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

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.

Figure 1

The asymmetric unit of the PupR-ASD crystal. The four PupR-ASD monomers in the asymmetric unit are displayed in ribbon. Monomer A is rendered in rainbow colors with blue at the N-terminus and red at the C-terminus with Monomer B in purple. Monomers C and D are displayed in shades of gray. The symmetric dimer comprises chains A and B; chains C and D are related by non-crystallographic symmetry to each other and to chains A and B. This and all molecular figures were made with PyMOL.

Figure 2

Electron density (blue mesh) for the conserved aromatic residues involved in stabilizing the PupR-ASD core. Helixes 1–3 of the PupR-ASD monomer form the core of the ASD, with off-centered parallel π-π stacking of His47 and Trp40, which is further stabilized by Trp20. The ASD backbone is displayed as a purple ribbon, with aromatic residues in stick, color-coded by atom type: N, blue; O, red; S, yellow and C, magenta.

Figure 3

Known ASD structures contain a conserved core helical bundle. Helices 1–3 of R. sphaeroides ChrR (blue; PDB entry 2Z2S; Campbell et al., 2007), M. tuberculosis RskA (green; PDB entry 4NQW; Shukla et al., 2014), M. tuberculosis RslA (salmon; PDB entry 3HUG; Thakur et al., 2010), and E. coli RseA (orange; PDB entry 1OR7; Campbell et al., 2003) were superimposed upon the PupR-ASD (purple).

Figure 4

SEC-SAXS analysis of PupR-ASD. A) The experimental scattering profile of the PupR-ASD (black) and the theoretical scattering profiles of the PupR-ASD dimer (green) and monomer (red) calculated from the X-ray crystal structure. B) The Guinier plot of the low q region of the X-ray scattering data. C) The distance distribution P(r) plot for the experimental data (black) and the theoretical curves calculated for the PupR-ASD monomer (red) and dimer (dimer). D) The Kratky plot calculated from the experimental scattering profile.

Figure 5

The PupR-ASD symmetric dimer interface. Residues selected for mutation to aspartic acid are shown in stick and colored by atom type as in Figure 2.

Figure 6

s_w isotherm for WT PupR-ASD created by integration of the c(s) peaks in the distribution between 0 and 4 S for the data recorded at 20°C, with error estimates calculated using SEDFIT.