Insights into Site‐1 Protease Recognition of a Sigma Regulator Involved in Cell Surface Signaling During Pseudomonas capeferrumPseudobactin BN7/8 Import

Abstract

Gram‐negative CSS pathways are composed of three key components, an outer membrane transducer that senses extracellular stimuli, an inner membrane sigma regulator responsible for transducing a signal from the periplasm to the cytoplasm, and an extracytoplasmic function (ECF) sigma factor which activates transcription of target genes (1). In our model system, the Pseudomonas capeferrumpseudobactin BN7/8 (Pup) import system, these proteins are PupB, PupR, and PupI, respectively. Our recent insights into this CSS system indicate the C‐terminal cell‐surface signaling domain (CCSSD) of PupR and the N‐terminal signaling domain (NTSD) of PupB form a stable complex in the periplasm (2). This complex stabilizes the PupR CCSSD prior to signal transduction (2). Subsequently, CSS signal transduction is activated by regulated intramembrane proteolysis (RIP) of the sigma regulator by an initial site‐1 protease followed by a site‐2 protease (3, 4). Recent data has implicated the C‐terminal processing protease, Prc (or Tsp), as the site‐1 protease in PseudomonasCSS (4). Therefore, our objective is to investigate the recognition and degradation of the PupR CCSSD by Prc in the presence and absence of the PupB NTSD utilizing both active protease and catalytically inactive mutants, Prc‐His6 S485A and Prc‐His6 K510A. We hypothesize that the site‐1 protease, Prc, will recognize and cleave the PupR CCSSD unless shielded by PupB NTSD in non‐signaling conditions. Here we present the 2.0 Å structure of Prc‐His6 S485A solved by X‐ray crystallography and additional structural information of Prc‐His6 K510A by size exclusion chromatography small angle X‐ray scattering. Furthermore, we characterize the interaction of Prc‐His6 K510A with MBP‐PupR CCSSD in the presence and absence of PupB NTSD by affinity pull‐down assays and isothermal titration calorimetry, verifying the tighter affinity of the PupR CCSSD for the PupB and supporting its potential shielding role. Additionally, we show proteolytic degradation of MBP‐PupR CCSSD by active Prc‐His6 and analyze the proteolytic fragments by SDS‐PAGE and mass spectrometry. Together these data provide new structural insights into how CSS is activated by RIP following extracellular stimulus. Thus, we support a new CCS activation model wherein the sigma regulator is shielded from Prc by interacting with the transducer NTSD until an event alters this interaction allowing RIP.1. N. Noinaj, et al., TonB‐Dependent Transporters: Regulation, Structure, and Function. Annual Review of Microbiology 64, 43‐60 (2010).2. J. L. Jensen, et al., Structural basis of cell surface signaling by a conserved sigma regulator in Gram‐negative bacteria. Journal of Biological Chemistry (2020).3. R. C. Draper, et al., Differential proteolysis of sigma regulators controls cell‐surface signalling in Pseudomonas aeruginosa. Mol Microbiol 82, 1444‐1453 (2011).4. K. C. Bastiaansen, et al., The Prc and RseP proteases control bacterial cell‐surface signalling activity. Environ Microbiol 16, 2433‐2443 (2014).