A Type-II Thymidine Kinase from Thermotoga maritima: KineticCharacterization, Substrate Binding, and Oligomerization Public
Lichter, Joseph (2009)
Published
Abstract
Abstract
Thymidine kinases (TKs) catalyze the phosphorylation of thymidine to form thymidylate. TKs also activate nucleoside analog (NA) pro-drugs used in anti-cancer and anti-viral therapies. In this dissertation, the characterization of a novel TK from the hyperthermophilic eubacterium Thermotoga maritima is described, with focus on the kinetics, substrate binding, and protein oligomerization.
Due to amino acid sequence, structure and substrate specificity, TmTK is characterized as a type-II TK. Catalysis at 37 °C is suboptimal compared to the activity at 82 °C yet the kinetic parameters change at the elevated temperature, demonstrating a greater specificity for thymidine (as compared to AZT) and positive cooperativity with ATP. Analysis of activation of other NAs at 37 °C, suggests the possibility for a common structural intermediate in the reaction pathway, potentially as a result of protein conformational changes. Fluorescence experiments further suggest temperature dependent conformational changes in TmTK.
A systematic study of substrate binding on protein conformation identifies a thymidine dependent formation of the C-terminal lasso region, while ATP binding changes both the tertiary and quaternary structure. Single tryptophan mutants engineered in the c1/3 region of the protein confirm ATP-dependent conformational changes. Engineering of disulfide bridges between the two 1 helices at the presumed dimer II interface disabled ATP binding and catalytic competency. From the substrate binding experiments, a two-state model is proposed whereby an expansion along the dimer II interface will turn a closed/inactive protein into an open/active state.
The crux of the two-state model is the presumption that TmTK exists as a tetramer in solution. Inconsistencies between predicted tetramer molecular weight (MW) and size exclusion chromatography (SEC) estimated MW led to an investigation of the oligomeric state. Mutagenesis studies suggest the dimer I ("strong") interface is part of the solution state structure. Crosslinking studies conducted on wild type and mutants were conducted and conclusions are drawn for potential monomer-dimer and monomer-dimer-tetramer equilibriums.
Table of Contents
Chapter 1: Introduction ............................................................................................... 1 1.1 Deoxyribonucleic acid (DNA).................................................................................. 1 1.2 Biosynthesis of deoxyribonucleotide triphosphates.................................................. 2 1.2.1 De novo pathway................................................................................................ 2 1.2.2 Salvage pathway ................................................................................................ 3 1.2.3 Regulation of dNTP levels and disease associated with dNTP imbalance....... 5 1.2 Deoxynucleoside kinase (dNK) ................................................................................ 6 1.3 Thymidine Kinases (TK) .......................................................................................... 8 1.3.1 Human thymidine kinase 1 ................................................................................ 8 1.3.2 Human thymidine kinase 2 .............................................................................. 11 1.3.3 Other eukaryotic, bacterial, and viral TKs....................................................... 11 1.3.4 Two types of TKs (type I and type II) ............................................................. 14 1.4 Medical significance and applications .................................................................... 15 1.4.1 Cancer marker.................................................................................................. 16 1.4.2 Nucleoside analog pro-drug activation ............................................................ 16 1.4.3 Suicide gene therapy ........................................................................................ 18 1.5 Many mesophilic TKs, Few thermophilic TKs....................................................... 19 1.6 Thermotoga maritima.............................................................................................. 21 1.6.1 T. maritima genome ......................................................................................... 22 1.7 Dissertation project aims and goals ........................................................................ 23
Chapter 2: Expression and Kinetic Characterization of a Thymidine Kinase from Thermotoga maritima .................................................................................................. 24
2.1 Introduction............................................................................................................. 24 2.2 Materials and methods ............................................................................................ 25 2.2.1 Isolation of thymidine kinase gene .................................................................. 25 2.2.2 Genetic complementation test.......................................................................... 26 2.2.3 Protein Overexpression of His-tagged protein................................................. 26 2.2.4 Purification of His-tagged protein ................................................................... 27 2.2.5 Overexpression and purification of native, untagged protein.......................... 28 2.2.6 Secondary structure and thermal denaturation................................................. 28 2.2.7 Spectrophotometric enzyme activity assay...................................................... 29 2.2.8 Radiometric enzyme activity assay.................................................................. 30 2.2.9 Fluorescence spectroscopy............................................................................... 30 2.3 Results and discussion ............................................................................................ 31 2.3.1 TmTK has in-vivo thymidine kinase activity .................................................. 31 2.3.2 TmTK is a highly thermostable type-II thymidine kinase ............................... 33 2.3.3 TmTK in-vitro catalytic performance .............................................................. 36 2.3.3.1 Natural substrate activity at 37°C (Spectrophotometric assay) ................ 36 2.3.3.2 Natural substrate specific at 82°C............................................................. 37 2.3.3.3 Broad nucleoside analog activation at 37 °C ............................................ 40 2.3.3.4 Temperature-activity profile reveals change in activation energy............ 42 2.3.4 Fluorescence spectroscopy suggest protein conformational changes .............. 43 2.4 Conclusion .............................................................................................................. 46
Chapter 3: Tertiary and quaternary conformational changes in TmTK as a function of substrate binding ..................................................................................... 51
3.1 Introduction............................................................................................................. 51 3.2 Materials and methods ............................................................................................ 53 3.2.1 Crystallization and structure determination ..................................................... 53 3.2.2 Site-directed mutagenesis ................................................................................ 53 3.2.3 Disulfide linkage experiments ......................................................................... 54 3.2.4 Enzyme kinetics ............................................................................................... 55 3.2.5 Fluorescence spectroscopy............................................................................... 55 3.3 Results and discussion ............................................................................................ 56 3.3.1 Structural changes seen upon thymidine and ATP binding ............................. 56 3.3.2 Tryptophan fluorescence monitors ATP induced β-hairpin movement........... 59 3.3.3 ATP binding induces a conformational change in the quaternary structure .... 63 3.3.4 Trapping the closed conformation by disulfide linkage................................... 65 3.4 Conclusion .............................................................................................................. 71
Chapter 4: Investigating the Oligomeric State of TmTK........................................ 75 4.1 Introduction............................................................................................................. 75 4.2 Materials and Methods............................................................................................ 77 4.2.1 Site directed mutagenesis................................................................................. 77 4.2.2 Protein overexpression and purification .......................................................... 78 4.2.3 hTK1 gene isolation and protein overexpression/purification......................... 79 4.2.4 Enzyme kinetics ............................................................................................... 79 4.2.5 Size exclusion chromatography ....................................................................... 80 4.2.6 Chemical crosslinking...................................................................................... 80 4.3 Results and discussion ............................................................................................ 81
4.3.1 Native TmTK elutes as a 67 kDa protein......................................................... 81 4.3.2 Disruptional mutagenesis indicates the dimer I interface is crucial for oligomerization ......................................................................................................... 87 4.3.3 Amine reactive crosslinking produces a 60 kDa product.............. .................. 93 4.3.4 Sulfhydryl reactive crosslinking ...................................................................... 97 4.4 Conclusion .............................................................................................................. 99
Chapter 5: Conclusions and Future Perspectives.................................................. 103 References.................................................................................................................. 107
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