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University of Cincinnati UNIVERSITY OF CINCINNATI Date: 10-Nov-2009 I, Keith J Gaddie , hereby submit this original work as part of the requirements for the degree of: Doctor of Philosophy in Molecular, Cellular & Biochemical Pharmacology It is entitled: Structural Elements that Regulate Interactions between the Extracellular and Transmembrane Domains of Human Nucleoside Triphosphate Student Signature: Keith J Gaddie This work and its defense approved by: Committee Chair: Terence Kirley, PhD Terence Kirley, PhD Scott Belcher, PhD Scott Belcher, PhD Paul Rosevear, PhD Paul Rosevear, PhD John Maggio, PhD John Maggio, PhD Ronald Millard, PhD Ronald Millard, PhD 11/23/2009 294 Structural Elements that Regulate Interactions between the Extracellular and Transmembrane Domains of Human Nucleoside Triphosphate Diphosphohydrolase 3 A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (Ph.D.) in the Department of Pharmacology and Cell Biophysics of the College of Medicine 2009 By Keith J. Gaddie B.S. Fisk University, 2003 Committee Chairperson: Terence L. Kirley, Ph.D. Abstract The nucleoside triphosphate diphosphohydrolases (NTPDases) are a family of constitutively expressed, endogenous nucleotidases, some of which regulate purinergic signaling by divalent cation-dependent hydrolysis of nucleotides acting as agonists at purinergic receptors. Due to the scarcity of specific inhibitors and genetically modified animals, the functions of most individual NTPDases are poorly understood and still under investigation. However, the NTPDases have been implicated in many biological and physiological processes, including secretion, cell adhesion, pain perception, cancer and malignant transformation, adenosine recycling, and platelet aggregation. Membrane-bound NTPDase3 expressed on the cell surface has a large extracellular domain, a “linker region”, and a transmembrane domain. Evidence exists for interactions between the transmembrane domain and the active site lobes that govern the function of rat NTPDase1. However, the specific mechanism(s) regulating this cross-talk for NTPDases, as well as how the N- and C-terminal transmembrane helices in NTPDase3 interact within and between monomers to mediate oligomerization and modulation of enzymatic activity is still unknown. The roles of the conserved proline residues of human NTPDase3, located in the “linker region” that connects the N- and C-terminal transmembrane helices with the extracellular active site, were examined by proline to alanine substitutions coupled with single cysteine substitutions strategically placed in the transmembrane domain to serve as cross-linking “sensors” of helical interactions. Mutation of several proline residues resulted in decreased nucleotidase activities and some “uncoupled” the effect of ATP binding on TMD movements. The data suggest a role for proline residues 53 and 481 in the linker region of human NTPDase3 for “coupling” iii nucleotide binding and hydrolysis at the enzyme active site to movements and/or rearrangements of the transmembrane helices necessary for optimal nucleotide hydrolysis. To investigate the structural/functional roles of the conserved polar residues in the transmembrane helices of human NTPDase3, each was singly mutated to alanine. All mutants were properly glycosylated and had specific activities similar to wild-type, except Q44A. The Q44A mutation decreased specific activities by approximately 50% - 70%, and nearly eliminated Triton X-100 detergent inhibition. The same conserved polar residues were mutated to cysteine, singly and in pairs, to allow a disulfide cross-linking strategy to map potential inter- and intra- molecular hydrogen bond interactions. The results support the centrality of Q44 for the strong inter-molecular interactions driving the association of the N-terminal domains of two NTPDase3 monomers in a dimer, while S39 and T495 may contribute to helical interactions involved in forming higher order oligomers. These results suggest a model for putative hydrogen bond interactions of the conserved polar residues in the transmembrane domain of native, dimeric NTPDase3 that are important for protein expression, activity, and susceptibility to membrane perturbations. This dissertation provides additional insights to the structural elements that regulate the enzymatic activity of the NTPDases. With the recent elucidation of the crystal structure of the extracellular portion of rat NTPDase2, the major remaining structural questions regarding the cell membrane NTPDases are how the TM helices interact and how these interactions are coupled to modulation of enzyme activity and NTPDase function. This dissertation addresses these questions for human NTPDase3, and suggests that similar structure-function relationships are relevant to the family of NTPDases controlling purinergic signaling. iv Acknowledgements My graduate experience was a big adjustment, a learning process of my strengths and weaknesses, and a time where I was always pushed to excel to new heights. Luckily, I was surrounded by talented and accomplished individuals who cared about my scientific future and were willing to share their expertise as I traveled this challenging journey. First, I would like to express my sincere gratitude to my mentor and advisor, Dr. Terry Kirley. Dr. Kirley possesses so many qualities of a great scientist, disciplined, the ability to pay a great attention to detail, and excellent time management skills. In addition, through our casual conversations and some observation, he is an excellent family man. Dr. Kirley was very instrumental in developing my skills as a research scientist and he always encouraged me to work and think in an independent manner, but more importantly, to pay attention to the small details. This has allowed me to grow as a scientist and to think critically about my data. I am truly indebted to Dr. Kirley for all his hard work, support, and guidance. In addition, I owe thanks to my committee members, Drs. Scott Belcher, John Maggio, Ronald Millard, and Paul Rosevear, for providing constructive criticism and insight over the years. Their input and suggestions were instrumental in shaping the progression of my research projects. I would also like to thank all the members of the Kirley laboratory for their assistance and guidance throughout the years. When I first joined the laboratory, Dr. Patrick Crawford was very instrumental in teaching me the basic laboratory protocols and worked closely with me on his project regarding the NTPDase3 splice variant, NTPDase3β. Dr. Vasily Ivanenkov instructed and motivated me to interpret my data from several points of view. In addition, he discovered that Tween 20 stimulated NTPDase3 activity and I applied in my dissertation v research. I would also like to thank Dr. Min Yang and Clifford Cookman for all of their assistance during their time in the laboratory. I would like to thank everybody in the department of Pharmacology, especially Nancy Thyberg, Damita Jo Harris, and Carol Ross for always handling the seen and unseen obstacles of graduate school from a paperwork point of view. Finally, I am so thankful for having a supportive family (Godsister’s Stephanie Ford and Janelle Henson) and friends (Linda Smith and Dr. Bradley Sheares) who never gave up on me along the way. I would not have successfully completed my studies if it were not for my mom (Pamela J. Gaddie) and dad’s (Keith Taylor) unconditional love, my aunts’ (Brenda, Dee Dee, LaVada, and Margo) unwavering support, as well as my best friend’s (Derrick G. Demmons) and roommate’s (Mark A. Edmonson) encouraging words. vi Table of Contents Page Abstract iii Acknowledgements v Table of contents vii List of figures and tables xiii List of abbreviations xvii Chapter I: Introduction 1 1. Evolutionary Appearance of Nucleosides 1 1.1. Nucleotide structure 2 1.2. Nucleotide synthesis 2 1.2.1. De novo purine nucleotide synthesis 2 1.2.2. De novo pyrimidine nucleotide synthesis 4 2. The Concept of Purinergic Receptor Signaling 8 3. ATP: The Neurotransmitter 12 4. Purinergic Receptors 15 4.1. P1 (Adenosine) Receptors 18 4.2. P2X Receptors 18 4.3. P2Y Receptors 19 5. Functional Significance of Purinergic Receptor Signaling 20 6. Ecto-nucleotidases 21 6.1. Alkaline phosphatase (AP) 21 6.2. Ecto-5’-nucleotidase/CD73 23 vii 6.3. Ecto-nucleotide phosphodiesterase/pyrophosphatase (E-NPP) 23 6.4. Nucleoside Triphosphate Diphosphohydrolase (NTPDase) 24 7. Molecular Cloning, Sequencing, Expression, and Characterization of the NTPDases 26 7.1. The membrane-bound, cell surface NTPDases (NTPDase1-3 and 8) 26 7.2. The intraluminal NTPDases (NTPDase4-7) 27 8. The Biochemical and Structural Characterization of the NTPDases 30 8.1. Enzymology of the NTPDases 30 8.2. Inhibitors of the NTPDases 31 8.3. Site-directed mutagenesis and computational comparative protein modeling of the membrane-bound, cell surface NTPDases 36 8.3.1. The structural features of the extracellular domain of the NTPDases 38 8.3.2. The structural features of the transmembrane domain of the NTPDases 41 9. Functional Significance of the NTPDases 44 10. Dissertation Scope and Objectives 46 Chapter II: Methods and Materials 49 1. General Methods and Materials 49 1.1. Purification of oligonucleotides for site-directed mutagenesis 49 1.2. Site-directed mutagenesis of NTPDase3 50 1.3. Bacterial transformation and inoculation of PCR products 50 1.4. Isolation of DNA from XL1-Blue
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