MOLECULAR DRIVERS OF SPECIFICITY IN HUMAN RIBONUCLEOTIDE REDUCTASE by ANDREW JOHN KNAPPENBERGER Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation advisor: Dr. Michael E. Harris Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY May, 2017 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Andrew John Knappenberger Candidate for the degree of Doctor of Philosophy*. (signed) Committee Chair Hung-Ying Kao Committee Member Chris Dealwis Committee Member Michael Harris Committee Member Arne Rietsch Committee Member Martin Snider (date) March 10, 2017 *We also certify that written approval has been obtained for any proprietary material contained therein. Copyright © 2017 by Andrew Knappenberger All rights reserved Table of Contents Acknowledgements ............................................................................................................ ix Chapter 1 : Introduction .................................................................................................... 13 Molecular recognition by enzymes is central to life ..................................................... 14 Recognition of multiple substrates and regulation by allostery are common features of enzymes .................................................................................................................... 16 Ribonucleotide reductase is an essential enzyme for all known cellular organisms .... 18 Loop 2 is central to specificity regulation in ribonucleotide reductase ........................ 21 Measuring multiple substrate kinetics through internal competition permits comprehensive description of enzymatic specificity .................................................... 22 Chapter 2 : Nucleoside Analogue Triphosphates Allosterically Regulate Human Ribonucleotide Reductase and Identify Chemical Determinants That Drive Substrate Specificity ......................................................................................................................... 28 Abstract ...................................................................................................................... 29 Introduction ................................................................................................................... 30 Results and discussion .................................................................................................. 42 Conclusions ................................................................................................................... 70 Materials and methods .................................................................................................. 72 Chapter 3 : Phylogenetic Comparative Sequence Analysis and Functional Studies of Mutant Enzymes Reveal Compensatory Amino Acid Substitutions in Loop 2 of Human Ribonucleotide Reductase .................................................................................... 77 i Abstract ......................................................................................................................... 78 Introduction ................................................................................................................... 79 Results and discussion .................................................................................................. 85 Conclusions ................................................................................................................. 119 Materials and methods ................................................................................................ 122 Chapter 4 : Summary and Future Directions .................................................................. 126 Summary ..................................................................................................................... 126 Future directions ......................................................................................................... 131 Bibliography ................................................................................................................... 137 ii List of Tables Table 3-1. Numerical values from activity assays in Figure 3-6. .............................. 107 Table 3-2. Numerical values from activity assays in Figure 3-9. .............................. 116 iii List of Figures Figure 1-1. Structure of eukaryotic R1. ....................................................................... 25 Figure 1-2. Conformational changes in loop 2. ............................................................ 26 Figure 2-1. Structure of human ribonucleotide reductase and location of the S-site and C-site. ................................................................................................... 35 Figure 2-2. Comparison of RR loop 2 across species. ................................................. 37 Figure 2-3. Measurements of RR specificity from this and other studies. ................. 39 Figure 2-4. Comparison of eukaryotic and bacterial RRs with dGTP or dTTP bound in the S-site. .......................................................................................................... 40 Figure 2-5. Application of internal competition to measurement of hRR specificity. ............................................................................................................... 56 Figure 2-6. Measurement of native hRR and ScRR specificities. ............................... 58 Figure 2-7. The specificities directed by a series of pyrimidine effector analogs. ..... 60 Figure 2-8. Structures of eukaryotic RR bound to S-site ligands. .............................. 62 Figure 2-9. The specificities directed by a series of purine effector analogs. ............ 64 Figure 2-10. Competition between purine effector analogs and the natural S-site effector dTTP. ................................................................................................................. 66 Figure 2-11. Effects of the D287A substitution on hRR substrate recognition. ........ 68 Figure 3-1. Three-dimensional structure of hRR. ........................................................ 83 Figure 3-2. Sequence conservation in eukaryotic ribonucleotide reductase and the extent of variation in loop 2. .................................................................................. 100 Figure 3-3. Loop 2 sequences from Figure 3-2C mapped onto a eukaryotic tree of life. .............................................................................................................................. 102 iv Figure 3-4. Crystal structure of hR1 bound to dTTP and GDP. .............................. 103 Figure 3-5. Circular dichroism (CD) of hRR variants. ............................................. 104 Figure 3-6. Activity and specificity of the hRR variants. .......................................... 105 Figure 3-7. Size exclusion chromatography (SEC) of hRR variants in the presence of dGTP, dTTP or ATP. ............................................................................... 109 Figure 3-8. SEC profiles of wild-type and P294K hRR large subunit in the presence of 3 mM ATP. ................................................................................................ 111 Figure 3-9. Activity and specificity of N291G mutant hR1 in the presence of 2- aminopurine-drTP, dITP, and dZeb. .......................................................................... 112 Figure 3-10. Activity and specificity of the hRR variants in the presence of both ATP and dGTP/dTTP................................................................................................... 114 Figure 3-11. Correspondence between the present in vitro experiments and “natural experiments” of evolution. .................................................................... 118 v List of Abbreviations • RR – ribonucleotide reductase. • DNA – deoxyribonucleic acid. • dNTP – deoxyribonucleotide diphosphate. • ATP – adenosine triphosphate. • dATP – deoxyadenosine triphosphate. • dGTP – deoxyguanosine triphosphate. • dTTP – deoxythymidine triphosphate. • tRNA – transfer ribonucleic acid. • ATC – aspartate transcarbamylase. • CTP – cytidine triphosphate. • SAMHD1 – SAM domain and HD domain-containing protein 1. • HIV – human immunodeficiency virus. • ADP – adenosine diphosphate. • CDP – cytidine diphosphate. • GDP – guanosine diphosphate. • UDP – uridine diphosphate. • R1 – ribonucleotide reductase large subunit. • R2 – ribonucleotide reductase small subunit. • C-site – catalytic site, the active site of ribonucleotide reductase. • S-site – specificity site. • A-site – activity site. vi • dNDP – deoxynucleotide diphosphate. • hRR – human ribonucleotide reductase. • ScRR – S. cerevisiae ribonucleotide reductase. • AMPPNP – adenylylimidodiphosphate. • PDB – Protein Data Bank. • HPLC – high-performance liquid chromatography. • TmRR – Thermotoga maritima RR. • EcRR – E. coli RR. • aa – amino acid. • hR1 – human ribonucleotide reductase large subunit. • 5FdUTP – 5-fulorodeoxyuridine triphosphate. • dUTP – deoxyuridine triphosphate. • dZeb – deoxyzebularine triphosphate. • 2-aminopurine-drTP – 2-aminopurine-deoxyribose triphosphate. • N2dATP – 2-amino-deoxyadenosine triphosphate. • dITP – deoxyinosine triphosphate. • hRRM1 – human ribonucleotide reductase large subunit. • hRRM2 – human ribonucleotide reductase small subunit. • ScRR1 – S. cerevisiae ribonucleotide reductase large subunit. • ScR2R4 – S. cerevisiae ribonucleotide reductase small subunit. • DTT – dithiothreitol.