Biophysical Characterization and Structural Insights Into the Complex between the Extracytoplasmic Function Sigma Factor, PupI, with the Antisigma Domain of the Sigma Regulator, PupR, from Pseudomonas capeferrum

Authors

Tajnin Sultana, North Dakota State University--Fargo
Robin N. Kutsi, North Dakota State University--Fargo
Alexander Tischer, Division of Hematology, Department of Internal Medicine, Mayo Clinic
Siri Froiland, North Dakota State University--Fargo
Elizabeth L. Gertz, North Dakota State University--Fargo
Jaime L. Jensen, North Dakota State University--Fargo
Matthew Auton, Division of Hematology, Department of Internal Medicine, Mayo Clinic
Sangita C. Sinha, North Dakota State University--Fargo
Christopher L. Colbert, North Dakota State University--Fargo

Document Type

Article

Publication Title

ASC Omega

Abstract

Gram-negative bacteria utilize siderophores to scavenge for environmental iron. Pseudomonas capeferrum produces the siderophores pseudobactin BN7/8, which trigger cell surface signaling (CSS). Iron import CSS pathways consist of an outer membrane TonBdependent transporter, an inner membrane sigma regulator, and a cytoplasmic sigma factor that activates transcription of target genes. The P. capeferrum sigma regulator, PupR, has an N-terminal antisigma domain (ASD). Previously, the PupR ASD was purified as a dimer in the absence of its cognate sigma factor, PupI. Here we use pulldown assays to demonstrate that His6-PupIFL interacts with the PupR ASD as well as with homodimer-disrupting mutants. We show that the PupR ASD and PupI bind with a dissociation constant of 180 nM using microscale thermophoresis. Circular dichroism spectroscopy confirms that each protein and the complex is well-folded, with predominantly α-helical secondary structure and there is no significant change in secondary structure upon complex formation. A high-confidence AlphaFold 3 predicted structure of the PupI:PupR ASD heterodimer complex indicates that the PupR ASD interacts with the C-terminal region of PupI. This model is supported by Hydrogen−Deuterium Exchange-Mass Spectrometry data (HDX-MS), which shows reduced solvent exchange in residues predicted to be in the complex interface. This model of the complex was validated by showing that PupI residues 99−173 bind to PupR. Further, a double mutation in the PupR ASD that maps to the heterodimer interface disrupts complex formation. These findings provide novel insights into how a sigma regulator interacts with its cognate sigma factor during iron import CSS.

DOI

https://doi.org/10.1021/acsomega.5c08813

Publication Date

12-19-2025

Comments

Published online: December 19, 2025

Published in print: January 13, 2026

Recommended Citation

Tajnin Sultana, Robin N. Kutsi, Alexander Tischer, Siri Froiland, Elizabeth L. Gertz, Jaime L. Jensen, Matthew Auton, Sangita C. Sinha, and Christopher L. Colbert. ACS Omega 2026 11 (1), 1717-1726 DOI: 10.1021/acsomega.5c08813