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MIAMI UNIVERSITY the Graduate School MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Tzvia Ilana Springer Candidate for the Degree Doctor of Philosophy ______________________________________ Natosha L. Finley, Advisor ______________________________________ Rachael Morgan-Kiss ______________________________________ Joseph M. Carlin, Reader ______________________________________ D.J Ferguson, Reader ______________________________________ Jack C. Vaughn, Graduate School Representative ABSTRACT STRUCTURAL CHARACTERIZATION OF HUMAN CALMODULIN AND ITS ROLE IN ACTIVATING BORDETELLA PERTUSSIS ADENYLYL CYCLASE TOXIN, CYAA by Tzvia I. Springer Bordetella pertussis is a human pathogen that causes whooping cough, a respiratory disease found within the human respiratory tract. Although vaccines are readily available, whooping cough continues to be a significant threat to infants and children worldwide. Bordetella pertussis requires a plethora of virulence factors, including its adenylyl cyclase toxin (ACT), CyaA, to initiate an infection and cause disease. The adenylate cyclase domain of CyaA (CyaA-ACD) requires host calmodulin (CaM) for activation, utilizing a unique mechanism that involves both a Ca2+ dependent and independent mode of activation. Currently, there is no high-resolution structure available of CaM-bound CyaA-ACD and the mechanism of activation remains elusive. CaM is a Ca2+ sensing host protein that contains both an N- and C-terminal domain connected by a flexible linker. Although studies highlight tight C-terminal CaM (C-CaM) interactions with CyaA-ACD, physiological studies demonstrate the importance for intact CaM to fully activate CyaA-ACD. Our main purpose was to determine specific intermolecular interactions between N- terminal CaM (N-CaM) and CyaA-ACD that led to activation, and contrast how this interaction may differ from other CaM-dependent bacterial adenylyl cyclase toxins. Our findings demonstrate unique site-specific interactions between CyaA-ACD and N-terminal CaM. Specifically, metal-binding properties at site I and II in N-CaM are affected when regions mapping to the CyaA-ACD β-hairpin catalytic loop are mutated through site-directed mutagenesis. Moreover, mutations mapping to metal binding sites I and II in N-CaM reduce both CyaA-ACD binding affinity and cAMP production without compromising structural stability of CaM. Taken together, this research shows a novel way that a CaM-dependent bacterial ACT such as CyaA-ACD has evolved to regulate its activation during a host infection. STRUCTURAL CHARACTERIZATION OF HUMAN CALMODULIN AND ITS ROLE IN ACTIVATING BORDETELLA PERTUSSIS ADENYLYL CYCLASE TOXIN, CYAA A DISSERTATION Presented to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology by Tzvia I., Springer The Graduate School Miami University Oxford, Ohio 2016 Dissertation Director: Natosha L. Finley © Tzvia Ilana Springer 2016 TABLE OF CONTENTS Chapter 1: Introduction ............................................................................................................... 1 1.1Adenylyl cyclases ...................................................................................................................... 2 1.1.1 Bacterial adenylyl cyclases ........................................................................................ 2 1.1.2 Bordetella pertussis ................................................................................................. 11 1.1.3 CyaA structure and function .................................................................................... 11 1.1.4 Bacillus anthracis .................................................................................................... 15 1.1.5 Edema factor structure and function ........................................................................ 15 1.2 Metal binding proteins ............................................................................................................ 19 1.2.1 Calmodulin and its Ca2+ bound structure ................................................................. 19 1.2.2 Calmodulin and its Mg2+ bound structure ................................................................ 22 1.3 Study of soluble protein utilizing solution NMR spectroscopy .............................................. 26 1.3.1 Solution state NMR techniques ............................................................................... 26 1.3.2 TROSY-based NMR experiments ........................................................................... 26 1.3.3 15N Solution state spectroscopy ............................................................................... 27 1.3.4 13C solution state spectroscopy ................................................................................ 27 1.4 Dissertation goals and hypotheses .......................................................................................... 30 1.5 References ............................................................................................................................... 32 Chapter 2: Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin ........................................................................ 36 2.1 Abstract ................................................................................................................................... 37 2.2 Introduction ............................................................................................................................. 39 2.3 Materials and methods ............................................................................................................ 40 2.3.1 Protein expression, purification, and complex formation ........................................ 40 2.3.2 NMR experiments .................................................................................................... 41 2.3.3 DLS experiments ..................................................................................................... 42 2.4 Results and discussion ............................................................................................................ 43 2.4.1 Conformational modulation of CaM upon CyaA-ACD association ........................ 43 2.4.2 Configuration of CaM’s metal-binding loops in the CyaA-ACD bound state ........ 50 2.4.3 Global conformation and thermal stability of CaM/CyaA-ACD complexes ........... 54 iii 2.5 References ............................................................................................................................... 58 Chapter 3: Interaction with adenylate cyclase toxin from Bordetella pertussis affects the metal binding properties of calmodulin .................................................................................... 61 3.1 Abstract ................................................................................................................................... 64 3.2 Introduction ............................................................................................................................. 65 3.3 Materials and methods ............................................................................................................ 68 3.3.1 Sample preparation .................................................................................................. 68 3.3.2 Nuclear magnetic resonance spectroscopy (NMR) .................................................. 69 3.3.3 Dynamic light scattering experiments ..................................................................... 69 3.3.4 Native PAGE ........................................................................................................... 69 3.4 Results and discussion ............................................................................................................ 70 3.4.1 Metal binding in N-terminal CaM upon CyaA-ACD association ........................... 70 3.4.2 Global conformational changes in metal-loaded CaM/CyaA-ACD complexes ...... 79 3.5 References ............................................................................................................................... 87 Chapter 4: Conclusions .............................................................................................................. 90 4.1 CyaA-ACD binding modulates CaM conformation ............................................................... 91 4.2 Disruption in inter-domain communication in CaM impacts effector binding ....................... 92 4.3 Future directions ..................................................................................................................... 94 4.4 References ............................................................................................................................... 95 Appendix A: Structural characterization of cardiac myosin binding protein-C .................. 97 A.1 Introduction ............................................................................................................................ 97 A.2 A hypertrophic cardiomyopathy-associated MYBPC3 mutation common in populations of South Asian descent causes contractile dysfunction ..................................................................... 99 A.2.1 Abstract ................................................................................................................. 102 A.2.2 Introduction ..........................................................................................................
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