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This content will become publicly available on September 29, 2016

Title: Mechanistic Implications of the Unique Structural Features and Dimerization of the Cytoplasmic Domain of the Pseudomonas Sigma Regulator, PupR

Gram-negative bacteria tightly regulate intracellular levels of iron, an essential nutrient. To ensure this tight regulation, some outer membrane TonB-dependent transporters (TBDTs) that are responsible for iron import stimulate their own transcription in response to extracellular binding by an iron-laden siderophore. This process is mediated by an inner membrane sigma regulator protein (an anti-sigma factor) that transduces an unknown periplasmic signal from the TBDT to release an intracellular sigma factor from the inner membrane, which ultimately upregulates TBDT transcription. Here we use the Pseudomonas putida ferric-pseudobactin BN7/BN8 sigma regulator, PupR, as a model system to understand the molecular mechanism of this conserved class of sigma regulators. We have determined the X-ray crystal structure of the cytoplasmic anti-sigma domain (ASD) of PupR to 2.0 Å. Size exclusion chromatography, small angle X-ray scattering, and sedimentation velocity analytical ultracentrifugation, all indicate that in contrast to other ASDs, the PupR-ASD exists as a dimer in solution. Mutagenesis of residues at the dimer interface identified from the crystal structure disrupts dimerization and protein stability, as determined by sedimentation velocity analytical ultracentrifugation and thermal denaturation circular dichroism spectroscopy. Lastly, these combined results suggest that this type of inner membrane sigma regulator may utilize an unusual mechanismmore » to sequester their cognate sigma factors and prevent transcription activation.« less
Authors:
 [1] ;  [2] ;  [3] ;  [4] ;  [2] ;  [1] ;  [1]
  1. North Dakota State Univ., Fargo, ND (United States)
  2. National Inst. of Health (NIH), Bethesda, MD (United States). National Inst. of Biomedical Imaging and Bioengineering
  3. Argonne National Lab. (ANL), Argonne, IL (United States)
  4. Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Publication Date:
OSTI Identifier:
1280870
Grant/Contract Number:
AC02-06CH11357; 1R03 NS090939; 2P20 RR015566; S10 RR029205; MCB-1413525; FAR0025216; 1R15 GM113227; 1S10OD018090-01; 9P41 GM103622; P30 GM103332; P41 GM103403
Type:
Accepted Manuscript
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 54; Journal Issue: 38; Journal ID: ISSN 0006-2960
Publisher:
American Chemical Society (ACS)
Research Org:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Org:
USDOE Office of Science (SC); National Inst. of Health (NIH)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES