ABSTRACT GAP ENGINEERING TO RESTORE GTP HYDROLYSIS TO ONCOGENIC KRAS MUTANTS by Benjamin A. Fenton The Kras gene encodes a small GTPase that is involved in pathways regulating cell growth and proliferation. Bound to GTP, Kras sends growth signals until the GTP is hydrolyzed. Hydrolysis is catalyzed by GTPase Activating Protein (GAP). Activating mutations in Kras are highly correlated to cancer development due to uncontrolled growth. Any mutation in the 12th amino acid of Kras (glycine) cause steric alterations in the catalytic site of GAP that prevent catalysis of GTP hydrolysis. Kras is a widely-studied signaling protein, and currently there are no clinical inhibitors. This project will attempt to redesign the catalytic site of GAP to circumvent the steric interference caused by G12 mutations, thus allowing hydrolysis to occur and signaling to be turned off. Recombinant proteins were purified from E. coli, and GAP mutants were screened for catalytic activity using a colorimetric GTPase assay. GAP ENGINEERING TO RESTORE GTP HYDROLYSIS TO ONCOGENIC KRAS MUTANTS A Thesis Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry and Biochemistry by Benjamin A. Fenton Miami University Oxford, Ohio 2014 Advisor______________________________ Dr. Michael A. Kennedy Reader______________________________ Dr. Ann E. Hagerman Reader______________________________ Dr. David L. Tierney Reader______________________________ Dr. Michael W. Crowder Table of Contents Title Page i List of Tables v List of Figures vi Acknowledgements vii Chapter 1: Introduction 1.1 The Kirsten Rat Sarcoma Viral Oncogene Homolog 2 1.2 Mechanism of GTP Hydrolysis 5 1.3 Oncogenic Mutations in Kras 8 1.4 Project Goals 8 1.5 Chapter 1 References 10 Chapter 2: Gene Cloning and Site-Directed Mutagenesis of Kras and rasGAP 2.1 Introduction 14 2.2 Methods 15 2.2.1 Primer Design 15 2.2.2 Gene Amplification 16 2.2.3 Endonuclease Digestion 19 2.2.4 Ligation 20 2.2.5 Transformation and Plasmid Isolation 20 2.2.6 Colony Screening and Sequencing 21 2.3 Results and Discussion 22 2.3.1 Gene Amplification and Mutagenesis 22 2.3.2 Endonuclease Digestion of Insert and Vector 22 2.3.3 Colony Screening and Sequencing 22 2.4 Conclusions 23 2.5 Chapter 2 References 29 ii Chapter 3: Expression, Purification, and Characterization of Kras and rasGAP 3.1 Introduction 32 3.2 Methods 34 3.2.1 Transformation, Expression, and Cell Lysis 34 3.2.2 Nickel Affinity Purification 35 3.2.3 Kras Expression in Minimal Media 35 3.2.4 Crystal Screening 36 3.2.5 Kras Nucleotide Binding State Determination 36 3.3 Results and Discussion 37 3.3.1 Protein Expression and Purification 37 3.3.2 Thrombin Cleavage 37 3.3.3 Binding State Determination by 1H-15N HSQC NMR 43 3.4 Conclusions 43 3.5 Chapter 3 References 48 Chapter 4: Measuring GAP-Mediated Catalysis of GTP Hydrolysis by Kras 4.1 Introduction 50 4.2 Methods 52 4.2.1 Kras GTP Loading and Desalting 52 4.2.2 Optimization of a Colorimetric GTPase Assay 54 4.3 Results 55 4.3.1 Kras GTP Loading and Desalting 55 4.3.2 Establishment of a Standard Curve 59 4.3.3 GTPase Activity of WT and G12A Kras with WT GAP 59 4.3.4 GAP Mutant Screening for Restoration of Rapid GTP Hydrolysis 63 4.4 Conclusions 68 4.5 References 69 Chapter 5: Conclusions and Future Directions iii 5.1 General Remarks 72 5.2 Conclusions 72 5.2.1 Cloning and Mutagenesis of Kras and GAP 72 5.2.2 Expression and Purification of Kras and GAP 72 5.2.3 Optimizing a Phosphate Assay to Monitor GTP Hydrolysis 73 5.4.4 Screening Engineered GAP Mutants for Restoration of GTPase Catalysis 73 5.3 Future Directions 73 5.4 Closing Remarks 75 5.5 References 75 iv List of Tables Table 2-1: Successfully cloned genes and mutants 28 Table 3-1: Successfully expressed proteins 44 v List of Figures Figure 1-1: Diagram of Kras signaling 3 Figure 1-2: Cycle of activation and deactivation of Kras 4 Figure 1-3: Crystal structure of Kras 6 Figure 1-4: Diagram of the active site of Kras in complex with GAP 7 Figure 2-1: Primers used for cloning and mutagenesis 17 Figure 2-2: Amplification of target genes 24 Figure 2-3: Amplification of genes for overlapping PCR mutagenesis 25 Figure 2-4: Digestion of pET28 vector plasmid 26 Figure 2-5: Restriction enzyme screening for successful ligation and transformation 26 Figure 2-6: Amino acid sequences of Kras and GAP gene products 27 Figure 3-1: Wild-type Kras purified from E. coli forms a dimer 38 Figure 3-2: Elution profile of wild-type Kras 39 Figure 3-3: Wild-type GAP purification 40 Figure 3-4: Purification of Kras mutants 41 Figure 3-5: Purification of GAP mutants 42 Figure 3-6: 1H-15N HSQC spectrum of wild-type Kras 45 Figure 3-7: 1H-15N HSQC spectrum showing single conformational state 46 Figure 3-8: Rapid conversion to GDP state of Kras upon addition of GAP 47 Figure 4-1: Mechanism of the GTPase colorimetric assay 53 Figure 4-2: Elution traces from PD-10 Desalting Columns 56 Figure 4-3: 1H-15N HSQC confirmation of GTP loading 57 Figure 4-4: 1H-15N HSQC representation of reaction time course 58 Figure 4-5: Standard curve for the PiColorLock Gold phosphate assay 60 Figure 4-6: GTPase activity of wild-type Kras with and without GAP 61 Figure 4-7: GTPase activity of G12A mutant Kras with and without GAP 62 Figure 4-8: GTP hydrolysis of Kras after two minutes 64 Figure 4-9: Activity of GAP mutants on wild-type Kras 66 Figure 4-10: Activity of GAP mutants on G12A Kras 67 vi Acknowledgements I would like to thank my advisor Dr. Kennedy for his assistance and guidance throughout my career at Miami, starting from my first month here as a freshman. My time in his lab has been filled with incredible opportunities that I doubt I would have found elsewhere. I would also like to thank my graduate committee for their valuable insights and ideas, and assistance with my thesis. Next, I want to thank Dr. Ni, my mentor in the lab who taught me all of the procedures and instrumentation used in the lab, and who was always available to help troubleshoot and talk things through. I want to thank Dr. Ramelot as well, for her expertise and training on all of my NMR experiments. I also want to thank the graduate members of the Kennedy research group, for giving me a supportive and enjoyable lab environment. The undergraduate team that I started out a part of and ended up leading has been instrumental in this project. I want to thank the current team, Jackie Ehrman, Amielia Adams, Aaron Keck, Peter McCandless, Katy McGill, Colleen O’Neil, Nathan Burns, and Brett VanCauwenbergh, as well as past members of the team for their time and effort. I would like to thank my friends and family for their support, especially my parents, who have helped make all of this possible. Finally, I want to dedicate this thesis in memory of my grandmother, Marilyn Fenton, who we lost to cancer during my sophomore year of college. vii Chapter 1: Introduction 1 1.1 The Kirsten Rat Sarcoma Viral Oncogene Homolog The Kirsten Rat Sarcoma Viral Oncogene Homolog (Kras) protein is member of the Ras superfamily of small GTPases. Characterized by a catalytic G domain, the Ras superfamily includes Ras, Rap, Rho, Ran, and many others. Kras, and more specifically the Kras4B variant that is highly expressed in human cells, is one of the most well-studied members of the family. Kras plays a role in signal transduction and acts as an on/off switch depending on its nucleotide binding state. When bound to GTP, Kras signaling is switched on, and Kras can interact with downstream effectors such as PI3K, MAPK, and Raf, ultimately resulting in the production of transcription factors that lead to cell growth and proliferation (1,2) (Figure 1-2). Kras has a degree of intrinsic GTPase activity and slowly hydrolyzes the bound GTP into GDP. When the GTP is hydrolyzed and the resulting phosphate is released, Kras undergoes a conformational change that stops interaction with effector proteins, turning off signal transduction. Eventually, GDP is released from Kras, and GTP, which is more abundant in the cell, will bind, resetting the cycle (3). The Kras protein is farnesylated at the C terminus and localized to the cytosolic face of the plasma membrane, where it regulates many signal transduction pathways (4). The nucleotide-mediated signal transduction switch is further mediated by GTPase Activating Proteins (GAPs) and Guanine Nucleotide Exchange Factors (GEFs). GAPs catalyze the hydrolysis of GTP, increasing the reaction rate by six orders of magnitude. The binding of GAP rapidly switches Kras signaling off by speeding up GTP hydrolysis (5-7). Conversely, the GEFs, such as Son of Sevenless (SOS), catalyze dissociation of GDP from Kras, which causes signaling to be switched on when a new molecule of GTP binds (8). Ras activation by GEF occurs through a transduction pathway associated with the epidermal growth factor receptor (EGFR) (3). Extracellular signals bind to receptors on the EGFR, which transmits the signal across the membrane and activates GEF proteins, that then activate Kras. As mentioned above, active Kras triggers the activation of serine/threonine kinase cascades, which result in cell growth.