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PhoQ: Structural and Mechanistic Investigations into an Important Bacterial Sensor Kinase Graham David Clinthorne University of Pennsylvania,
[email protected]
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PhoQ: Structural and Mechanistic Investigations into an Important Bacterial Sensor Kinase Abstract
Two-component systems represent the dominant mechanism for cellular signal transduction in prokaryotes. One particular system, PhoQ, has been the focus of our interest because it is the switch that controls virulence in Salmonella and other pathogenic gram negative bacteria. Certain domains of PhoQ and other twocomponent systems have been studied extensively and this research has yielded several structures with atomic level resolution. However, no complete structure or experimentally-based model has been forthcoming and the precise mechanism by which these diverse systems transmit signals from the exterior of the cell to the interior has remained elusive. We have undertaken a study to examine the E coli PhoQ from a structural and mechanistic viewpoint. We discovered a transmembrane polar residue that is conserved among many twocomponent systems that is critical for signaling in PhoQ. Critically, this feature is shared between PhoQ and the only two-component protein for which a transmembrane domain structure has been solved at the atomic level, HtrII. We undertook a disulfide scanning mutagenesis experiment to probe the structure of the transmembrane domain of PhoQ. We found evidence that PhoQ shares a similar topology with HtrII, especially the presence of a hemi-channel--a striking feature characterized by a group of tightly packed helices that sharply diverge at one end of the bundle to form a pocket that allows water molecules to penetrate into the core of the protein. We used the inferred homology between HtrII and PhoQ to create a model of the TM domain of PhoQ. Through statistical analysis of our Cys-crosslinking data, we determined that our data represented the average of different conformations of PhoQ brought about by changes in signaling state. We determined that the data was best explained by two models of PhoQ, which represent independent signaling states. This discovery represents the first evidence of a two-state model of activation of a Two-component protein, which we present alongside the first full-length model of a sensor kinase. Degree Type
Dissertation Degree Name
Doctor of Philosophy (PhD) Graduate Group
Pharmacology First Advisor
William F. DeGrado Second Advisor
Mark D. Goulian Keywords
histidine kinase, mechanism, PhoQ, structure, two-component system, two-state model
This dissertation is available at ScholarlyCommons: http://repository.upenn.edu/edissertations/624
Subject Categories
Biochemistry | Biophysics | Molecular Biology
This dissertation is available at ScholarlyCommons: http://repository.upenn.edu/edissertations/624
PHOQ: STRUCTURAL AND MECHANISTIC INVESTIGATIONS INTO AN IMPORTANT BACTERIAL SENSOR KINASE Graham D. Clinthorne A DISSERTATION in Pharmacology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2012
Supervisor of Dissertation William F. DeGrado Professor, Department of Pharmaceutical Chemistry University of California, San Francisco
Graduate Group Chairperson Julie Blendy Professor of Pharmacology Perelman School of Medicine Dissertation Committee Doron Greenbaum, Assistant Professor of Pharmacology Mark Goulian, Professor of Biology Mark Lemmon, Professor of Biochemistry and Biophysics Jun Zhu, Professor of Microbiology
Co-Supervisor of Dissertation Mark D. Goulian Professor, Department of Biology University of Pennsylvania
PHOQ: STRUCTURAL AND MECHANISTIC INVESTIGATIONS INTO AN IMPORTANT BACTERIAL SENSOR KINASE
COPYRIGHT 2012 Graham David Clinthorne This work is licensed under the Creative Commons AttributionNonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/
DEDICATION I dedicate this work to my wife, Megan. Megan, without your unwavering love, support and unflappable confidence in me I would never have found it in myself to complete this work. I could never thank you enough for the efforts you made to ensure my success.
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ACKNOWLEDGMENT
The Author would like to acknowledge the help of the following individuals who contributed to the success of this work by offering guidance, support, discussions, reagents, and the like: Bill DeGrado, Mark Goulian, Andrew Lippa, Tim Myashiro, Cinque Soto, Ilan Samish, Brett Hanningan, Gabriel Gonzalez, Geronda Montalvo, Chiam Schramm, Bruk Mensa, Gregory Caputo, Jun Wang. I would also like to thank contributors to this work and fellow authors Massimiliano Bonomi, Riccardo Pellarin, and Andrej Sali whose hard work, insight, and methods of statsitcal analysis have made the interpretation of this complex data possible and has elevated the impact of this work tremendously. Special thanks go to Shalom D. Goldberg whose previous work in the PhoQ system laid the foundations for this work, and who personally helped me develop the assays and procedures herein. Also, special thanks go to Ivan Korendyvich who- along with Dr. Goldberg, helped me grow as a scientist and a person.
Finally I would like to express my gratitude to Dr. Mark Goulian who provided support for me and my research in ways too numerous to recount here and without whom this work would certainly never have come to fruition.
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ABSTRACT PhoQ: Structural and Mechanistic investigations into an important bacterial sensor kinase Graham D. Clinthorne William F. Degrado Two-component systems represent the dominant mechanism for cellular signal transduction in prokaryotes. One particular system, PhoQ, has been the focus of our interest because it is the switch that controls virulence in Salmonella and other pathogenic gram negative bacteria. Certain domains of PhoQ and other two-component systems have been studied extensively and this research has yielded several structures with atomic level resolution. However, no complete structure or experimentally-based model has been forthcoming and the precise mechanism by which these diverse systems transmit signals from the exterior of the cell to the interior has remained elusive. We have undertaken a study to examine the E coli PhoQ from a structural and mechanistic viewpoint. We discovered a transmembrane polar residue that is conserved among many two-component systems that is critical for signaling in PhoQ. Critically, this feature is shared between PhoQ and the only two-component protein for which a transmembrane domain structure has been solved at the atomic level, HtrII. We undertook a disulfide scanning mutagenesis experiment to probe the structure of the transmembrane domain of PhoQ. We found evidence that PhoQ shares a similar topology with HtrII, especially the presence of a hemi-channel—a striking feature characterized by a group of tightly packed helices that sharply diverge at one end of the bundle to form a pocket that allows water molecules to penetrate into the core of the protein. We used the inferred homology between HtrII and PhoQ to create a model of the TM domain of PhoQ. Through statistical analysis of our Cys -crosslinking data, we determined that our data represented the average of different conformations of PhoQ brought about by changes in signaling state. We determined that the data was best explained by two models of PhoQ, which represent independent signaling states. This discovery represents the first evidence of a two-state model of activation of a Two-component protein, which we present alongside the first full-length model of a sensor kinase.
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TABLE OF CONTENTS
PHOQ: STRUCTURAL AND MECHANISTIC INVESTIGATIONS INTO AN IMPORTANT BACTERIAL SENSOR KINASE ........................................................... I DEDICATION PAGE ........................................................................................................ III ACKNOWLEDGMENT..................................................................................................... IV ABSTRACT......................................................................................................................... V TABLE OF CONTENTS.................................................................................................... VI LIST OF TABLES ............................................................................................................. IX LIST OF ILLUSTRATIONS................................................................................................ X CHAPTER 1: INTRODUCTION TO TWO-COMPONENT SIGNALING ......................... 1 Background....................................................................................................................................................................... 1 What is Known About PhoQ ......................................................................................................................................... 5 The Sensor Domain..................................................................................................................................................... 6 The Transmembrane domain ................................................................................................................................... 9 The HAMP domain....................................................................................................................................................12 The Kinase Domain ...................................................................................................................................................16 Partner Recognition..................................................................................................................................................17 Autophosphorylation and Phosphotransfer Reaction .......................................................................................19 The role of the ATP lid..............................................................................................................................................21 Cis or Trans?...............................................................................................................................................................22 The phosphatase reaction .......................................................................................................................................24 Mechanisms of Signal Transduction ......................................................................................................................25 Small peptide regulators of PhoQ..........................................................................................................................28
vii What Questions will be asked with this work? ......................................................................................................30
CHAPTER 2: IDENTIFICATION OF A CONSERVED SIGNALING COMPONENT: POLAR RESIDUES IN THE TRANSMEMBRANE DOMAIN ......................................... 31 Identification of polar residues in PhoQ that are required for signali ng in vivo .............................................31 In vitro investigations into the activity of the N202A mutant.............................................................................35 Functional recapitulation of the WT PhoP/Q system in the N202A background ............................................36 Saturation mutagenesis at position 202 ...............................................................................................................36 Nearby TM2 residues replaced with Asn..............................................................................................................37 Residues of TM1 replaced with Asn ......................................................................................................................39 Conclusions.....................................................................................................................................................................41
CHAPTER 3: STRUCTURAL AND MECHANISTIC INVESTIGATIONS INTO THE TRANSMEBRANE DOMAIN OF PHOQ: EVIDENCE FOR A TWO-STATE MODEL OF ACTIVATION ................................................................................................................... 47 Cys scanning mut agenesis ...........................................................................................................................................47 Discussion of method ...............................................................................................................................................47 Results.........................................................................................................................................................................51 Homology modeling and sequence threading with HtrII .....................................................................................56 Inter-Subunit Crosslinking ...........................................................................................................................................58 Bayesian modeling of Cys crosslinking dat a............................................................................................................63 Introduction to macromolecular modeling..........................................................................................................64 The initial state ..........................................................................................................................................................65 Single state modeling...............................................................................................................................................66 A two state model.....................................................................................................................................................68 A Thr ee state model .................................................................................................................................................73 Discussion of Structures...............................................................................................................................................75
CHAPTER 4: MUTATIONAL ANALYSIS OF THE CONNECTOR DOMAINS THROUGH HELIX-DISRUPTING SUBSTITUTIONS.................................................... 86 Effect of Cys Mutations on Activity ...........................................................................................................................86 Ala and Phe scanning mut agenesis ...........................................................................................................................88 Discussion .......................................................................................................................................................................95
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CONCLUDING REMARKS............................................................................................... 98 BIBLIOGRAPHY ............................................................................................................104 INDEX .............................................................................................................................110
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LIST OF TABLES Table 1: Distances from the homologous residues in HtrII and corresponding fraction crosslinking between residue 207 and the TM1 residue, as shown in Figure 32 F. ....................................... 62 Table 2: Properties of the 10 most populated clusters found with single-state modeling: cluster population, χ2 and likelihood of the cluster center, cluster average and cluster best. ............... 66 Table 3: RMSD from known crystallographic structures of periplasmic domains, for model found in single- and two-states modeling......................................................................................... 68 Table 4: Properties of the 10 most populated clusters found by two-states modeling: cluster population, χ2 and likelihood of the cluster center, cluster average and cluster best. ............... 70 Table 5: Properties of the 10 most populated clusters found in three-states modeling: cluster population, χ2 and likelihood of the cluster center, cluster average and cluster best. ............... 74 Table 6: Summary of the effects of mutations on the activity of PhoQ under activating conditions. Asterisks indicate p values
