University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2015 Elucidating the Energetics of Bacterial Signal Transduction: Insights From Phoq Kathleen Susan Molnar University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, and the Biophysics Commons Recommended Citation Molnar, Kathleen Susan, "Elucidating the Energetics of Bacterial Signal Transduction: Insights From Phoq" (2015). Publicly Accessible Penn Dissertations. 1097. https://repository.upenn.edu/edissertations/1097 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1097 For more information, please contact [email protected]. Elucidating the Energetics of Bacterial Signal Transduction: Insights From Phoq Abstract Bacteria transduce signals across the membrane using two-component systems, consisting of a membrane-spanning 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 Second Advisor Mark Goulian Keywords Coupled equilibria, Disulfide-crosslinking, Histidine kinase, PhoQ, Transmembrane, Two-component signaling Subject Categories Biochemistry | Biophysics This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1097 ELUCIDATING THE ENERGETICS OF BACTERIAL SIGNAL TRANSDUCTION: INSIGHTS FROM PHOQ Kathleen S. Molnar A DISSERTATION in Biochemistry and Molecular Biophysics Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2015 Supervisor of Dissertation Co-Supervisor of Dissertation ______________________________ ______________________________ William F. DeGrado, Ph.D. S. Walter Englander, Ph.D. Professor of Pharmaceutical Chemistry Professor of Biochemistry and Biophysics Graduate Group Chairperson ______________________________ Kim A. Sharp, Ph.D. Associate Professor of Biochemistry and Biophysics Dissertation Committee Mark Goulian, Ph.D. Mark A. Lemmon, Ph.D. Edmund J. and Louise W. Kahn Endowed Term George W. Raiziss Professor of Professor of Biology, Physics and Astronomy Biochemistry and Biophysics Bohdana M. Discher, Ph.D. James Shorter, Ph.D. Research Assistant Professor of Associate Professor of Biochemistry and Biophysics Biochemistry and Biophysics Yoshitomo Hamuro, Ph.D. Adjunct Associate Professor of Biochemistry and Biophysics ELUCIDATING THE ENERGETICS OF BACTERIAL SIGNAL TRANSDUCTION: INSIGHTS FROM PHOQ COPYRIGHT 2015 Kathleen S. Molnar This work is dedicated to my parents, Arpad and Susana Molnar, who are the source of inspiration and courage to dream big and work hard. iii Acknowledgements This work was completed within the walls of two great institutions, with the collaboration of many scientists and the support of even more friends. I have limited space to express my gratitude to this deserving group. I thank my thesis advisor, Bill DeGrado, for all that he provided, namely, his time, his lab, and his grilled salmon. I especially appreciate how supportive Bill has been through the many challenges I faced scientifically as well as circumstantially. I admire how he balances scientific passion with a passion for enjoying life. This balance extends also to the members of DeGrado lab who were thoughtful critics and editors as well as fantastic friends. I must thank several past and present lab members by name – Yoshitomo Hamuro, Shalom Goldberg, Graham Clinthorne, Manasi Bhate, Bruk Mensa – because their suggestions and contributions were crucial to the completion of this work. I also thank my committee – Mark Goulian, Walter Englander, Mark Lemmon, and Jim Shorter – not only for their scientific critiques and compelling suggestions, but for their patience and understanding. My committee chair, Mark, has also been a great collaborator and I thank him and his lab for being such a superb resource. I thank my co-advisor, Walter, for his support that was felt even across the country. Finally, I thank Yoshitomo Hamuro and Bohdana Discher for agreeing to join the committee as additional examiners; your efforts are greatly appreciated. I thank the Sali lab for their collaboration and I greatly appreciate their quick email responses. My heartfelt gratitude goes to my extremely supportive family; my parents, to whom I dedicate this thesis, and my sister, Elizabeth Molnar, who helped me in countless ways over the years. Finally, I thank Matthew Puster for his exuberant encouragement, his unwavering support, and especially his delicious breakfast sandwiches. iv ABSTRACT ELUCIDATING THE ENERGETICS OF BACTERIAL SIGNAL TRANSDUCTION: INSIGHTS FROM PHOQ Kathleen S. Molnar Dr. William F. DeGrado Dr. S. Walter Englander Bacteria transduce signals across the membrane using two-component systems, consisting of a membrane-spanning 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. v 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. vi Table of Contents Acknowledgements ......................................................................................................................... iv Abstract ............................................................................................................................................ v List of Tables ..................................................................................................................................