Biophysical Characterization and Structural Insights Into the Complex between the Extracytoplasmic Function Sigma...

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 TonB-dependent 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-PupI^FL 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.