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A Thesis Entitled Phor, Phop and Mshc A Thesis entitled PhoR, PhoP and MshC: Three essential proteins of Mycobacterium tuberculosis by Erica Loney Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Chemistry __________________________________ Dr. Donald R. Ronning, Committee Chair __________________________________ Dr. John J. Bellizzi, Committee Member __________________________________ Dr. Ronald Viola, Committee Member __________________________________ Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo May 2014 Copyright 2014, Erica Loney This document is copyrighted material. Under copyright law, no parts of this document may be produced without the expressed permission of the author. An Abstract of PhoR, PhoP and MshC: Three essential proteins of Mycobacterium tuberculosis by Erica Loney Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Chemistry The University of Toledo May 2014 The tuberculosis (TB) pandemic is responsible for 1.6 million deaths annually, most of which occur in developing nations. TB is treatable, though patient non- compliance, co-infection with HIV, and the long, 6-9 month treatment regimen have resulted in the emergence of drug-resistant TB. For these reasons, the development of novel anti-tuberculin drugs is essential. Three proteins – PhoR, PhoP, and MshC – of Mycobacterium tuberculosis (M.tb), the causative agent of TB, are the focus of this thesis. The PhoPR two-component system is a phosphorelay system responsible for the virulence of M.tb. The histidine kinase PhoR responds to a yet-unknown environmental stimulus and autophosphorylates a conserved histidine. The phosphate is transferred to an aspartate of the response regulator PhoP, which then forms a head-to-head homodimer and initiates the transcription of 114 virulence genes. Inhibiting PhoR or PhoP would rendered M.tb avirulent and increase its susceptibility to other drugs. For our PhoPR project, PhoR was separated into its extracellular (PhoRex) and intracellular (PhoRin) domains. We attempted to crystallize PhoRex and PhoRin, though currently no crystals iii have been achieved. Using the malachite green assay PhoRin was confirmed to spontaneously dephosphorylate. The DNA binding activity of PhoP was assessed using fluorescence polarization, though no binding was observed. To observe the complexation of PhoRin and PhoP, a pull-down method was developed using the non-hydrolyzable ATP analog, adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP). A stable PhoRin-AMP- PNP-PhoP complex was not identified. Future work is necessary for the crystallization of PhoRex and PhoRin. A new DNA binding assay is also necessary to elucidate PhoP kinetics. Obtaining these data will give significant insight to potential inhibitors for these proteins. Mycothiol is the low molecular weight thiol responsible for protection against oxidative stress and electrophilic toxins in actinomycetes. Previous studies suggest MSH is critical for the survival of M.tb, leading to its attractiveness as a potential drug target. MshC is the penultimate enzyme of the mycothiol (MSH) biosynthetic pathway, catalyzing the ATP-dependent ligation of cysteine and glucosamine inositol in a Bi Uni Uni Bi Ping Pong mechanism. An incomplete 1.6 Å crystal structure of M. smegmatis MshC has previously been solved using trypsin digestion and a cysteine-adenylate analog, 5-O-[N-(L-cysteinyl)-sulfamonyl]adenosine (CSA). Steady state and pre-steady state kinetics for M. smegmatis MshC have also been determined. For this project, we attempted to crystallize the full-length M. smegmatis MshC using Km values of ATP, L- cysteine, and the potential inhibitor glucosazide inositol. Thus far, crystallization attempts have not been successful. We also attempted to develop a high-throughput assay for drug screening. Using the quinaldine red assay, the specific activity of MshC was determined to be 2.40x10-2 µmol/min∙mg. While the high-throughput assay is not yet iv complete, the foundation has been developed for future optimization and usage. As a final goal, we desired to express M.tb MshC in E. coli codon-optimized competent cells. This venture has not yet proved successful. Expression of M.tb MshC will allow for crystallization attempts of this protein as well as the determination of kinetic data. v Acknowledgements I first wish to express my sincere gratitude to my advisor, Dr. Donald R. Ronning, for his wisdom, kindness, and limitless patience throughout my research journey. Thank you for being such a great mentor and for teaching me so much. The experience I have gained in your lab has been and will be significant for my future development as a scientist. I truly appreciate your guidance. I would like to extend my gratitude to my committee members, Dr. John J. Bellizzi and Dr. Ronald Viola for their endless support and advice while I prepared my thesis. I could not have chosen a better committee! To my labmates, both past and present, thank you so much for sharing your knowledge and for being such an awesome team to work with! I had a wonderful time with everyone. I would like to specifically acknowledge Vidhi, Lorenza, and Jared for showing me the ropes and keeping me saturated with valuable advice. I would also like to thank Dr. Peter Andreana and Krishnakant Patel for their contributions, both conceptually and synthetically, to the MshC project. Thank you to Jen, Steven, and Rachel for providing levity and endless support. Your friendship is invaluable. Finally, to all my family and friends outside the university, thank you for seeing me through this journey and putting up with me during the rough lab days and long lab nights. To my mom and dad, my brothers Matthew and Charles, and my partner Clay, thank you from the bottom of my heart. I could not have done this without all of you. vi Table of Contents Abstract.............................................................................................................................iii Acknowledgements...........................................................................................................vi Table of Contents.............................................................................................................vii List of Tables......................................................................................................................x List of Figures....................................................................................................................xi List of Equations.............................................................................................................xiii List of Abbreviations......................................................................................................xiv List of Symbols................................................................................................................xvi 1 Introduction............................................................................................................1 1.1 The Tuberculosis Burden...................................................................................1 1.1.1 Introduction to the Tuberculosis infectious process...........................1 1.1.2 History of treatment regimens............................................................2 1.1.2.1 The BCG vaccine.................................................................2 1.1.2.2 Development of first and second line drugs........................3 1.1.2.3 Co-infection with HIV and emergence of drug resistance...3 1.2 Roles of the PhoPR two-component system in Mycobacterium tuberculosis...4 1.1.1 Introduction to two-component systems.............................................4 1.1.2 PhoR: histidine kinase.........................................................................5 1.1.3 PhoR: current structural knowledge....................................................5 vii 1.1.4 PhoP: response regulator ....................................................................6 1.1.5 PhoP: current structural knowledge....................................................7 1.3 Roles of MshC in the mycothiol biosynthetic pathway of Mycobacterium tuberculosis................................................................................................11 1.3.1 Mycothiol biosynthetic pathway.......................................................11 1.3.2 MshC: cysteine ligase.......................................................................13 1.3.3 MshC: current structural knowledge.................................................14 1.4 Project goals.....................................................................................................17 1.4.1 PhoPR project goals..........................................................................17 1.4.2 MshC project goals...........................................................................17 2 Materials and Methods........................................................................................19 2.1 Molecular cloning, expression, and purification of PhoP, PhoR, and MshC..19 2.1.1 Molecular cloning.............................................................................19 2.1.2 Protein expression.............................................................................22 2.1.3 Protein purification...........................................................................24 2.2 Mass spectrometry analyses.............................................................................27
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