5CAM

Crystal Structure of the Cytoplasmic Domain of the Pseudomonas putida Anti-sigma Factor PupR (SeMet)

Summary for 5CAM

• Entry DOI: 10.2210/pdb5cam/pdb
• Descriptor: PupR protein (2 entities in total)
• Functional Keywords: bacterial proteins, membrane proteins, cytoplasm, sigma factor, transcription factors, gene expression regulation, transcription
• Biological source: Pseudomonas putida
• Total number of polymer chains: 4
• Total formula weight: 38996.98

Authors

Jensen, J.L.,Colbert, C.L. (deposition date: 2015-06-29, release date: 2015-09-09, Last modification date: 2024-11-06)

Primary citation

Jensen, J.L.,Balbo, A.,Neau, D.B.,Chakravarthy, S.,Zhao, H.,Sinha, S.C.,Colbert, C.L.
Mechanistic Implications of the Unique Structural Features and Dimerization of the Cytoplasmic Domain of the Pseudomonas Sigma Regulator, PupR.
Biochemistry, 54:5867-5877, 2015

PubMed Abstract: Gram-negative bacteria tightly regulate intracellular levels of iron, an essential nutrient. To ensure this strict control, 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. These combined results suggest that this type of inner membrane sigma regulator may utilize an unusual mechanism to sequester their cognate sigma factors and prevent transcription activation.

PubMed: 26313375
DOI: 10.1021/acs.biochem.5b00826

Experimental method

X-RAY DIFFRACTION (2.171 Å)