Abstract:

<p>Cell-surface signaling (CSS) in Gram-negative bacteria involves highly conserved regulatory pathways that optimize gene expression by transducing extracellular environmental signals to the cytoplasm via inner-membrane sigma regulators. The molecular details of ferric siderophore-mediated activation of the iron import machinery through a sigma regulator are unclear. Here, we present the 1.56 Å resolution structure of the periplasmic complex of the C-terminal CSS domain (CCSSD) of PupR, the sigma regulator in the Pseudomonas capeferrum pseudobactin BN7/8 transport system, and the N-terminal signaling domain (NTSD) of PupB, an outer-membrane TonB-dependent transducer. The structure revealed that the CCSSD consists of two subdomains: a juxta-membrane subdomain, which has a novel all-β fold, followed by a secretin/TonB, short N-terminal subdomain at the C-terminus of the CCSSD, a previously unobserved topological arrangement of this domain. Using affinity pull-down assays, isothermal titration calorimetry, and thermal denaturation circular dichroism spectroscopy; we show that both subdomains are required for binding the NTSD with micromolar affinity and that NTSD binding improves CCSSD stability. Our findings prompt us to present a revised model of CSS wherein the CCSSD:NTSD complex forms prior to ferric-siderophore binding. Upon siderophore binding, conformational changes in the CCSSD enable regulated intramembrane proteolysis of the sigma regulator, ultimately resulting in transcriptional regulation.</p>