Elucidating the Energetics of Bacterial Signal Transduction: Insights From Phoq

Bacteria transduce signals across the membrane using two-component systems, consisting of a membranespanning sensor histidine kinase and a cytoplasmic response regulator. The histidine kinase, PhoQ, serves as a master regulator of virulence response in S. typhimurium and E. coli. It also is inhibited by divalent cations, particularly Mg2+. While the periplasmic sensor domain of this protein has a unique function, the cytoplasmic portion of this modular protein is made of structurally conserved domains found in many other bacterial sensor kinases. Signal transduction through these conserved domains is thought to be universal; however, the structural and energetic rearrangements that occur during signaling have generated numerous models. Through Bayesian inference we constructed a two-state model based on cysteine crosslinking data and homologous crystal structures. These two signaling states differ in membrane depth of the periplasmic acidic patch as well as the reciprocal displacement of diagonal helices along the dimer interface. Comparative studies of multiple histidine kinases suggest that diagonal displacement of helices is a common mode of signal transduction. A similar scissor-like model was previously ruled out in CheA-linked chemoreceptors; therefore, this new evidence suggests that sensor His-kinase and CheA-linked receptors possess different signaling mechanisms. To unify the various signaling mechanisms that exist for the different protein domains, we built a thermodynamic model based on Linked Equilibrating Domains (LED). We used this model to quantitatively interpret functional data of single-point Ala, Phe and Cys mutants throughout the signal transducing regions of PhoQ. Data from 35 mutants, including both activating and deactivating phenotypes, were globally fit using LED, and gross features such as Vmax and Kd were related to more nuanced population distributions and thermodynamic coupling. LED analysis highlights the principles by which individual signaling domains can be connected to create a functional signal transducer. These principles allow us to quantitatively explain signaling in histidine kinases and are likely to be broadly applicable to many other signal transduction proteins. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biochemistry & Molecular Biophysics First Advisor William F. DeGrado This dissertation is available at ScholarlyCommons: http://repository.upenn.edu/edissertations/1097 Second Advisor Mark Goulian