| dc.description.abstract | Cell surface signaling (CSS) allows Gram-negative bacteria to transcriptionally regulate gene expression in response to external stimuli. CSS pathways involve three key components: an outer membrane transducer for sensing the extracellular stimuli; an inner membrane sigma regulator for relaying the signal; and a cytoplasmic sigma factor, which activates transcription of target genes. The goal of this research was to structurally and biophysically characterize events leading to the processing of the sigma regulator that results in transcription activation. Our model systems are the Pseudomonas capeferrum BN7/8 (Pup) and Escherichia coli ferric citrate (Fec) uptake pathways.
We detail the X-ray crystal structure of the N-terminal signaling domain (NTSD) of the transducer, PupB, complexed with the C-terminal cell-surface signaling domain (CCSSD) of the sigma regulator, PupR. Stabilization of the PupR CCSSD by the PupB NTSD provides a rationale for the formation of a preformed CSS complex. Additionally, we probed the FecR CCSSD and FecA NTSD interaction and observed similarities. We found the FecA NTSD complexes with the FecR CCSSD and stabilizes the domain in nonsignaling conditions indicating a conserved mechanism.
Further, we show that access to the PupR CCSSD is only possible in the absence of the PupB NTSD. Pulldown assays, isothermal titration calorimetry, protease assays, and mass spectrometry analysis demonstrate the site-1 protease, Prc, only recognizes and degrades PupR in the absence of the PupB NTSD. X-ray crystal structures of Prc mutants and potential product peptides reveal transitions between “closed” and “open” conformations as well as catalytic intermediates in the protease active site. Size exclusion small angle X-ray scattering data confirms the Prc conformations in solution and an elongated molecular envelope of the Prc:PupR complex. Together this provides new structural insights into protease activation during CSS.
Finally, we studied the TonB C-terminal domain of P. capeferrum by size exclusion small angle X-ray scattering. Our results indicate it forms a monomeric structure in solution.
Overall, our results indicate there is a conserved CSS pathway that has been characterized by our individual signal transduction states. Thus, we have provided novel implications in ferric siderophore uptake and the mechanism of iron import mediated CSS. | en_US |
