Hammerstrom, Troy G., Horton, Lori B., Swick, Michelle C., Joachimiak, Andrzej, Osipiuk, Jerzy, & Koehler, Theresa M. Crystal structure of B acillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity: AtxA multimerization, phosphorylation and activity. United States. https://doi.org/10.1111/mmi.12867
Hammerstrom, Troy G., Horton, Lori B., Swick, Michelle C., Joachimiak, Andrzej, Osipiuk, Jerzy, and Koehler, Theresa M. 2014. "Crystal structure of B acillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity: AtxA multimerization, phosphorylation and activity". United States. https://doi.org/10.1111/mmi.12867.
@article{osti_1339284,
title = {Crystal structure of B acillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity: AtxA multimerization, phosphorylation and activity},
author = {Hammerstrom, Troy G. and Horton, Lori B. and Swick, Michelle C. and Joachimiak, Andrzej and Osipiuk, Jerzy and Koehler, Theresa M.},
abstractNote = {The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (HisAsp) and phosphoablative (HisAla) amino acid changes for activity in B.anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.},
doi = {10.1111/mmi.12867},
url = {https://www.osti.gov/biblio/1339284}, journal = {Molecular Microbiology},
issn = {0950-382X},
number = 3,
volume = 95,
place = {United States},
year = {Tue Dec 30 00:00:00 EST 2014},
month = {Tue Dec 30 00:00:00 EST 2014}
}

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