T細胞的蛋白酪胺酸磷酸水解酶 (TCPTP, PTPN2) 是在人體細胞中普遍表達的一種非受體型蛋白酪胺酸磷酸水解酶，在不同的細胞間室中有多種不同的作用受質。它調控關鍵訊息傳遞路徑，並與各種癌症生成、發炎反應以及其他人類疾病的發生息息相關。因此，了解TCPTP活性調控的分子機制對於開發針對TCPTP的治療方法至關重要，然而以結構基礎來詮釋TCPTP活性調控機制仍然難以捉摸。在本研究中，我們結合生物物理學以及生物化學的研究方法，進行全面性結構分析，闡明TCPTP活性調控的分子機制。
由於TCPTP和PTP1B在PTP家族中是最接近的同源物，可以假設此兩種磷酸水解酶的活性調控是相似的。因此，我們首先透過X 射線晶體學來探討TCPTP的活性調控是否也存在在PTP1B的變構位點。在解析度分別為1.7Å及1.9Å的TCPTP晶體結構中，我們都觀察到C 端的螺旋 α7。螺旋 α7在PTP1B上是具有功能性且被確定為其變構開關，然而過往研究並未解析螺旋 α7在TCPTP中的功能。此論文中，我們首次證明螺旋 α7發生截斷或刪除時，TCPTP的催化效率會下降約四倍。整體來說，我們的結果證明螺旋 α7的變構角色在TCPTP活性調控之功能與PTP1B相似，且強調螺旋 α7和主要的催化區域的協調對於TCPTP的有效催化功能是必要的。
根據晶體結構的觀察分析，我們提出更進一步的問題: 如果TCPTP和PTP1B的活性催化調控相似，那該如何區分兩者之間活性調控的專一性? 此一問題的釐清對開發TCPTP的藥物有其必要，因此我們繼續專注地研究TCPTP非催化的C側尾端的活化調控。先前的研究已提出TCPTP被自身的C端滅活的假設，但如何造成此結果則仍未知。此外，如果TCPTP表現後無活性，那其如何在細胞內被激活?為了回答這些問題，我們使用核磁共振 (NMR)光譜學、小角度 X 射線散射 (SAXS)以及化學交聯與質譜偶合 (CX-MS)為主要的工具來闡示TCPTP的尾端無結構序列做為分子內自動抑制其酵素活性機制的主要工具。然而，這並不是靠靜態作用造成，而是C端尾部在活化位點周圍移動，以動態遮擋TCPTP的基質，就像是汽車的”擋風玻璃雨刷”一般的機制。 再者，TCPTP活化是藉由細胞內的競爭來達成，意即Integrin-alpha1無結構尾端序列取代了TCPTP的活性抑制尾端，導致TCPTP的完全活化。我們的工作不僅定義了調控TCPTP活性獨特的機制，同時揭露了兩個極度相近的PTPs (PTP1B與TCPTP) 利用其尾端無結構序列經由截然不同的機制調控其酵素活性。這種獨特的調控機制可以用以發展針對TCPTP專一的治療方式。

T-Cell Protein Tyrosine Phosphatase (TCPTP, PTPN2) is a non-receptor type protein tyrosine phosphatase that is ubiquitously expressed in human cells and targets a broad variety of substrates across different subcellular compartments. It is a critical component of various key signaling pathways that are directly associated with the formation of cancer, inflammation, and other diseases. Thus, understanding the molecular mechanism of TCPTP activity regulation is essential for the development of TCPTP based therapeutics. In spite of that, the structural basis for the regulation of TCPTP’s activity has remained elusive. In this study by using a combination of biophysical and biochemical methods such as nuclear magnetic resonance (NMR), X-ray crystallography, small-angle X-ray scattering (SAXS), chemical cross-linking coupled with mass spectrometry (CX-MS), and enzymatic activity we have carried out comprehensive structural characterization, elucidating the underlying regulatory mechanism of TCPTP’s catalytic activity.
Since TCPTP and PTP1B are the closest homologs within the PTP family, it has been hypothesized that activity of both the phosphatases is regulated in a similar manner. Thus, through X-ray crystallography, we investigated whether the activity of TCPTP could also be regulated by the same allosteric site that comparably exists in PTP1B. We determined two crystal structures of TCPTP at 1.7 Å and 1.9 Å resolution that includes helix α7 at the TCPTP C-terminus. Helix α7 has been functionally characterized in PTP1B and was identified as its allosteric switch. However, its function in TCPTP has been unknown. Here, we demonstrate that truncation or deletion of helix α7 reduced the catalytic efficiency of TCPTP by ~four-fold. Collectively, our data demonstrated an allosteric role of helix α7 in the regulation of TCPTP’s activity similar to its function in PTP1B, and highlights that the coordination of helix α7 with the core catalytic domain is essential for the efficient catalytic function of TCPTP.
Following the observation from crystal structural analysis, we wondered if the catalytic activity of TCPTP and PTP1B is regulated similarly, then how could we specifically tune the activity of TCPTP, which is critically required for the development of TCPTP-based therapeutics. With this objective in mind, we went ahead to investigate if the non-catalytic C-terminal tail of TCPTP regulates the catalytic activity specifically. Indeed, a previous study has suggested that under basal conditions, TCPTP is inactivated by its own C-terminal tail, but how this inactivation is achieved has been unknown. Furthermore, if it is inactive then how can it be activated inside a cell. To answer these questions, we used nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), and chemical cross-linking coupled with mass spectrometry (CX-MS) as major tools to show that the C-terminal intrinsically disordered tail of TCPTP functions as an intramolecular autoinhibitory element that controls the TCPTP catalytic activity. However, this is not achieved by completely blocking the active site, but rather the C-terminal tail moves around the active site and dynamically occludes substrates from the TCPTP active site which is akin to a ‘windshield wiper’ in a car. Activation of TCPTP is achieved by cellular competition, i.e., the intrinsically disordered cytosolic tail of Integrin-α1 displaces the TCPTP autoinhibitory tail, allowing for the full activation of TCPTP. Taken together our work not only defines the completely unique mechanism by which TCPTP is regulated but also reveals that the intrinsically disordered tails of two of the most closely related PTPs (PTP1B and TCPTP) autoregulate the catalytic activity of their cognate PTPs via entirely different mechanisms, which can be exploited to develop TCPTP based specific therapeutics.

Table of Contents
摘要 I
Abstract III
Acknowledgment V
Table of Contents VIII
List of Abbreviations XV
1. INTRODUCTION 1
1.1 Background and significance of protein tyrosine phosphorylation. 2
1.2 Classification of PTPs. 3
1.3 Molecular mechanism of classical PTP substrate dephosphorylation. 6
1.4 Regulatory mechanism of PTP substrate dephosphorylation. 8
1.5 T cell protein tyrosine phosphatase (TCPTP). 9
1.6 TCPTP controls multiple signaling pathways associated with various diseases. 10
1.7 TCPTP acts as a double-edged sword in cancer development and treatment. 11
1.8 Regulation of TCPTP catalytic activity. 12
1. 9 Key questions being addressed by this study. 13
2. MATERIALS AND METHODS 15
2.1 Construct preparation. 16
2.2 Site-directed mutagenesis. 16
2.3 Protein expression and purification. 17
2.3.1 Protein expression and purification for biophysical and biochemical analysis. 17
2.3.2 Protein expression and purification for NMR analysis. 18
2.4 Protein crystallization and data collection. 19
2.5 Crystal structure determination. 20
2.6 NMR spectroscopy. 20
2.7 SAXS data collection and processing. 21
2.8 Chemical cross-linking. 22
2.8.1 LC-MS analysis for BS3 cross-linked TCPTP. 22
2.8.2 LC-MSn analysis for DSSO cross-linked sample. 23
2.8.3 Cross-linking data analysis and software parameters (PD-XLinkX). 24
2.9 HDX-MS analysis. 25
2.10 Fluorescent size exclusion chromatography (FSEC). 26
2.11 Bio-layer interferometry analysis (BLI). 27
2.12 Size-exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS) analysis. 27
2.13 Thermal shift Assay. 28
2.14 Enzymatic activity using pNPP as a substrate. 28
2.14.1 Kinetic analysis using pNPP as a substrate. 28
2.14.2 Comparative phosphatase activity analysis using pNPP as substrate 29
2.15 Peptide Synthesis. 30
2.16 Enzymatic activity using phospho-peptide as a substrate. 30
2.16.1 EC50 assays. 30
2.16.2 Kinetic analysis using phospho-peptide as a substrate. 31
2.16.3 Comparative phosphatase activity analysis using phospho-peptide as a substrate. 31
2.17 Activity assay using DiFMUP as substrate. 32
2.18 Development of TCPTP auto-inhibitory model. 32
2.19 Data deposition. 33
3. RESULTS 34
3.1 Crystal structure of TCPTP catalytic domain reveals additional insight in WPD loop, E-loop, and helix α7 compared to the previously published structure. 35
3.2 Crystal packing of TCPTP led to pseudo-substrate-bound conformation, mimicking the mechanism of substrate recruitment. 37
3.3 Structural and functional characterization of TCPTP helix α7. 38
3.4 Removal of helix α7 hamper the enzymatic activity of TCPTP by reducing the catalysis power as well as substrate affinity. 40
3.5 In solution biophysical characterization of TCPTP catalytic domain 40
3.6 Helix α7 is a unique structural element that seems to be present only in TCPTP and PTP1B. 42
3.7 Regulation of TCPTP catalytic activity by non-catalytic C-terminal tail. 43
3.8 Structural characterization of TCPTP reveal C-terminal tail is intrinsically disordered and adopt an ensemble of conformation. 44
3.9 TCPTP’s C-terminal tail directly interact with the catalytic domain to inhibit its enzymatic activity. 46
3.10 NMR analysis reveals C-terminal residue 344-385 constitute the autoinhibitory domain of TCPTP. 48
3.10.1 TCPTPCAT, TCPTPTail, and TCPTPTailRK backbone resonance assignment. 48
3.10.2 Identification of interacting residues in TCPTPTail. 49
3.11 NMR analysis reveals C-terminal autoinhibitory domain bind TCPTPCAT via two surfaces that are distal from the active site. 51
3.12 Molecular mechanism of TCPTP autoinhibition. 52
3.13 Activation of TCPTP’s catalytic activity by ITGA1. 54
3.14 Molecular mechanism of TCPTP activation by ITGA1. 56
3.15 Truncation of C-terminal autoinhibitory domain led to change in conformation and activation of TCPTP. 58
3.16 Activation of TCPTP by collagen-binding integrin. 59
4. DISCUSSION 60
4.1 Necessity of TCPTP’s structural insight in activity regulation. 61
4.2 Molecular mechanism of helix α7 mediate activity regulation in TCPTP. 62
4.3 Allosteric pocket formed by helix α7 in TCPTP and PTP1B differ by surface charge. 64
4.4 Intrinsic disordered C-terminal tail distinctively regulates the catalytic activity of TCPTP. 64
4.5 Mechanism of TCPTP activation. 66
4.6 Reduced ITGA1 expression in cancerous cell might be averting TCPTP mediated suppression of RTKs pathway. 67
4.7 Strategy for TCPTP drug development. 67
5. FIGURES 69
Figure 1. Crystal packing of TCPTP molecules in the asymmetric unit 70
Figure 2. Crystal packing of TCPTP molecules in asymmetric unit illustrates multimerization of TCPTP. 71
Figure 3. Overview of TCPTP secondary structure elements, defined by the crystal structures of TCPTP1-314 and TCPTP1-302. 73
Figure 4. Comparison of present and earlier TCPTP crystal structures reveals new insights into WPD loop, E-loop, and helix α7. 74
Figure 5. TCPTP dimer formed in crystal packing mimics a phosphatase-substrate complex. 75
Figure 6. TCPTP dimer formed in crystal packing mimics a phosphatase-substrate complex. 77
Figure 7. Structural characterization of helix α7 in TCPTP crystal structure. 79
Figure 8. Allosteric role of helix α7 in regulating TCPTP catalytic activity. 80
Figure 9. Structural characterization of helix α7 in PTP1B crystal (PDB: 5K9W). 81
Figure 10. Allosteric role of helix α7 on TCPTP kinetic parameter. (A) 82
Figure 11. Kinetic data demonstrating structural coordination of helix α7 is essential for the efficient enzymatic activity of TCPTP. 83
Figure 12. Biophysical characterizations of TCPTP variants. 84
Figure 13. Structural analysis of catalytic domains in the PTP superfamily: 85
Figure 14. Distinct variation at the allosteric site of TCPTP and PTP1B. 87
Figure 15. Sequence alignment of TCPTP and PTP1B. 89
Figure 16. Autoinhibition of TCPTP catalytic activity by C-terminal tail. 90
Figure 17. Examining the influence of PTP1B C-terminal tail on PTP1B catalytic activity. 91
Figure 18. Examining the influence of TCPTP C-terminal tail on TCPTP catalytic activity by using phosphopeptides derived from the real substrate (EGFR). 92
Figure 19. Structural characterization of TCPTP by NMR. 93
Figure 20. Characterization of TCPTP and TCPTPCAT by SAXS. 95
Figure 21. Structural characterization of TCPTP by SAXS demonstrates ensemble of conformation adopted by TCPTP in solution. 96
Figure 22. TCPTP CX-MS analysis demonstrates that the dynamic C-terminal tail binds specifically to the TCPTPCAT domain. 97
Figure 23. MS spectra of the cross-linked peptide from intramolecular cross-linking analysis of TCPTP. 99
Figure 24. Intermolecular interaction between TCPTPCAT and TCPTPTail detected by CX-MS. 100
Figure 25. MS spectra of the cross-linked peptide from inter-molecular cross-linking analysis of TCPTPCAT and TCPTPTail. 102
Figure 26. Annotated 2D [1H, 15N] TROSY spectrum of the TCPTP catalytic domain. 103
Figure 27. Annotated 2D [1H, 15N] HSQC spectrum of the TCPTPTail (A) and TCPTPTailRK (B). 104
Figure 28. Intermolecular interaction between TCPTPCAT and TCPTPTail by NMR. 105
Figure 29. Surface charge distribution on TCPTP catalytic domain indicates area near cross-linked residue is negatively charged. 107
Figure 30. Identification of TCPTP C-terminal tail binding site to the TCPTP catalytic domain. 108
Figure 31. The N-surface binding site on TCPTP catalytic domain facilitates autoinhibition. 110
Figure 32. Identification of key residue within L1 loop of N-surface for binding to C-terminal tail. 111
Figure 33. Switch charge mutation in L5 loop to consolidate charge-charge interaction is critical for TCPTPTail binding to TCPTPCAT. 112
Figure 34. Structural model of TCPTP autoinhibition. 113
Figure 35. Activation of TCPTP by ITGA1. 114
Figure 36. FSEC demonstrates ITGA1 peptide form complex with TCPTP and TCPTPCAT. 115
Figure 37. Identification of ITGA1 binding site to the TCPTP catalytic domain. 116
Figure 38. Integrin α1 (ITGA1) and TCPTP C-terminal tail bind to the same site on TCPTP catalytic domain. 118
Figure 39. Integrin α1 (ITGA1) activates TCPTP by displacing the TCPTP C-terminal tail. 119
Figure 40. HDX-MS relative deuterium uptake heat map in TCPTP and TCPTP+ITGA1_FCT complex. 121
Figure 41. Peptide coverage map for TCPTP and TCPTP+ITGA1_FCT complex from HDX-MS analysis. 122
Figure 42. Validation of ITGA1 binding site on TCPTPCAT in full-length TCPTP. 123
Figure 43. Partial truncation of TCPTP C-terminal autoinhibitory binding region is sufficient to activate TCPTP 124
Figure 44. Activation of TCPTP due to partial truncation of C-terminal autoinhibitory binding region occurred due to change in conformation of TCPTP. 125
Figure 45. TCPTP is distinctively activated by Integrin cytoplasmic tail with positive charge residue clusters. 126
Figure 46. TCPTP and Integrin alpha-1 (ITGA1) expression analysis in cancers based on TCGA data. 128
Figure 47. Summary/Conclusion: TCPTP autoinhibition and activation is driven by intrinsically disordered protein:protein interactions. 130
6. TABLES 131
Table 1. Data collection and refinement statistics of X-ray Crystal. 132
Table 2. Enzymatic activity assay (Michaelis-Menten analysis) using substrate pNPP. 133
Table 3. Enzymatic activity assay (Michaelis-Menten analysis) using substrate pEGFR peptide-1 derived from cytoplasmic tail of EGFR. 134
Table 4. Enzymatic activity assay (Michaelis-Menten analysis) using substrate pEGFR peptide-2 derived from the EGFR’s kinase domain activation loop. 135
Table 5. SAXS data collection and scattering derived parameter for TCPTP variants. 136
Table 6: TCPTP cross-linking analysis (intramolecular): 138
Table 7: Intermolecular cross-linking analysis between TCPTPCAT and TCPTPTail 140
Table 8: Primers used in construct preparation. 142
7. REFERENCES 145
8. APPENDIX 154

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