Biophysical Characterization of Protein Interactions by ITC and NMR Xiaochen Lin University of Connecticut - Storrs, [email protected]

Biophysical Characterization of Protein Interactions by ITC and NMR Xiaochen Lin University of Connecticut - Storrs, Xiaochen.2.Lin@Gmail.Com

University of Connecticut OpenCommons@UConn

Doctoral Dissertations University of Connecticut Graduate School

4-29-2016 Biophysical Characterization of Protein Interactions by ITC and NMR Xiaochen Lin University of Connecticut - Storrs, [email protected]

Follow this and additional works at: https://opencommons.uconn.edu/dissertations

Recommended Citation Lin, Xiaochen, "Biophysical Characterization of Protein Interactions by ITC and NMR" (2016). Doctoral Dissertations. 1058. https://opencommons.uconn.edu/dissertations/1058

Biophysical Characterization of Protein Interactions by ITC and NMR

Xiaochen Lin, PhD

University of Connecticut, 2016

Proteins are crucial in virtually all cell activities. The vast majority of proteins interact with other molecules to function properly. The investigation of biomolecular interactions calls for structural and thermodynamic information acquired by a variety of experimental techniques and analytical methods. This dissertation is mainly concentrated on two frequently used and powerful tools, Isothermal

Titration Calorimetry (ITC) and Nuclear Magnetic Resonance (NMR) spectroscopy, to investigate protein interactions. p52 Shc, a well-studied cellular adaptor protein, is a positive regulator in the mitogen-activated protein kinases pathway. We have confirmed that Shc interacts with both, integrin tyrosine(s)-phosphorylated cytoplasmic tails and phosphotidylinositol lipids, through the same

Phospho-Tyrosine Binding (PTB) domain. The binding to phosphorylated peptides is enthalpy driven and tighter, while Shc interactions with PtdIns are entropy driven and much weaker. Shc interactions with PtdIns and β3-derived peptides are only weakly competitive with partially overlapping bindings sites. We also observed thermodynamic indications for potential intramolecular interactions within Shc, which could be perturbed by the binding to the phosphorylated receptor. BTN3A1, an immunoglobulin superfamily member, was found recently to be critical in Vγ9Vδ2 T cell activation. We described a phosphoantigen prodrug approach with great potency in Vγ9Vδ2 T cell stimulation. Our NMR and ITC results revealed the intracellular binding of BTN3A1 to phosphoantigens. In addition, we have provided biophysical evidence indicating the involvement of membrane proximal region in phosphoantigen binding and potential structural changes within BTN3A1 intracellular region. By focusing on p52 Shc and BTN3A1, I hope to demonstrate how a combination of structural and thermodynamic knowledge can be utilized to answer many important questions in signaling transduction close to the cell membrane.

Biophysical Characterization of Protein Interactions by ITC and NMR

Xiaochen Lin

B.S., China Pharmaceutical University, 2009

A Dissertation

Submitted in Partial Fulfillment of the

Requirements for the Degree of

Doctor of Philosophy

at the

University of Connecticut

2016

Xiaochen Lin

2016

APPROVAL PAGE

Doctor of Philosophy Dissertation

Biophysical Characterization of Protein Interactions by ITC and NMR

Presented by Xiaochen Lin, B.S.

Major Advisor ______Olga Vinogradova

Associate Advisor ______Debra Kendall

Associate Advisor ______Andrei Alexandrescu

Associate Advisor ______Andrew Wiemer

University of Connecticut 2016

iii

Table of Contents

Table of Contents ...... iv Acknowledgements ...... vi List of Figures ...... viii List of Tables ...... x List of Abbreviations ...... xi Chapter 1 ITC in Protein Interaction Study ...... 1 Principles of ITC ...... 1 Data Analysis of ITC ...... 3 Practical Considerations in an ITC Experiment ...... 7 Chapter 2 NMR spectroscopy in Protein Interaction Study ...... 10 Overview of NMR Spectroscopy ...... 10 Principles of NMR Spectroscopy...... 11 Chemical Shifts in Protein NMR ...... 13 1H-15N HSQC 2D Experiments ...... 14 Chapter 3 p52 Shc in Signaling...... 15 Shc Family ...... 15 Shc in MAPK Pathway ...... 16 Shc in PI3K/AKT Pathway ...... 17 Chapter 4 Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling ...... 21 Introduction ...... 21

Shc Interaction with Phosphatidylinositols Tested Using Ins(1,4,5)P3 ...... 24

Shc Interaction with Phosphatidylinositols Analysed by ITC Using PtdIns(4)P1 and PtdIns(4,5)P2 ...... 27 PtdIns Binding Sites on Apo Shc PTB Surface Partially Overlap with Integrin Binding Sites ...... 29 Story of Integrin Interactions with the Shc - PTB Domain versus Full Length Shc . 32

PtdIns Only Weakly Compete with β3-Peptides for Interaction with Shc ...... 34 Discussion ...... 38 Research Design and Methods ...... 42 Materials and Reagents ...... 42 Cloning and Protein Purification ...... 42 ITC Experiments ...... 44 15N-HSQC Titration by NMR ...... 45

Chapter 5 Vγ9Vδ2 T cells in the Immune System ...... 46 Overview of the Immune System in Humans ...... 46 Lymphocytes: B Cells and T Cells ...... 47 Vγ9Vδ2 T Cells ...... 50 Activation of Vγ9Vδ2 T Cells by Phosphoantigens ...... 51 BTN3A1 in Activation of Vγ9Vδ2 T Cells...... 55 Chapter 6 Do Phosphoantigens Bind to the Extracellular IgV Domain of BTN3A1? ...... 57 The Extracellular Binding Model of IgV-Phosphoantigen ...... 57 Crystal Structures of IgV Complexed with Phosphoantigens...... 57 Mass Spectroscopy and SPR Analysis of the IgV-Phosphoantigen Interaction ..... 60 Chapter 7 Intracellular Binding of BTN3A1 to Phosphoantigens ...... 62 A POM-protected Phosphoantigen is Highly Potent in Vγ9Vδ2 Stimulation...... 62 The BTN3A1 PRY/SPRY (B30.2) Domain Directly Binds Phosphoantigens...... 65 The Intracellular Domain of BTN3A1 Undergoes Conformational Changes after Binding to Phosphoantigens...... 75 Discussion ...... 78 Research Design and Methods ...... 79 Cloning and Purification of BTN3A1 Constructs ...... 79 ITC Experiments ...... 80 15N-HSQC Titration by NMR ...... 80 Author Contributions ...... 80 Chapter 8 Conclusions ...... 82 References ...... 83

Acknowledgements

It has been a long journey and an unforgettable experience for me to pursue a doctorate degree as an international student. There was doubt, frustration, rebound and joy along the way. Looking back to the naïve young man who just stepped out from the airplane coming to the United States, I am now sure he did not realize even ten-percent of what was ahead of him.

It has been not only a journey to learn as a student but also a journey to mature as a man. It would be an impossible task for me to finish my PhD study without the help from many people in my life.

I would like to thank my advisor, Dr. Olga Vinogradova. I want to convey my sincere gratitude to her not only for her guidance in science but also her help in life. Managing a lab with limited resources in a highly competitive field, she showed us how to be creative, persistent and resilient, and most of all, that science should never be compromised. There is still a great deal of knowledge I have not learned from her, especially in NMR. Next, I want to thank Dr. Andrew Wiemer for the opportunity to work on the BTN3A1 project. The collaboration with Wiemer lab has been a wonderful experience and I am truly thankful for what I have learned from him in the area of Vγ9Vδ2 T cell. I would also like to acknowledge my committee members, Dr. Debra Kendall for her useful suggestions in academic writing and Dr. Andrei

Alexandrescu for his instructions in NMR. In addition, I truly thank all the former and current members also my friends in Vinogradova lab, Robbins Puthenveetil, Priya Katyal, Khiem

Nguyen and Dr. Lalit Deshmukh, and our NMR facility manager, Dr. Vitaliy Gorbatyuk.

I would like to convey my gratitude to my family especially my parents and my wife. My father, Mr. Rong Lin, who never had an opportunity for higher education due to the social restrictions back then, is the most self-motivated person I ever met. His guidance and encouragement was my source of power when facing difficulties. He has been and will always

Acknowledgements

be the role model I look up to. My mother, Mrs. Nianzhi Wu, is the greatest mother in my eyes.

There is no word for me to truly describe how grateful I am for her unconditional love and givings. Years away from my family have made me realize how much they mean to me. Last but not least, I want to thank my beautiful wife, Lu Huo. It has been more ten years since we met but my memory of the first day I saw her is still vivid and lovely. From Nanjing to America, both pursuing a PhD degree, our journey to reach here was a bumpy road and almost a miracle.

I cannot tell how lucky I feel to have her by my side in almost all the important moments in my life. I am complete only with my wife.

vii

List of Figures

Figure 1. A schematic representation of Nano ITC from ta instruments ...... 2 Figure 2. An example of itc raw data and processed data with a fitted independent model with 1:1 stoichiometry ...... 6 Figure 3. Simulated itc data to find the optimal concentrations for the reactants ...... 8 Figure 4. Spin-1/2 nuclei in an external magnetic field ...... 11 Figure 5. T1 (spin-lattice) and T2 (spin-spin) relaxation (from blue to red) ...... 13 Figure 6. The protein architecture of p52 Shc ...... 16 Figure 7. Illustration of Erk and β3CT competing over the same binding site on Shc PTB .... 18 Figure 8. ITC results showing a direct interaction between Shc SH2 domain and p-1433ζ phosphopeptide at 37 °C ...... 20 Figure 9. Structures of PtdIns molecules used in the study ...... 24 Figure 10. ITC and NMR studies on the interaction of Shc PTB domain and Ins(1,4,5)P3...... 26 Figure 11. ITC and NMR studies on the interaction of Shc PTB domain and PtdIns ...... 28 Figure 12. PtdIns(5)P1 does not show to bind to Shc PTB domain at 25 °C in ITC ...... 29 Figure 13. 15N-HSQC spectra showing chemical shifts perturbations in a classical PTB binding pocket, the second binding site and F30 ...... 31 Figure 14. (a) Bi-β3 peptide and (b) c-β3 peptide binding to Shc PTB domain at 25 °C...... 33 15 Figure 15. N-HSQC spectra of Shc PTB pre-satured by Ins(1,4,5)P3 (at protein-PtdIns ratios of 1:0, 1:3, 1:5 and 1:15) with bi-β3 added (at a 1:2 protein-to-peptide ratio) ...... 36 Figure 16. Full length Shc binding to bi-β3 in the absence (upper) and presence (lower) of Ins(1,4,5)P3 at 25 °C ...... 38 Figure 17. A model for Shc-mediated integrin signaling at the cytosolic face of the plasma membrane ...... 41 Figure 18. Chromatograms from Size-exclusion chromatography in a Superdex-75 column and SDS-PAGE gel pictures of full-length Shc, Shc PTB domain and SH2 domain ...... 44 Figure 19. The innate and adaptive immune response ...... 46 Figure 20. Cell-mediated immune response and antibody response ...... 47 Figure 21. Humoral immune response by B cells activated in presence of helper CD4+ T cells ...... 48 Figure 22. Helper CD4+ T cells vs. cytoxic CD8+ T cells in antigen recognition ...... 49 Figure 23. Comparison of the signals required to activate a helper T cell and a B cell ...... 50 Figure 24. αβ TCR vs. Vγ9Vδ2 TCR ...... 51 Figure 25. Isoprenoid biosynthesis in bacterial and human ...... 52 Figure 26. Structure of zoledronate ...... 54 Figure 27. Structure of BrHPP ...... 54 Figure 28. Protein architecture of B7 family and butyrophilin superfamilies ...... 56 Figure 29. Electron density of BTN3A1 IgV-phosphoantigen structures constructed from PDB entries: 4JKW and 4K55 showing a poor fitting for both IPP and HMBPP ...... 58 Figure 30. PEG molecules in a circular shape with a Lysine residue in the center (PDB entry: 3FFR) ...... 59 Figure 31. PEG molecules showing a better fitting than HMBPP (PDB entry: 4K55) and IPP (PDB entry: 4JKW) ...... 60 Figure 32. Chemical structures of phosphoantigens used in the study ...... 63 Figure 33. Bis-POM prodrug compound 1b functions as a direct phosphoantigen with elevated potency relative to compound 1a and HMBPP ...... 64 Figure 34. Evidence for the absence of major 18 intramolecular interactions between the 68- residue membrane-proximal region and PRY/SPRY domain ...... 66

viii

List of Figures

Figure 35. Binding of HMBPP and Compound 1a to the Intracellular Domain of BTN3A1 .... 67 Figure 36. Evidence for the absence of interaction between HMBPP and the extracellular IgV domain of BTN3A1 ...... 68 Figure 37. 1H-15N HSQC titration of HMBPP into the full-length intracellular domain and the PRY/SPRY domain of BTN3A1...... 69 Figure 38. Binding of C-HDMAPP to the BTN3A1 full intracellular domain ...... 71 Figure 39. Binding of HMBPP to the BTN3A1 full intracellular domain in the presence of IPP ...... 72 Figure 40. 1H-15N HSQC titration of IPP and compound 1a into the full-length intracellular domain of BTN3A1 (BFI) ...... 75 Figure 41. Structural comparison between free BTN3A1 B30.2 domain (gray, PDB entry: 4N7I) and B30.2-cHDMAPP complex (green, PDB entry: 4N7U) ...... 76 Figure 42. Structures of BFI with (green) and without (gray) HMBPP by SAXS ...... 78

List of Tables

Table 1. Thermodynamic parameters for the simulated reaction ...... 7 Table 2. Concentrations used to generated simulated ITC data in Figure 3 ...... 9 Table 3. Summary of the thermodynamic data for Shc SH2 interaction with p-1433ζ phosphopeptide at 37 °C ...... 20 Table 4. ITC data for Shc PTB domain binding to PtdIns at 25°C ...... 29 Table 5. Summary of the thermodynamic data for Shc interaction with β3 integrin derived mono- and bi-tyrosine-phosphorylated peptides acquired from the ITC measurements at two temperature points using full length Shc and PTB domain constructs...... 33 Table 6. Summary of the thermodynamic data for Shc interaction with β3 integrin derived mono- and bi-tyrosine-phosphorylated peptides acquired from the ITC measurements at 12°C in the presence and the absence of Ins(1,4,5)P3 ...... 37 Table 7. Activity of compounds for expansion of Vγ9Vδ2 T Cells from human peripheral blood mononuclear cells and inhibition of cell viability ...... 63 Table 8. Summary of the thermodynamic data derived from the ITC measurements using different ligands ...... 68 Table 9. Primers used for BTN3A1 constructs ...... 79

List of Abbreviations

PtdIns: Phosphatidylinositol NMR: Nuclear Magnetic Resonance spectroscopy ITC: Isothermal Titration Calorimetry Shc: Src Homology Collagen PTB domain: Phospho-Tyrosine Binding domain MAPK: Mitogen-Activated Protein Kinase β3CT: β3 integrin Cytoplasmic Tail SH2: Src Homology 2 Kd: dissociation constant bi-β3: β3 integrin derived bi-phosphorylated (Y747 and Y759) peptide pY: phosphorylated tyrosine PtdIns(4)P1: Phosphatidylinositol-4-phosphate PtdIns(4,5)P2: Phosphatidylinositol-4,5-diphosphate Ins(1,4,5)P3: D-myo-Inositol-1,4,5-triphosphate PtdIns(5)P1: Phosphatidylinositol-5-phosphate c-β3: β3 integrin derived peptide phosphorylated at C-terminal tyrosine (Y759) SDS-PAGE: Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis 15N-HSQC: 1H-15N Heteronuclear Single Quantum Correlation Spectroscopy SAXS: Small-Angle X-ray Scattering APC: Antigen-Presenting Cell MHC: Major Histocompatibility Complex BTN: Butyrophilin TCR: T Cell Receptor IPP: isopentenyl diphosphate HMBPP: (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate GDP: guanosine triphosphate

ITC in Protein Interaction Study

Chapter 1 ITC in Protein Interaction Study

Principles of ITC

In biology, the answers to many important questions about biomolecular interactions are closely related to a change in heat or enthalpy occurring during the process. ITC directly measures the heat change absorbed from the surroundings for an endothermic process or released to the surroundings for an exothermic process, which makes it an ideal technique to study biomolecular interactions. ITC is a powerful tool to study biomolecular interactions by depicting the complete thermodynamic profile including the binding affinity (Ka), the enthalpy

(ΔH) and entropy (ΔS) changes in a single experiment for interaction of interest (1).

ITC measures biochemical reactions at constant temperature. Modern ITC only requires low quantity of samples and is capable of studying interactions with a dissociation constant (Kd) in a range from nM to mM. Figure 1 is a schematic representation of NanoITC calorimeter from TA Instruments. In the assembly of ITC reside the sample and reference cells.

When an ITC experiment is undergoing, the sample cell contains one of the reactant of the reaction of interest and the reference cell contains a solution which has a similar heat capacity of the buffer used for the sample in the sample cell. Prior to the start of the experiment, the other reactant in exactly the same buffer is loaded into the titration syringe. The syringe is then mounted onto the burette handle and inserted into the ITC instrument. During an experiment, the burette handle rotates and spins the syringe to ensure fast and adequate mixing of the two reactants. The stepwise injection of ITC is controlled by the software where the user can specify the settings for the experiment such as temperature, injection volume and stirring speed.

ITC in Protein Interaction Study

Figure 1. A schematic representation of Nano ITC from ta instruments. Figure taken from Nano Isothermal Titration Calorimeter (Nano ITC) Getting Started Guide, Revision A by TA Instruments.

The mechanism of differential power compensation is utilized to achieve the high sensitivity and short response time (2). Currents are applied to both cells during the experiment to maintain them at constant temperature set by the user. The injection of one reactant in the injection syringe to the other reactant induces heat released to the surrounding environment

ITC in Protein Interaction Study

(an exothermic reaction) or absorbed from surrounding environment (an endothermic reaction).

The heat change in the sample cell causes the change in the current applied to the sample cell resulting a voltage difference between the two cells. In this manner, the energetics of isothermal biochemical reactions is converted to electrical signals.

Data Analysis of ITC

The determination of the parameters of the thermodynamic profiles of the reaction can be derived from the basic equilibrium thermodynamics and Gibbs free energy equation (2, 3).

To demonstrate of the analysis of ITC data, we consider the simplest scenario, in which a macromolecule (such as proteins and nucleic acids) binds to its ligand (such as small molecules) with 1:1 stoichiometry. The association of the macromolecule with the ligand gives us a reaction:

푀 + 퐿 ⇌ 푀퐿 (1) where M is the macromolecule, L is the ligand and ML is the macromolecule-ligand complex.

For this reaction, the equilibrium association constant (Ka) is defined as:

[푀퐿] 1 퐾푎 = = (2) [푀][퐿] 퐾푑 where [ML], [M], [L] are the concentrations of free ML, M and L, respectively, and Kd is the dissociation constant of the equilibrium. Due to the mass balance of the system, we have the following relations among the free, bound and total concentrations for M and L:

[푀] = [퐿] = [푀퐿] (3) 퐵 퐵

[푀] = [푀] + [푀퐿] (4) 푇

[퐿] = [퐿] + [푀퐿] (5) 푇

ITC in Protein Interaction Study

where [M]B and [L]B are the concentrations of bound M and L, respectively, and [M]T and [L]T are the concentrations of total M and L, respectively. The Gibbs free energy can be derived from the following two equations:

Δ퐺 = −푅푇푙푛퐾푎 (6)

Δ퐺 = Δ퐻 − 푇Δ푆 (7)

ITC directly measures the heat (qi) released or absorbed from the reaction triggered by

th the i titration of L into M in the reaction cell. Meanwhile, qi is directly proportional to the characteristic enthalpy change (ΔH) of the reaction and increase in [L]B:

푞푖 = 푣 × Δ퐻 × Δ[퐿]푖 (8)

th where v is the volume of the reaction cell and ΔLi is the increase in [L]B thanks to the i injection of L. In our simplest case where the macromolecule has only one binding site, the Equation (8) becomes:

퐾푎[퐿]푖 퐾푎[퐿]푖−1 푞푖 = 푣 × ΔΗ × [푀]푇 × ( − ) (9) 1 + 퐾푎[퐿]푖 1 + 퐾푎[퐿]푖−1

th where [L]i is the concentration of free ligand after the i injection. However, the Equation (8) is still not in its implementable form and it needs to be rewritten in terms of the total ligand concentration. The total cumulative heat, Q, is directly proportional to [L]B (hence [ML] due to

Equation (3)) as in:

푄 = 푣 × Δ퐻 × ∑ Δ[퐿]푖 = 푣 × Δ퐻 × [퐿]퐵 = 푣 × Δ퐻 × [푀퐿] (10) which gives us the following equation:

[푀퐿] 푄 = (11) [푀]푇 푣 × Δ퐻 × [푀]푇

From Equations (2) to (5), we also obtain:

ITC in Protein Interaction Study

[푀퐿] 퐾 [퐿] = 푎 (12) [푀]푇 1 + 퐾푎[퐿]

[푀퐿] [푀]푇 = [푀퐿] + [푀] = [푀퐿] + (13) 퐾푎[퐿]

Thus, we have a quadratic equation for [ML]:

2 1 [푀퐿] + [푀퐿] (−[푀]푇 − [퐿]푇 − ) + [푀]푇[퐿]푇 = 0 (14) 퐾푎

The only real root for Equation (13) can solved and substituted in to Equations (5), (10) and

(11). Eventually, we establish the implementable form to determine Ka, ΔH from the non-linear least-squares analysis of the dependent, Q, versus the known independent variable, usually

[L]T/[M]T. The precise relation is:

2 2 1/2 (1 + [푀]퐾푎 + 퐾푎[퐿]푇) − [(1 + [푀]퐾푎 + 퐾푎[퐿]푇) − 4[푀]퐾푎 [퐿]푇] 푄 = (15) 2퐾푎 푣 × Δ퐻

Figure 2 demonstrates the parameters calculation from the data acquired by ITC. The upper panel shows the raw data collected in an ITC experiment. Each peak is the heat change induced by an injection. The peaks pointing different directions (up or down) indicates the reaction being endothermic or exothermic. The lower panel represents the integration of the row data with the fitted curve for an exothermic protein-ligand interaction with 1:1 stoichiometry.

Each data point on the plot of the lower panel is calculated from the area under peak for the corresponding peak in the row data. On the X-axis, the cumulative total ligand-protein ratios are calculated after each injection.

ITC in Protein Interaction Study

Figure 2. An example of itc raw data and processed data with a fitted independent model with 1:1 stoichiometry

After the fitting, the important thermodynamic parameters are reflected on the sigmoidal curve (Figure 2). The distance between the two horizontal asymptotes of the curve represents the enthalpy change, ΔH, of the reaction. The stoichiometry of the reaction is obtained by determining where the midpoint is on the X-axis for the sigmoidal curve. The slope at this point

ITC in Protein Interaction Study

specifies the binding affinity, Ka, of the reaction. In addition, Equations (6) and (7) enable us to obtain the Gibbs free energy change, ΔG, and entropy change, ΔS, for the reaction of interest.

Practical Considerations in an ITC Experiment

ITC is popular in biophysical studies due to its simplicity and capability in determining a complete thermodynamic profile for the interaction of interest in short time (4). ITC instruments directly measure the energy associated with a chemical reaction in a stepwise fashion (1). The reaction cell contains one of the reactants, usually macromolecules such as proteins and nucleic acids. The other reactant of the reaction, usually with better stability and solubility at higher concentrations, such as small molecules, is loaded into the injection syringe and its injection is controlled by the ITC software.

ITC is a highly sensitive technique to study biochemical reactions and it is easy to use.

ITC experiments do not need any special modifications for the reactants and can be done using spectroscopically silent samples in a wide range biologically relevant conditions (such as pH, buffer and temperature). However, there are precautions a researcher needs to pay attention to in order to obtain the optimal results.

For simplicity, we focus the association of a protein with its ligand of a small molecule with 1:1 stoichiometry (Table 1). An optimal binding curve from ITC should contain the two

“platform” phases in the beginning and in the end of the curve while the transition phase is not too steep or flat to obtain an accurate estimate of the slope at the midpoint as shown in Figure

3A.

Kd (μM) ΔH (kJ/mol) TΔS (kJ/mol) stoichiometry

1 50 15.75 1 Table 1. Thermodynamic parameters for the simulated reaction

ITC in Protein Interaction Study

Figure 3. Simulated itc data to find the optimal concentrations for the reactants

ITC is able to study biochemical reactions with a Kd in a range from nanomolar to millimolar. However, there are still limitations. We can determine if a reaction is suitable for

ITC based on a parameter known as:

푐 = 퐾푎 × [푀] (16) where Ka is the reciprocal of Kd and [M] is the concentration of macromolecule sample. An initial value of Kd can be obtained from other techniques or a test ITC run in order to calculate the value for c as in Equation 16. Modern ITC instruments are capable of directly measure reactions with 10 < c < 1000 (Table 2). A c value lower than 10 gives a transition too flat to

ITC in Protein Interaction Study

determine ΔH (Figure 3D). On the other hand, a c value greater than 1000 gives a transition too steep, which leaves few points to accurately determine the slope at the midpoint (Figure

3E).

Experiment [L] (μM) [M] (μM) c [L]/[M] Comment

A 150 25 25 6 Optimal B 300 25 25 12 Saturated too fast C 75 25 25 3 Saturation not achieved D 15 2.5 2.5 6 c < 10 E 7500 1250 1250 6 c > 1000 Table 2. Concentrations used to generated simulated ITC data in Figure 3

In order to achieve the optimal binding curve, the reactant is usually 5-10 times more concentrated than the other reactant in the sample cell (Figure 3, Table 2). Thus it is reasonable to have the reactant, which has better solubility and stability under the experiment condition, in the injection syringe. In case of our protein-ligand interaction example, it is a common practice to prepare the ligand of small molecule at a higher concentration. The exact ratio can be determined in simulation based on the volumes of the injection syringe and the sample cell of a specific ITC model available.

NMR spectroscopy in Protein Interaction Study

Chapter 2 NMR spectroscopy in Protein Interaction Study

Overview of NMR Spectroscopy

In biology, in order to understand how macromolecules (such as proteins and nucleic acids) function, related structural information is essential. ITC is popular in biophysical studies due to its simplicity and capability in determining a complete thermodynamic profile for interaction of interest in short time (4). NMR spectroscopy, on the other hand, focuses on structural information at the atomic level and is particularly valuable in protein dynamics studies.

Along with X-ray crystallography, NMR spectroscopy has become one of the two primary techniques to solve three-dimensional structures of macromolecules including protein and nucleic acids. In addition, it is also widely used to reveal the structural details of biomolecular binding events such as protein-protein and protein-ligand interactions (5). Unlike X-ray crystallography, NMR can monitor biomolecular interactions in solution at a near-physiological condition.

Over the past few decades, the range of biological system suitable for NMR has been remarkably expanded by the improvement in instrumentation, application of multidimensional techniques and other methodological advances (6-8). NMR has been employed successfully in structural determination of large-sized proteins (9) and membrane proteins (10). In the dissertation, we mainly utilized two-dimensional NMR experiments, especially 1H-15N HSQC, to study a variety of protein interactions and protein conformational changes upon ligand binding.

NMR spectroscopy in Protein Interaction Study

Principles of NMR Spectroscopy

The fundamental principle of NMR spectroscopy comes from the behavior of nuclei in an external magnetic field. In the scope of our discussion here, we will focus on spin-1/2 nuclei

(including 1H, 13C and 15N). When an external magnetic field is applied to the nuclei, the nuclei are distributed into higher and lower spin energy states. As described by the Boltzmann distribution, there are only slightly more nuclei at lower energy state than higher energy state.

The energy difference between the two states, ΔE, is proportional to the strength of the external magnetic field:

휇퐵 Δ퐸 = 0 (1) 퐼 where µ is the magnetic moment of the nucleus, B0 is the strength of applied external magnetic field and I is the spin number of the nucleus (I = ½ for spin-1/2 nuclei). The magnetic moment,

µ, is positively related to the gyromagnetic ratio, γ, of the nucleus:

훾퐼ℎ 휇 = (2) 2휋 where h is Planck constant.

Figure 4. Spin-1/2 nuclei in an external magnetic field. E represents the energy difference between the two states and B0 is the strength of applied external magnetic field. Figure taken from https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/nmr/nmr1.htm.

NMR spectroscopy in Protein Interaction Study

When a nucleus is irradiated with radio frequency (RF) energy corresponding exactly to the difference between the two energy states, nuclei at lower energy state absorb the energy and are excited to the higher energy state resulting in NMR signals. The process of nuclei in excitation return to the lower energy state is called relaxation in NMR. Relaxation also describes how NMR signals deteriorate with time. The rate of relaxation can be used to define how fast NMR signals become gradually weaker and broader and is vital in NMR spectroscopy.

There are two major relaxation processes: spin-lattice relaxation and spin-spin relaxation. The spin-lattice relaxation is a process in which the nuclei emits energy to the surrounding environment. T1 is often used to describe how long this process takes place and is also the decay constant for the recovery of the longitudinal component (parallel to the direction of B0 in Figure 5A) of the nuclear spin magnetization in excitation to the equilibrium state. The spin-spin relaxation, on the other hand, is a process in which a nucleus interacts with neighboring nuclei resulting in a decaying transverse nuclear spin magnetization due to decoherence. The over-population of the excited state does not change in the spin-spin, or T2, relaxation but the life time of the NMR signal from the transverse nuclear magnetization

(perpendicular to the direction of B0 in Figure 5B) becomes shorter. T1 relaxation is responsible for the weakening and T2 is accountable for the broadening in NMR signals.

NMR spectroscopy in Protein Interaction Study

Figure 5. T1 (spin-lattice) and T2 (spin-spin) relaxation (from blue to red). The Z-axis represent the direction of B0. Figure modified from http://www-mrsrl.stanford.edu/~brian/bloch/.

Chemical Shifts in Protein NMR

The magnetic field at the nucleus is not equal to the applied external magnetic field due to the nuclear shielding phenomena in which the electrons circulating around a nucleus creates a secondary induced magnetic field. The induced magnetic field can oppose or align with the external magnetic field causing a shift in the resonant frequency of a nucleus in a magnetic field. After standardized by a reference, the chemical shift is defined as the relative resonant frequency of a nucleus to the reference, which is independent of the strength of the external magnetic field. In chemistry, chemical shifts are particularly helpful for structural determination of small molecules.

In protein NMR, the backbone chemical shift assignments of 13Cα, 13Cβ, 13C’, 15N, 1Hα and 1HN, as the first step in NMR structure determination process, can often be carried out rapidly using moderate amount of sample. NMR chemical shifts, which are highly sensitive to local structure, provides us with a broad range of structural factors including secondary

NMR spectroscopy in Protein Interaction Study

structure, backbone and sidechain conformations and hydrogen bonding (11, 12). Thus, the structural information encoded in chemical shift values should not be overlooked. In addition, assorted protein dynamics studies require the prerequisite assignment of the chemical shifts of backbone atoms. Notably, the exploration of the structure-chemical shift relationship has been an active area and advancing rapidly. Direct utilization of backbone chemical shifts has been shown to considerably improve the accuracy of the protein structures predicted from homology/de novo modeling (13, 14). The capability of predicting high-quality protein structures based on sequence homology and experimental NMR backbone chemical shift data may represent a new direction for high-throughput NMR structure determination.

1H-15N HSQC 2D Experiments

Among a variety of 2D heteronuclear NMR experiments (such HSQC and HMQC), 1H-

15N HSQC is one of the most frequently used experiments in protein NMR (15). 1H-15N HSQC experiments require 15N protein samples which are easy to produce and inexpensive compared to 13C labeled samples. Due to its better sensitivity, 1H-15N HSQC can be acquired in a short period of time compared other multidimensional NMR experiments. 1H-15N HSQC correlates the chemical shifts of the amide protons with the covalently bound 15N amide nitrogens. In a

1H-15N HSQC spectrum, each peak represents a bonded amide N-H pair with the corresponding coordinates of the chemical shifts of the 1H and 15N. 1H-15N HSQC spectra can be considered as the “finger-print” of the protein and are often collected first in the structural determination process. The perturbation of chemical shifts in 1H-15N HSQC spectra can serve as indicators for protein interactions. With the assignment of the spectrum, the perturbations can be mapped upon the protein structure hence reveal the binding sites on the protein for its ligand.

14 p52 Shc in Signaling

Chapter 3 p52 Shc in Signaling

Shc Family

Shc was one of the first characterized cellular adaptor proteins and a key node in the activation of MAPK signaling in response to growth factors, cytokines, antigens, integrin activation and mechanical force (16-18). By complexing with a wide range of membrane receptors, the Shc family stands as a first point of signaling network convergence in cell by transducing signals from the cell surface to DNA in the nucleus. The Shc family has significant physiological impact on animal development and maintenance (16). Furthermore, Shc has been implicated to be crucial in cancers including prostate (19) and breast cancer (20-22), and many other diseases (16). In human, the p52 isoform of Shc is ubiquitously expressed but also highly regulated. Given its pivotal role in cell regulation, vast in vivo and in vitro studies have been conducted in last two decades to decode the molecular mechanism of Shc in signaling.

However, a number of new findings lend strong evidence to notion that Shc plays a versatile role in signal transduction including its inhibitory function in the MAPK signaling pathway (23) and its positive regulation in the PI3K/AKT pathway (21, 24).

All members in the Shc family share a PTB-CH1-SH2 core architecture (Figure 6) and are only distinguished by the difference in the length of their N-termini (16). In addition to its C- terminal SH2 domain, which is capable of binding tyrosine-phosphorylated targets, the N- terminal PTB domain was later characterized as a novel type of protein motif, which also binds tyrosine-phosphorylated targets. Nonetheless, the two independent pTyr-docking domains,

PTB and SH2, recognize pTyr-containing sequences in two distinct manners. In case of the

PTB domain, the classical pTyr-binding site defines its specificity by selectively recognizing the residues N-terminal to the pTyr residue (25), especially the sequence Φ-X-Asn-Pro-X-pTyr

15 p52 Shc in Signaling

(Φ represents a hydrophobic residue); however, the specificity of SH2 is primarily determined by residues C-terminal to the pTyr residue and prefers the sequence pTyr-Φ-X-Ile/Leu/Met

(26). Between the PTB and SH2 lies the highly flexible proline-rich CH1 region, which, once phosphorylated at Y239/240 (27, 28) and Y317 (29, 30), offers the recognition motifs to the downstream partner Grb2 to activate the MAPK pathway.

Figure 6. The protein architecture of p52 Shc. The yellow circles represent the tyrosines in the CH1 region which undergo phosphorylation during the activation of Shc.

Shc in MAPK Pathway

The canonical view of Shc in the MAPK pathway is in linking stimulated receptors to the activation of the downstream MAPK pathway (17). Generally, activated and tyrosine- phosphorylated receptors recruit Shc through the PTB or SH2 domain of Shc. Interestingly, the PTB domain, not SH2, is predominantly chosen by membrane receptors. Therefore so far most in vivo and in vitro studies on Shc have focused on the PTB domain. Recruitment of Shc to the activated receptors leads to phosphorylation in the CH1 region of Shc at Y239/240 and Y317

(27, 29). The phosphorylated tyrosine residues form two consensus binding motifs to downstream adaptor protein Grb2. The formation of Shc-Grb2 complex leads to the activation of Ras/MAPK and results in the phosphorylation of the terminal kinase Erk by Mek in the MAPK pathway (16). In summary, traditionally defined as a positive mediator in pTyr-based signaling pathways, Shc links stimulated membrane receptors (via PTB or SH2) to downstream signaling

16 p52 Shc in Signaling

pathways (via CH1) activating Ras/MAPK. Though remarkable progress has been achieved in our knowledge of Shc, many intriguing studies have led to a perception that Shc functions in more ways than its role as a positive regulator in the MAPK pathway.

Shc in PI3K/AKT Pathway

By using a combination of in vivo and in vitro methods, Suen and co-workers demonstrated that the PTB domain of Shc exhibits direct binding to the N-terminal lobe of Erk kinase in non-stimulated cells (23). This interaction was suggested to have inhibitory effects on Erk’s activity by restraining its nuclear translocation and impeding Erk-dependent gene transcription. The binary Shc-Erk complex is disrupted once the CH1 region of Shc is tyrosine- phosphorylated resulting in a potential conformational change within Shc. The release of Erk from the Shc-Erk complex makes Erk susceptible for activation. Those exciting findings strongly indicate a new inhibitory role of Shc in the MAPK pathway. Combined with its orthodox positive regulatory function, a dual role of Shc in MAPK is suggested: in non-stimulated cells,

Shc suppresses the activation of MAPK by coupling with Erk and restricting its kinase activity; in the presence of extracellular stimuli, Shc is recruited by various receptors and phosphorylated in the CH1 region, ultimately leading to the activation of MAPK. Integrins, a major class of transmembrane receptors which mediate cell-cell and cell-extracellular matrix interactions (31), are among the broad range of receptors recruiting Shc via the PTB domain

(32, 33). Previously, we have identified a classical binding site and a novel binding site on PTB for β3CT (34). Surprisingly, the suggested binding site on PTB for Erk overlaps with the novel

747 binding site for pY of β3CT (Figure 7). It would be very intriguing to investigate how Erk and

β3CT compete over the same binding site on the Shc PTB domain.

17 p52 Shc in Signaling

Figure 7. Illustration of Erk and β3CT competing over the same binding site on Shc PTB. The structure of the PTB domain (light yellow) in complex with bi-β3 (green) is generated from PDB entry 2L1C. The amino acids suggested to directly bind Erk are colored in blue (23). The 747 region resides in the long loop which contributes to form the novel binding site for pY in β3CT.

As Shc has been defined as a major signaling axis that activates the MAPK signaling pathways, several studies have shown its importance in the PI3K/AKT pathway (21, 24). The

PI3K/AKT pathway makes significant contributions to many aspects of cell growth and survival

(35). Class 1A PI3Ks (hereafter referred to as PI3Ks) comprise a regulatory/inhibitory subunit p85 and a catalytic subunit p110. When activated, PI3Ks, via its p85 subunit, are recruited to the plasma membrane by various receptors and produce phosphatidylinositol-(3,4,5)- trisphosphate (PIP3) as a secondary messenger (35). AKT has been identified as the major positive downstream mediator of the PI3K signaling pathway. Gu and co-workers performed in vitro studies demonstrating Shc’s positive function in the activation of PI3K/AKT pathway

(24). They found that by forming the complex of Shc-Grb2-Gab2, CH1-phosphoyrlated Shc, as a part of the complex, is linked to the p85 subunit of PI3K and eventually promotes the activation of downstream AKT. This new mechanism of Shc was believed to be crucial in the activation of the PI3K/AKT mediated by receptors lacking direct binding sites for the p85 subunit of PI3K. 14-3-3 proteins, a class of ubiquitously expressed phosphoserine/threonine binding modules and known to bind a wide range of diverse signaling proteins, serve as positive regulators of Akt downstream signaling (36). Recently, the Tyr179 of 14-3-3 was found

18 p52 Shc in Signaling

to be phosphorylated in a cytokine-dependent manner and a direct interaction occurs between the SH2 domain of Shc and pY179 of 14-3-3ζ (37). Moreover, over-expression of the ζ isoform of the 14-3-3 protein family was found to be associated with increased AKT phosphorylation in human breast tumors (38). Later on, by employing transgenic mice expressing Shc with non- functional SH2 domain, Ursini-Siegel and co-workers showed the SH2-dependent Shc signaling in the activation of PI3K/AKT is crucial for breast tumor outgrowth and survival (21).

Thus, it is highly likely that Shc, via its CH1 and/or SH2, plays a significant role in the PI3K/AKT pathway.

We have used a phosphopeptide encompassing pY179 of 14-3-3ζ (p-1433ζ:

174FSVFY(pY)EILNSPEK186) in our ITC studies to confirm the presence of a direct interaction between isolated Shc SH2 domain and pY179-14-3-3ζ. The thermodynamic parameters of the interactions revealed a binding affinity in the low μM range and an enthalpy and entropy- favorable process (Figure 8 and Table 3).

19 p52 Shc in Signaling

Figure 8. ITC results showing a direct interaction between Shc SH2 domain and p-1433ζ phosphopeptide at 37 °C

Kd (µM) ΔH (kJ/mol) -TΔS (kJ/mol) ΔG (kJ/mol) 6.28 ± 0.209 -6.45 ± 0.531 23.5 ± 4.56 -30.0 ± 4.03 Table 3. Summary of the thermodynamic data for Shc SH2 interaction with p-1433ζ phosphopeptide at 37 °C

In summary, as a convergence point for many signaling pathways, Shc has been believed to fulfill an up-regulatory function in the MAPK pathway. A growing number of independent studies have suggested the biological significance of Shc is not limited to this. In the MAPK pathway, Shc may also act to prohibit undesirable activation in the non-stimulated cell by complexing with Erk kinase (23). Outside the MAPK pathway, Shc seems to be crucial in the up-regulation of the PI3K/AKT pathway involving membrane receptors lacking a binding site for p85 subunit of PI3K (21, 24). Increasing studies have shown the necessity to better understand the diversity of Shc’s functionality in signaling.

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

Chapter 4 Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc

Mediated Integrin Signaling

Based on Lin, X. and Vinogradova, O., 2015. Phospho-Tyrosine (s) vs. Phosphatidylinositol Binding in Shc Mediated Integrin Signaling. American journal of molecular biology, 5(2), p.17.

Introduction

Integrin mediated signaling events control numerous developmental, physiological and pathological processes in multicellular organisms. Although significant progress has been achieved in the understanding of integrin activation (39, 40), the early intracellular events following the integrin mediated extracellular matrix engagement are not well characterized structurally. To illuminate the potential mechanisms of integrin selectivity in the recognition of proximal effectors at different stages of cell spreading, we have studied the effect of phosphorylation on the conformation, membrane insertion, and target binding capability of the cytoplasmic tail of the major platelet integrin αIIbβ3 (41, 42). Here we extend this investigation by presenting our new work on deciphering the role that PtdIns lipids might share with phosphotyrosine(s) in integrin signaling.

Amongst the number of potential chemical modifications correlated with the activated state of integrin receptor, it is the tyrosine(s) phosphorylation of the cytoplasmic tail that has been proven crucial for the outside-in signaling events (43-47). Shc p52 isoform, more specifically, its PTB domain, has been identified as the primary signaling partner for the tyrosine(s) phosphorylated β3CT (47, 48). This cytoplasmic adaptor protein, known to mediate

MAPK signaling pathway (49), needs to be localized at the cytosolic face of the plasma membrane in order to be readily recruited by a number of different receptors (50). Two of Shc’s three distinct domains, PTB and SH2, have the potential to bind phospholipids and, thus, might

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

assist Shc translocation to the lipid bilayer (51, 52). Though only the PTB, and not the SH2 domain, is shown to interact with specific PtdIns (53). The purpose of the work presented here is to compare molecular details of the interaction between Shc and integrin to the binding mode between Shc and phospholipids.

Since their discovery about two decades ago, PTB domains have been identified and structurally characterized within numerous multi-domain proteins. The ways they interact with their targets are strikingly versatile. The phosphorylated tyrosine is not always required for the high affinity interactions, and, in some cases, the binding motif might not even contain the tyrosine residue at all (54). PTB domains in general are also famous for the significant variance in their ligand binding affinities, ranging from 0.1 to 100 μM (55). Shc PTB domain in particular exhibits a wide range of affinities to various tyrosine-phosphorylated binding partners. The tightest interaction, with a Kd of 0.19 µM, was measured by ITC for the nerve growth factor receptor TrkA derived peptide (56), correcting the originally over-estimated Kd value of 53 nM

(by Scatchard analysis of Surface Plasmon Resonance data) (57). Upon the structure determination of Shc PTB in complex with a β3 integrin derived bi-phosphorylated peptide, bi-

759 β3 (41), we have found that the C-terminal phosphotyrosine (pY ) of β3CT occupies the classical PTB binding pocket. The other phosphotyrosine (pY747) containing motif fits nicely into the grove formed between the second helix and the unusually long flexible loop (~24 residues) connecting the second beta strand with the second helix in PTB fold. In this interaction, a negatively charged phosphate group of pY747 forms a salt-bridge with a positively charged side chain of Shc R104. This second binding site is in close proximity to the Shc residue

R112, which is situated at the beginning of the PTB domain second beta strand. This R112, along with two lysines, K116 and K139, which also contain positively charged side chains, has been proposed earlier to mediate Shc PTB-phospholipids interaction (58). In addition, Zhou and co- workers have originally found that Shc PTB domain has higher specificities to acidic PtdIns,

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

particulalry PtdIns(4)P1 and PtdIns(4,5)P2 (Figure 9), over the other lipids that they have tested

(53). Furthermore, the binding of the full length Shc, enclosing the SH2 domain, to Ins(1,4,5)P3

(Figure 9), representing the polar head group of PtdIns(4,5)P2, has been reported to be much tighter than the binding of the isolated Shc PTB domain alone to PtdIns(4,5)P2 (59). Therefore, in order to investigate whether β3 integrin/phospholipids binding sites on Shc PTB surface indeed overlap, what the thermodynamic profiles of these interactions are, and how β3CT derived phosphopeptides might affect Shc’s ability to localize itself to the lipid bilayer we have initiated a through biophysical characterization of the above system. Here we present our data, acquired by ITC and NMR experiments, which allowed us to visualize PtdIns mediated Shc association with the lipid bilayer and how tyrosine(s)-phosphorylated integrin cytoplasmic tail could later replace PtdIns from their binding site on Shc surface. Out data also suggests that the interaction between the cytoplasmic tails of activated integrin receptors and Shc may result in the rearrangement/separation of Shc internal domains.

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

Figure 9. Structures of PtdIns molecules used in the study

Shc Interaction with Phosphatidylinositols Tested Using Ins(1,4,5)P3

We started our investigation by studying Shc binding to Ins(1,4,5)P3 using ITC. We were greatly surprised that we were not able to reproduce the data reported by George and co-workers, who have reported a tight dissociation constant (Kd of 0.7 µM) for the full length

Shc (59). No binding was found under any conditions tested (to have a better control on all possible variables, we have purchased Ins(1,4,5)P3 from the same supplier (Cayman Chemical) as reported in the paper. The conditions we tested include: i) Shc PTB domain at 12°C and

25°C in several different buffers (including exactly the same as the one described by George,

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

which is 50 mM Tris, pH 8.0, 200 mM NaCl, 1 mM Dithiothreitol); ii) Shc full length at 12°C and 25°C; and iii) Shc SH2 domain at 12°C and 25°C (typical raw titration data is shown as an example in the Figure 10a-c). In the attempt to interpret our negative findings, we hypothesized that the heat of the reaction was too small, actually below the detection range of our Nano ITC calorimeter. Thus, we turned to NMR as the method of choice due to its endogenous ability to capture the interactions with minimal or no enthalpy changes. In this case, we performed 15N-HSQC chemical shifts mapping experiments by titrating the

15 unlabeled Ins(1,4,5)P3 into the N-labled Shc PTB domain. Under the conditions tested (see

Materials and Methods for details), we only observed minimal chemical shift perturbations for the handful of residues in the 15N-HSQC spectra even at the maximum of lipid to protein ratio (15 to 1, Figure 10d). Uncertain about these findings, we decided to choose different lipid mimetics for further investigation.

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

Figure 10. ITC and NMR studies on the interaction of Shc PTB domain and Ins(1,4,5)P3. (a) Ins(1,4,5)P3 does not show to bind to Shc PTB domain at 25 °C in ITC. (b) Ins(1,4,5)P3 does not show to bind to full length Shc at 25 °C in ITC. (c) Ins(1,4,5)P3 does not show to bind to SH2 15 domain at 25 °C in ITC. (d) N-HSQC titration of PTB domain by Ins(1,4,5)P3. The effect from Ins(1,4,5)P3 only show up at a high PTB-to-Ins(1,4,5)P3 ratio (1:15) and is less pronounced compared to PtdIns(4,5)P2.

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

Shc Interaction with Phosphatidylinositols Analysed by ITC Using PtdIns(4)P1 and PtdIns(4,5)P2

With minimal success observing Shc interaction with Ins(1,4,5)P3, representing exclusively the polar head-groups of phosphatidylinositols, we wondered whether the aliphatic chains of the lipids were necessary for the measurability of the binding by ITC and NMR. Long lipid chains are not exactly soluble in aqueous solution. Thus, to perform the binding studies described below, we restricted the length of aliphatic chains to six carbons in dihexanoyl, or eight in dioctonoyl phosphatidylinositol derivatives and concentrated on PTB domain of Shc, previously shown to interact specifically with PtdIns(4)P1 and PtdIns(4,5)P2 (53). Binding affinities for the apo PTB domain, originally examined in the unilamellar liposomes centrifugation assay, were reported to be 52 μM for PtdIns(4)P1 and 140 μM for PtdIns(4,5)P2. Binding affinity of the

PTB domain complexed with TrkA peptide was reported to be even weaker, with a Kd of roughly

450 μM for both lipids.

First, we employed ITC to determine the binding affinities of the PTB domain to three different phosphatidylinositol containing soluble probes (see the Materials and Methods section for details). We measured a Kd of 95 µM and 125 µM for PtdIns(4)P1 and PtdIns(4,5)P2 respectively (titration curves and isotherms are presented in Figure 11a and Figure 11b). ITC was not able to detect the binding PtdIns(5)P1 (titration curve is shown Figure 12), thus confirming the specificity of interaction reported by Zhou and coworkers. The ITC measurements demonstrated similar binding affinity for PtdIns(4,5)P2 as compared to the affinity previously determined by centrifugation assay data analysis, though PtdIns(4)P1 appeared to bind about two times weaker than reported before (53). Small amounts of heat are consumed upon binding (in addition to the relatively large heat of solvation), demonstrating endothermic interactions at 25°C that are driven predominantly by entropy. Considering these data, it is not

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

entirely surprising that we were only able to observe minimal Shc PTB binding with Ins(1,4,5)P3 alone in ITC. The complete thermodynamic analysis of the above interactions is presented in the Table 4.

Figure 11. ITC and NMR studies on the interaction of Shc PTB domain and PtdIns. (a) PtdIns(4)P1 and (b) PtdIns(4,5)P2 binding to Shc PTB domain at 25 °C.

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

Figure 12. PtdIns(5)P1 does not show to bind to Shc PTB domain at 25 °C in ITC

Ligand Kd (µM) ΔH (kJ/mol) -TΔS (kJ/mol) ΔG (kJ/mol) PtdIns(4)P1 95.00 1.51 -24.46 -22.95 PtdIns(4,5)P2 125.1 1.94 -24.21 -22.27 PtdIns(5)P1 No binding Table 4. ITC data for Shc PTB domain binding to PtdIns at 25°C

PtdIns Binding Sites on Apo Shc PTB Surface Partially Overlap with Integrin

Binding Sites

To find out the molecular details of these weak Shc-PtdIns interactions, we next employed NMR and performed 15N-HSQC chemical shifts mapping experiments which are

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

especially useful to define weak binding (60). Two non-labeled PtdIns derivatives, PtdIns(4)P1 and PtdIns(4,5)P2, demonstrated to interact with Shc PTB by ITC experiments, were titrated into the samples containing 15N-labeled apo PTB domain. Chemical shift perturbations, associated with the probes binding, were monitored. Overall, the observed shifts in Shc PTB resonance frequencies were small. However, they were concentration dependent, reproducible, and similar among the two PtdIns tested. Furthermore, the same residues were affected as the ones in Ins(1,4,5)P3 titration described above (Figure 10d), but the shifts were more pronounced in case of PtdIns(4,5)P2, even at 5 to 1 lipid to protein ratio as compared of 15 to 1 ratio for

15 Ins(1,4,5)P3. The close-ups of N-HSQC spectra of PTB in the presence of PtdIns(4,5)P2, exemplifying the most shifted resonances, are shown in Figure 13. These chemical shifts perturbations occur at three major binding regions. The first one, represented by the residues

Q148, S149 and A168, overlaps with the classical phospho-tyrosine binding pocket of the protein- peptide binary complex. However, the canonical PTB fold cannot be formed without the stabilizing hydrophobic residue (pY-5) of the target peptide. Thus, in apo PTB domain

Ins(1,4,5)P3 could only establish intermediate dynamic contacts with the amine functional groups of R67 or Q148 side chains. The second binding site, including the residues L63, Q76 and S107, is formed at the interface of the second helix, the loop connecting the first beta strand with the second helix and the beginning of the second beta strand. This second PtdIns binding site is distinct from the second novel phospho-tyrosine binding site we previously described for Shc

PTB-bi-β3 complex (41), although it includes several overlapping residues. The third PtdIns binding site is restricted to a single but the most perturbed residue of the spectra, F30, which is located within the unstructured Shc PTB N-terminus. The chemical shift perturbations in 15N-

HSQC spectrum of apo Shc PTB upon PtdIns(4,5)P2 titration, plotted against the residue number, are presented in Figure 13. The inset demonstrates affected residues mapped onto PTB

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

surface, which is colored according to the absolute value of the corresponding chemical shift perturbations.

Figure 13. 15N-HSQC spectra showing chemical shifts perturbations in a classical PTB binding pocket, the second binding site and F30. Chemical shifts perturbations upon PtdIns(4,5)P2 binding to apo Shc PTB domain inset with surface mapping of PTB generated from PDB entry 1OY2. The surface is colored acorrding to the corresponding chemical shift perturbations, Delta (ppm), from yellow (smallest) to red (largest) and light gray indicating missing assignments. Delta (ppm) refers to the combined HN and N chemical shift changes, 2 2 1/2 obtained from the equation: Δδ(HN,N) = ((ΔδHN + 0.2(ΔδN) ) , where Δδ = δbound – δfree.

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

Story of Integrin Interactions with the Shc - PTB Domain versus Full Length Shc

Two of the Shc three distinct domains, PTB and SH2, have the potential to bind phospho-tyrosines and/or phospholipids. However, in all the experiments performed we did not observe any indications of Shc SH2 domain interaction with either PtdIns or β3 integrin derived tyrosine(s)-phosphorylated peptides. Thus, we focused our investigation on studying the PTB domain alone and comparing it to the full length Shc in order to understand the thermodynamic forces driving the interactions. We started with a bi-β3 peptide, which was used previously for the structural characterization of the Shc PTB/integrin binary complex (41). We found that at 25 °C Kd of this interaction was 5.1 μM (ΔG = -30.17 kJ/mol) and it was predominantly driven by enthalpy (ΔH = -22.98 kJ/mol). A representative calorimetric isotherm and the corresponding titration curve, depicted in Figure 14a, demonstrate that the interaction is exothermic at 25 °C, releasing the heat upon peptide binding with a stoichiometry of about

1. The driving force of this complex formation is very different from the one defined for the TrkA peptide (enthalpy vs. entropy), and it shows a higher specificity, even though the binding affinity is about twenty five times weaker (5.14 µM vs. 0.19 µM). We have speculated that the secondary binding site for pY747 within the complex might be responsible, at least in parts, for the difference. To find out whether this was the case, we ran ITC experiments for the smaller

759 c-β3 peptide containing only one terminal pY (Figure 14b). Our reasoning appeared to be wrong. The shorter (13 vs. 27 residues) mono-phosphorylated peptide had an even higher binding affinity with the Kd of 0.34 µM (ΔG = -36.86 kJ/mol) and an even larger favorable enthalpy term (ΔH = -25.41 kJ/mol). The thermodynamic analysis of the above interactions is presented in Table 5.

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

Figure 14. (a) Bi-β3 peptide and (b) c-β3 peptide binding to Shc PTB domain at 25 °C.

ΔH ΔCp -TΔS ΔG Shc construct Ligand T (°C) K (µM) d (kJ/mol) (kJ/mol∙K) (kJ/mol) (kJ/mol) 25 5.14 -22.98 -7.191 -30.17 bi-β3 -0.88 12 4.32 -11.52 -17.75 -29.27 PTB Domain 25 0.35 -25.41 -11.45 -36.86 c-β3 -0.09 12 1.05 -24.18 -8.446 -32.63

25 2.84 -28.82 -2.784 -31.60 bi-β3 -0.96 12 5.21 -16.27 -12.55 -28.82 Full Length 25 1.33 -7.40 -26.13 -33.53 c-β3 1.04 12 0.82 -21.03 -12.17 -33.20

Table 5. Summary of the thermodynamic data for Shc interaction with β3 integrin derived mono- and bi-tyrosine-phosphorylated peptides acquired from the ITC measurements at two temperature points using full length Shc and PTB domain constructs.

Next, we decided to find out how the Shc solvent-accessible surface area was affected during the reaction. This was accomplished by investigating the change in constant pressure heat capacity (ΔCp). ΔCp is given by the slope of the linear regression analysis of ΔH plotted

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

vs. temperature (Equation 17), and it is often independent of temperature within the narrow physiological range (reviewed in (61)). Thus, two temperature points, 12 °C and 25 °C, would provide an estimate for the slope. For binding reactions, negative ΔCp is expected to demonstrate the reduction in protein surface area in contact with the solvent. The larger and more negative values reflect a larger surface area buried upon complex formation. Through this correlation, structural information of the macromolecular complex could be inferred from the thermodynamic parameters. As expected, the longer bi-β3 peptide in complex with Shc

PTB domain demonstrated a more negative ΔCp of about -0.88 kJ/mol∙K as compared to the shorter c-β3 peptide represented by ΔCp of -0.09 kJ/mol∙K (see Table 5 for details). This correlates well with the larger solvent-accessible surface area buried upon binding of the longer peptide. As we already established that the Shc SH2 domain does not bind to either of the above peptides, we decided to confirm that the full length Shc behaves similarly to PTB domain alone. To our great surprise that was not the case. We found that while bi-β3 peptide in complex with full length Shc showed quite similar ΔCp of about -0.96 kJ/mol∙K, binding of c-β3 peptide resulted in a positive ΔCp of about 1.04 kJ/mol∙K. That means that upon complex formation with c-β3 peptide more of the Shc solvent-accessible surface area was exposed. The only reasonable explanation of this phenomenon that we can imagine is the unraveling of the intramolecular interactions between the domains within Shc itself.

휕ΔH Δ퐶 = (17) 푝 휕푇

PtdIns Only Weakly Compete with β3-Peptides for Interaction with Shc

We were also curious about the relation between binding sites for PtdIns and β3- peptides (bi-β3 and c-β3) on Shc PTB surface. If these sites do not overlap, the presence of β3- peptide should not interfere with the interaction between PTB and PtdIns; thus, PtdIns should

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

cause concentration-dependent perturbations in the spectra of the PTB-β3-peptides complex.

In case of a competitive binding between PtdIns and β3-peptides to PTB, since Shc’s affinity to bi-β3 and c-β3 is much higher in comparison to its affinity to phospholipids, it is reasonable to speculate that integrin tyrosine(s)-phosphorylated CTs can easily replace PtdIns from the partially overlapping binding sites on the Shc surface. We have proved the latter scenario by using NMR. First, we prepared protein samples of the PTB domain with PtdIns added at protein-PtdIns ratios of 1:0, 1:3, 1:5 and 1:15. Then bi-β3 or c-β3 was added to the sample at a

1:2 protein-to-peptide ratio and 15N-HSQC experiments were performed. The 15N-HSQC spectra of both Shc PTB-bi-β3 and Shc PTB-c-β3 complexes in the absence and the presence of PtdIns(4)P1, PtdIns(4,5)P2 or Ins(1,4,5)P3 overlap perfectly (an example of the overlapping spectra is presented in Figure 15). Therefore, we have confirmed that the binary interfaces between Shc PTB and phosphopeptides remained the same in the presence of phospholipids added in excess. Also, bound peptides did not allow PtdIns to occupy their partially accessible

(according to the chemical shifts mapping experiments summarized in Figure 13) potential binding sites on the surface of PTB domain. The remaining question was whether PtdIns could affect the thermodynamic profiles of Shc interaction with phosphorylated peptides. To test this, we investigated the binding of full length Shc and Shc PTB domain with bi-β3 and c-β3 peptides in the presence of Ins(1,4,5)P3 by ITC. As expected, our data indicate that the protein samples over-saturated by Ins(1,4,5)P3 at 3 to 1 lipid to protein ratio were not much different in Shc binding to either of the peptides. The most noticeable difference, observed in a single case, was the perturbation in the thermodynamic profile of the full-length Shc titrated by bi-β3, where the unfavorable 3 kJ/mol change in enthalpy was almost compensated by the favorable change in entropy term, resulting in a slight reduction of Kd value (see Table 6). The impact from Ins(1,4,5)P3 on the peptide interaction with Shc was also evidenced in this case by the appearance of small negative peaks ahead the dominating positive peaks in the titration curve (presented in Figure

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

16), when the reaction was close to saturation. In the absence of the lipid mimetic, the small negative peaks were not present. This phenomenon is quite specific, as no similar patterns were witnessed in any other ITC experiments performed for studying Shc- bi-β3/c-β3 binding.

Lastly, to complete the investigation and to confirm the inability of PtdIns to replace the phosphopeptides, we performed titrations of PtdIns into Shc PTB complexed with phosphorylated peptides (bi-β3 or c-β3, 2:1 peptide to protein ratio). We then monitored potential changes in HSQC spectra by NMR or in calorimetric isotherms by ITC. As expected, no indications of binding were observed by either method.

15 Figure 15. N-HSQC spectra of Shc PTB pre-satured by Ins(1,4,5)P3 (at protein-PtdIns ratios of 1:0, 1:3, 1:5 and 1:15) with bi-β3 added (at a 1:2 protein-to-peptide ratio). The lack of difference in the overlaid spectra indicates that integrin tyrosine(s)-phosphorylated β3 integrin CTs can easily replace PtdIns from the overlapping binding sites on the Shc surface.

Shc -TΔS Ligand K (µM) ΔH (kJ/mol) ΔG (kJ/mol) construct d (kJ/mol)

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

bi-β3 4.32 -11.52 -17.75 -29.27

bi-β3 + Ins(1,4,5)P3 3.65 -12.37 -17.30 -29.66 PTB Domain c-β3 1.05 -24.18 -8.45 -32.63

c-β3 + Ins(1,4,5)P3 1.12 -23.62 -8.84 -32.46

bi-β3 5.21 -16.27 -12.55 -28.82

bi-β3 + Ins(1,4,5)P3 4.38 -13.87 -15.37 -29.23 Full Length c-β3 0.82 -21.03 -12.17 -33.20

c-β3 + Ins(1,4,5)P3 0.87 -21.11 -11.96 -33.07

Table 6. Summary of the thermodynamic data for Shc interaction with β3 integrin derived mono- and bi-tyrosine-phosphorylated peptides acquired from the ITC measurements at 12°C in the presence and the absence of Ins(1,4,5)P3

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

Figure 16. Full length Shc binding to bi-β3 in the absence (upper) and presence (lower) of Ins(1,4,5)P3 at 25 °C

Discussion

Scaffold adaptor protein Shc can be recruited through numerous receptors, including integrins, growth factors, antigens, cytokines, G-protein-coupled, and hormone receptors (50).

Interactions of this cytoplasmic protein with specific phospholipids have been proposed as a mechanism for its translocation to the membrane (51, 52). Despite vast structural, functional,

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

and biophysical data characterizing Shc involvement in signal transduction from different receptors, its interactions with integrins or specific phospholipids are much less studied or understood. We have analyzed Shc binding to tyrosine(s) phosphorylated peptides derived from β3 integrin and PtdIns using ITC and solution NMR methods. Through these studies we have found that PtdIns only weakly compete with phosphorylated integrin cytoplasmic tail for

Shc PTB binding. This conclusion is based on several observations. First, Shc interactions with phosphopeptides are characterized by several orders of magnitude higher affinity than binding to PtdIns. Second, Shc interaction with peptides is enthalpy driven in contrast to the entropy driven interaction with phospholipids. Third, the overall conformational rearrangement of Shc PTB domain upon interaction with the phosphopeptides is coupled with the replacement of PtdIns from their binding sites on Shc surface. Lastly, PtdIns cannot replace phosphotyrosines from their PTB binding sites and the unoccupied surface is not sufficient to stabilize Shc PTB interaction with phospholipids.

The most interesting finding in this study comes from the thermodynamic analysis of mono- and bi-phosphorylated peptides binding to Shc PTB domain vs. the full length Shc.

Although there are not many differences in the ΔCp of bi-β3 binding, characterized by expected negative values of about 0.9 kJ/mol∙K for solvent-accessible surface area buried in both cases, there is a striking difference in ΔCp of c-β3 binding. While for PTB domain alone ΔCp is expectedly reduced (shorter peptide) but still negative, full length Shc binding is characterized by the positive ΔCp value of about 1.0 kJ/mol∙K. This suggests exposure of the originally buried solvent-accessible surface area, which is larger than the surface of interaction with mono- phosphorylated peptide, and this is only possible if the interactions among PTB, CH1 and SH2 domains within Shc have been disrupted. One of the potential rearrangement schemes for Shc domains upon binding different peptides is proposed in Figure 17. In this scenario, Shc is recruited to the membrane by the interaction of its PTB domain with PtdIns. While binding of

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

the bi-phosphorylated integrin releases PTB domain from the membrane, β3CT-PTB complex may still provide the surface area necessary for intra-molecular interaction with other domains of Shc. Contrarily, binding of mono-phosphorylated peptide might result in conformational rearrangements leading to a more “extended” Shc structure as PTB domain detaches from the lipid bilayer. As Shc is known to be a positive regulator in the MAPK pathway, the recruitment of Shc to the activated receptors leads to phosphorylation in the CH1 region at Y239/240 and Y317.

The phosphorylated tyrosine residues form two consensus binding motifs to the downstream adaptor protein Grb2 and the formation of Shc-Grb2 complex eventually leads to the activation of the MAPK pathway. We have established previously that integrin cytoplasmic tails are capable of accommodating different structural features depending upon their binding partner and/or the phosphorylation state (39, 41, 42, 62). Our new data suggests that they can also cause variable conformational rearrangements in their targets, as Shc seems to adapt different conformations when binds mono/bi-phosphorylated β3 cytoplasmic tail. The plausible biological significance of the dexterity in the conformation of Shc may contribute to differential phosphorylation in the CH1 region. Although both Y239/240 and Y317 are capable of serving as the binding site for Grb2, the biological outcomes emanating from the two phosphorylation sites can be significantly different (63). Deciphering this remarkable dexterity should definitely aid in better understanding of crucial bidirectional information flow through these distinct receptors.

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

Figure 17. A model for Shc-mediated integrin signaling at the cytosolic face of the plasma membrane. The cytoplasmic tails of integrin αIIb and β3 are shown in red and blue, respectively. The PTB domain and SH2 domain of Shc are shown in cyan and gray, respectively. The molecules of PtdIns(4,5)P2 and phosphotyrosines are colored acorrding to heteroatoms. This descriptive model was generated in Chimera (64) by using PDB entries: 1OY2, 1TCE, 1S4X, 2KNC, 1SHC, 2LJE, 2L1C, and Phosphorylethanolamine/Phosphocholine lipid membrane (65). (a) Shc is recruited to the membrane by the interaction of its PTB domain with PtdIns, integrin heterodimer is shown in latent state; (b) binding to the activated mono-phosphorylated β3CT via PTB results in the conformational rearrangements leading to a more “extended” full length Shc; (c) binding to the activated bi-phosphorylated β3CT releases PTB domain from the membrane, though β3CT- PTB complex may still provide the surface area necessary for the interaction with the other internal Shc domains.

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

To conclude, we have: i) confirmed that Shc interacts with both, integrin tyrosine(s)- phosphorylated cytoplasmic tails and PtdIns lipids, through the same PTB domain (another potential hub, SH2 domain, is not involved in these interactions); ii) found that phosphorylated peptides binding is enthalpy driven and tighter, while Shc interactions with PtdIns are entropy driven and are much weaker; iii) determined that Shc interactions with PtdIns and β3-derived peptides are only weakly competitive and are characterized by partially overlapping bindings sites; iv) observed thermodynamic indications for potential intramolecular interactions within

Shc, which could be perturbed by the binding to the phosphorylated receptor; and v) proposed a model for Shc-mediated integrin signaling through its recruitment to the lipid bilayer, which paves the foundation for the future experiments.

Research Design and Methods

Materials and Reagents

Ins(1,4,5)P3 was purchased from Sigma and Cayman Chemical. (dihexanoyl- or dioctonoyl-) PtdIns(4)P1, PtdIns(5)P1 and PtdIns(4,5)P2 were purchased from Cayman Chemical

(Figure 9). Phosphopeptides derived from β3CT (bi-β3:

736 762 750 762 RAKWDTANNPL(pY)KEATSTFTNIT(pY)RGT and c-β3: ATSTFTNIT(pY)RGT ) were synthesized by NEO-Peptide.

Cloning and Protein Purification

The Shc PTB domain (17-207) containing the pET15b vector was transformed into

Rosetta (DE3) cell line to pursue optimal expression levels (41). SH2 domain (380-473) was cloned into NdeI and XhoI sites in pET21b vector and then transformed into a BL21 (DE3) cell line. The vector of pET28a-full length Shc (1-473), generously provided by Dr. Ladbury

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

(University of Texas MD Anderson Cancer Center, TX), was expressed in BL21 (DE3) cell line as well. Typically, 10 ml overnight culture was used to inoculate 1 liter LB broth. Cells were grown at 37 °C until OD600 reached 0.6. At this point the culture was cooled to room temperature and then the overnight overexpression was induced by 1 mM isopropyl-β-D- thiogalactopyranoside. Harvested cells were kept at -20 °C for future purification or resuspended in lysis buffer supplemented with protease inhibitors. Cells were further lysed by french press and the cell debris was removed by centrifugation at 18,000 rpm at 4 °C. The N- terminal His-tag fused PTB domain and full length Shc, and the C-terminal His-tag fused SH2 domain were first purified under native conditions with Ni-NTA resin (QIAGEN) using the standard protocol followed by a buffer exchange to 50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM

β-mercaptoethanol in a Superdex-75 column (GE Healthcare) where all three recombinant proteins showed a proper folding in monomeric state (Figure 18). The overexpression of the target protein and the purified protein were confirmed by SDS-PAGE and the bands of the expected mass were observed. The determination of protein concentration by UV measurement was consistent with Bradford assay.

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

Figure 18. Chromatograms from Size-exclusion chromatography in a Superdex-75 column and SDS-PAGE gel pictures of full-length Shc, Shc PTB domain and SH2 domain. All three recombinant proteins are shown in monomeric state with a good purity after the purification process.

ITC Experiments

Calorimetric measurements of a full length Shc, PTB domain, and SH2 domain to β3 phosphopeptides or PtdIns were performed on a low volume Nano ITC (TA Instruments). All experiments were carried out in a buffer of 50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM β- mercaptoethanol (unless described otherwise) at 12 or 25 °C with a stirring speed of 250 rpm to minimize the precipitation of the protein. 300 second time intervals were set between injections. The concentration of protein samples was determined by UV measurements. The titrants (β3 phosphopeptides or PtdIns) were prepared in the same buffer by the dilution of

Phospho-Tyrosine(s) versus Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

higher concentration stocks. The concentrations of the protein ranged from 30 μM to 150 μM.

The concentration of protein was examined after each ITC experiment by UV to ensure the stability of proteins under the test condition. The analysis of the data was done with

NanoAnalyze Software (TA Instruments) suite using an “independent” model. In all cases, a stoichiometry of 1 ± 0.15 was revealed for the interaction between protein and β3 phosphopeptides/PtdIns.

15N-HSQC Titration by NMR

15N-labelled recombinant proteins were overexpressed in M9 medium supplemented

15 with NH4Cl as the sole nitrogen source. The same purification procedures as described above were followed. NMR samples of 100-200 µM were prepared in a buffer of 50 mM Tris, pH 7.5,

100 mM NaCl, 5 mM β-mercaptoethanol, 7% D2O, 1 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid as the internal standard. 15N-HSQC titration experiments were performed on a

Varian/Agilent Inova 600 MHz spectrometer equipped with inverse-triple resonance cryo-probe at 35 °C. All spectra were processed with NMRPipe (66) and analyzed in CCPNmr Analysis software suite (67).

Vγ9Vδ2 T cells in the Immune System

Chapter 5 Vγ9Vδ2 T cells in the Immune System

Overview of the Immune System in Humans

The immune system detects and fights against pathogens including foreign proteins, virus, bacteria and even cancer cells. The immune system in many species including humans can be classified into two sub-systems (Figure 19), non-specific or innate immune system, and specific or adaptive immune system. The innate immune system recognizes the conserved features of pathogens which successfully enters the organisms and is quickly activated to attack the invaders; however, it defends pathogens in a generic way and cannot develop immunological memory. Another function the innate immune system has is to work as APCs to activate the adaptive immune system. The adaptive immune system, on the other hand, is able creates immunological memory after the initial exposure to the pathogens and develops long-lasting immunity.

Figure 19. The innate and adaptive immune response. Figure taken from Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Figure 24-1, Innate and adaptive immune responses.

The adaptive immune system produces both humoral (antibody-mediated) response and cell-mediated response (Figure 20). In humoral immunity, extracellular antigens are

Vγ9Vδ2 T cells in the Immune System

targeted and antibodies are secreted to destroy the antigens. Cell-mediated immunity is triggered when intracellular infections occur and does not involve antibodies.

Figure 20. Cell-mediated immune response and antibody response. Figure taken from Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Lymphocytes and the Cellular Basis of Adaptive Immunity.

Lymphocytes: B Cells and T Cells

Lymphocytes are a type of white blood cells in immune system and responsible for adaptive immune responses to infections and other foreign substances, and host defense against diseases. There are two primary types of lymphocytes, B cells and T cells. B cells are largely involved in the humoral immune response (Figure 21). The activation of B cells is triggered by antigens usually in a T cell-dependent manner. B cells then proliferate and differentiate in to plasma cells producing an antibody targeting the specific antigen the B cell is exposed to. In addition, B cells can internalize and process antigens and then present the peptides to T cells using MHC II molecules. Thus B cells belong to the family of professional

APCs.

Vγ9Vδ2 T cells in the Immune System

Figure 21. Humoral immune response by B cells activated in presence of helper CD4+ T cells. Figure taken from Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune System in Health and Disease. 5th edition. New York: Garland Science; 2001. Chapter 9, The Humoral Immune Response.

T cells play a central role in the cell-mediated immune response. In the lineage of T cells, T cells expressing a αβ TCR, αβ T cells, constitute the majority of T cells in humans. αβ

T cells are among the best studied T cell lineage and often considered as “conventional”. αβ

T cells can be further categorized as either “helper CD4+ T cells”, which recognize antigens via MHC II (Figure 22) and help to activate the other immune cells (such B cells, Figure 23) by

Vγ9Vδ2 T cells in the Immune System

releasing cytokines, or “cytoxic CD8+ T cells”, which recognize antigens via MHC I and directly kill target cells including cancerous, infected and damaged cells (Figure 22). The recognition between αβ TCR and antigen peptides has low binding affinity and specificity.

Figure 22. Helper CD4+ T cells vs. cytoxic CD8+ T cells in antigen recognition. Figure take from reference (68).

Vγ9Vδ2 T cells in the Immune System

Figure 23. Comparison of the signals required to activate a helper T cell and a B cell. Figure taken from Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Helper T Cells and Lymphocyte Activation.

On the other hand, T cells expressing a γδ TCR, γδ T cells, comprising only a small percentage of the circulating T cells, are far less understood. The antigenic specificities and functional features of γδ T cells are being investigated actively and extensively (69-73). γδ T cells show innate cell-like features allowing its early activation induced by conserved stress- induced ligands. In addition, γδ T cells are capable of producing cytokines rapidly in case of tissue stress (74). Most of the molecular mechanism of the activation of γδ T cells remains enigmatic. Nonetheless, γδ T cells garner increasing interest due to its killer cell-like cytotoxic activity against cancer cells (74).

Vγ9Vδ2 T Cells

In humans and non-human primates, Vγ9Vδ2 T cells represents the majority of γδ T cells circulating in blood and account for between 1% and 10% of total blood T cells (69).

Vγ9Vδ2 T cells express a TCR containing a Vδ2 chain paired with a Vγ9 chain. Both Vδ2 and

Vγ9 genes are evolutionarily conserved in primates (75) and such a pairing only exists in humans and primates (76). The Vγ9Vδ2 T cells can rapidly acquire characteristics of professional APCs after activation (77). Vγ9Vδ2 T cells exhibit anti-microbial and anti-tumor properties (73), which make them a promising target for immunotherapies for a variety of diseases.

Structurally, Vγ9Vδ2 TCR has a smaller angle between Vγ and Cγ domains compared to αβ TCR (110° vs. 147°, Figure 24). The V domains of Vγ9Vδ2 TCR are generally similar to

αβ TCR; however the C domains are remarkably different, which may provide the molecular basis of a distinct recognition/signaling mechanism compared to αβ TCR.

Vγ9Vδ2 T cells in the Immune System

Figure 24. αβ TCR vs. Vγ9Vδ2 TCR. Figure modified from reference (78).

Activation of Vγ9Vδ2 T Cells by Phosphoantigens

Distinct from αβ T cells, γδ T cells can be activated in a MHC-independent manner. In humans, the activation of Vγ9Vδ2 T cells can be triggered by non-peptide phosphorous- containing small molecules also known as phosphoantigens. The Vγ9Vδ2 T cells are sensitive to the di-phosphorylated isoprenoid intermediates (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is produced by bacterial in the non-mevalonate pathway

(Figure 25), leading to Vγ9Vδ2 T cell anti-microbial functions. Cancer cells can produce another natural phosphoantigen, isopentenyl pyrophosphate (IPP), from the human mevalonate pathway (Figure 25). IPP can also activate Vγ9Vδ2 T cells resulting in rapid expansion and cytotoxicity against cancer cells.

Vγ9Vδ2 T cells in the Immune System

Figure 25. Isoprenoid biosynthesis in bacterial and human. Figure taken from reference (79).

Novel γδ T cells-based immunotherapeutical approaches have been proved promising in the treatment for a variety of diseases including cancers (73, 80). Here we summarized

Vγ9Vδ2 T cells in the Immune System

briefly the rationale of Vγ9Vδ2 T cells cells-based cancer immunotherapies (73, 80-83). First,

Vγ9Vδ2 T cells are capable of infiltrating tumors and accumulating at tumor sites. Secondly,

Vγ9Vδ2 T cells exhibit a potent cytotoxicity against a broad range of malignancies while sparing normal cells by secreting cytokines. Thirdly, the activation of γδ T cells can be triggered by exogenous supply of phosphoantigens or bioaccumulation of endogenous phosphoantigens (80).

A number of selective agonists of Vγ9Vδ2 T cells have been developed and are being tested under clinical trials for a variety of malignancies. As reviewed in reference (79), phosphoantigens to activate Vγ9Vδ2 T cells can be classified into two direct and indirect categories. Direct phosphoantigens, such as HMBPP and IPP and synthetic analogs from them, are able to directly bind to BTN3A1, a critical positive regulator in Vγ9Vδ2 T cell activation. In contrast, the mentality of using indirect phosphoantigens to activate Vγ9Vδ2 T cells is largely based on inhibition the human isoprenoid metabolism, particularly farnesyl diphosphate synthase, to elevate the level of the direct phosphoantigen of IPP (Figure 25).

Some FDA-approved bisphosphonates for the treatment of other diseases were found to have anti-tumor activities (84), which were believed to be associated with their antagonistic effect of Vγ9Vδ2 T cells. Zoledronate (Figure 26), a third-generation bisphosphonate drugs used for osteolysis and high blood calcium levels, is an effective farnesyl diphosphate synthase inhibitor (79). BrHPP, a synthetic analog of HMBPP, was found to trigger Vγ9Vδ2 T cell activation at nanomolar concentrations with minimal unspecific binding at micromolar concentrations (85). Zoledronate and BrHPP, representing indirect and direct phosphoantigens, respectively, have been evaluated in a handful clinical studies for a variety of cancers (79, 80). Although treatment Zoledronate or BrHPP with IL-2 have been shown to have good safety and some efficacy for certain malignancies, mixed results indicate phosphoantigens may have different stimulation patterns. Thus a better understanding of the

Vγ9Vδ2 T cells in the Immune System

underlying molecular mechanism how phosphoantigens cause activation of Vγ9Vδ2 T cells is undoubtedly crucial for future studies on phosphoantigens in Vγ9Vδ2 T cell-based immunotherapies.

Figure 26. Structure of zoledronate

Figure 27. Structure of BrHPP

Vγ9Vδ2 T cells in the Immune System

BTN3A1 in Activation of Vγ9Vδ2 T Cells

BTN3A1, the butyrophilin subfamily 3 member A1, is a B7 immunoglobulin superfamily protein. By far, 13 members of the butyrophilin family have been identified (86). Enriched content of butyrophilins was detected in milk lipid globules. Thus butyrophilins were originally believed to be important only in lactation and milk production (87). Due to their structural features of B7 superfmily of co-stimulatory molecules (Figure 28), and their expression in immune cells and cells closely interacting with immune cells, butyrophilines have been studied actively to explore their immunological functions as nicely reviewed in a recent reference (86).

B7 family members feature a protein architecture of extrcellular IgV and IgC domains, a transmembrane region and a short cytoplasmic tail (Figure 28). Butyrophilin and butyrophilin- like family members retain the same IgV-IgC extracellular structure but, distinct from other B7 family members, comprise a B30.2 (PRY/SPRY) intracellular domain following the transmembrane region (Figure 28).

Vγ9Vδ2 T cells in the Immune System

Figure 28. Protein architecture of B7 family and butyrophilin superfamilies. Figure taken from (86).

Recently, BTN3A1 has been identified as the binding partners for direct phosphoantigens (such as HMBPP and IPP), establishing its significance in Vγ9Vδ2 T cell activation. The fact that the direct interaction between BTN3A1 and phosphoantigens leads to the following Vγ9Vδ2 T cell activation has been recognized. Models of intracellular and extracellular binding of BTN3A1 to phosphoantigens have been proposed by different groups.

We used a phosphoantigen prodrug approach to demonstrate this strategy has superior potency in Vγ9Vδ2 T cell activation compared to other current phosphoantigens clinically used.

Furthermore, our biophysical data from NMR and ITC undoubtedly suggests an intracellular binding of BTN3A1 to phosphoantigens. With those findings, the underlying molecular mechanisms of phosphoantigen-induced Vγ9Vδ2 T cell activation has been advanced and a prodrug approach provides new possibilities for development of phosphoantigens for Vγ9Vδ2

T cell-based immunotherapies.

Do Phosphoantigens Bind to the Extracellular IgV Domain of BTN3A1?

Chapter 6 Do Phosphoantigens Bind to the Extracellular IgV Domain of

BTN3A1?

Critical review on reference (88)

The Extracellular Binding Model of IgV-Phosphoantigen

During the course of these studies, two groups reported binding of phosphoantigens to

BTN3A1 via conflicting mechanisms, extracellular vs. intracellular. Initially, De Libero and colleagues demonstrated extracellular binding (88), whereas a recent paper by Adams clearly showed intracellular binding (89). The exact binding site on BTN3A1 for phosphoantigens is a matter of debate. However, our biological and biophysical studies clearly suggest an intracellular interaction between BTN3A1 and phosphoantigens as also confirmed by other independent studies (Chapter 7).

De Libero and colleagues purposed a model where the extracellular IgV domain of

BTN3A1 binds phosphoantigens suggesting BTN3A1 functions as the antigen-presenting molecule for the activation of Vγ9Vδ2 T cells. In this study, biophysical evidence showing direct extracellular binding of BTN3A1 IgV domain to phosphoantigens are mainly from three methods: X-ray Crystallization, Mass spectroscopy and SPR analysis. Here is the argument that the results presented in the paper may be deceptive leading to the extracellular model.

Crystal Structures of IgV Complexed with Phosphoantigens

Two main structures, IgV-IPP (PDB entry: 4JKW) and IgV-HMBPP (PDB entry: 4K55), were determined by X-ray with a resolution of 2.0 Å. The protein did resemble an immunoglobulin V domain structure. However, after a closer investigation on the molecular

Do Phosphoantigens Bind to the Extracellular IgV Domain of BTN3A1?

details of the binding site, the electron density map shows an almost identical “donut” shape for both ligands, IPP and HMBPP. Both IPP and HMBPP have a structure distinct from a circular shape (Figure 32). Indeed, both ligands can only occupy half of the “donut” leaving a vacancy for the other half (Figure 29). This strongly suggests a poor fitting of the phosphoantigen onto the electron density map and leads to the question of what the ligand is exactly in the structures. Our hypothesis was that PEG molecules, the co-precipitant regent used for the crystallization, bind to the Lys39 residue of IgV domain. Another structure from

PDB clearly shows a PEG molecule can adopt a circular shape during crystallization and wrap itself around the side-chain of a Lys residue (Figure 30) (90, 91). Our speculations were confirmed by another independent research group (Figure 31) (89).

Figure 29. Electron density of BTN3A1 IgV-phosphoantigen structures constructed from PDB entries: 4JKW and 4K55 showing a poor fitting for both IPP and HMBPP

Do Phosphoantigens Bind to the Extracellular IgV Domain of BTN3A1?

Figure 30. PEG molecules in a circular shape with a Lysine residue in the center (PDB entry: 3FFR). Figure taken from Crystal structure of phosphoserine aminotransferase SerC (EC 2.6.1.52) (YP_677612.1) from CYTOPHAGA HUTCHINSONII ATCC 33406 at 1.75 A resolution (To be published).

Do Phosphoantigens Bind to the Extracellular IgV Domain of BTN3A1?

Figure 31. PEG molecules showing a better fitting than HMBPP (PDB entry: 4K55) and IPP (PDB entry: 4JKW). Figure taken from reference (89).

Mass Spectroscopy and SPR Analysis of the IgV-Phosphoantigen Interaction

In the investigation of the direct IgV-phosphoantigen interaction claimed by De Libero and colleagues (88), IPP and GDP (as a negative control) were chosen to perform the Mass

Spectroscopy study. Considering stronger affinity of HMBPP, it is surprising to see HMBPP was not tested for reasons not explicitly reported. The interaction between IgV and IPP with

1:1 stoichiometry was confirmed in an iso-trap mass spectrometer with electrospray ionization as the de-convoluted spectrum of IgV-IPP showed an increase of 244 Da which is close to the molecular mass of IPP, 246 Da. Considering that the Kd is in low-micromolar range, it is debatable if the non-covalently bound IPP can survive the ionization during the experiment.

Mass Spectroscopy may not be the perfect tool for this type of interactions as extreme cautions need to be taken in the search for the optimal conditions (92). In addition, few Mass

Spectroscopy data other than the de-convoluted spectra was provided to support the result.

The kinetics of IgV-IPP and IgV-HMBPP interactions were assessed using SPR by De

Libero and colleagues (88). A stronger binding was suggested for HMBPP than IPP, with association rate constants of 76.6 M-1s-1, 746.9 M-1s-1 and dissociation rate constants of 5.35

× 10-3 s-1, 2.28 × 10-3 s-1 for IPP and HMBPP, respectively. The equilibrium binding constants

(Kd) were calculated to be 69.6 µM for IPP and 3.06 µM for HMBPP. However, one of disadvantages of SPR is that it cannot effectively distinguish specific from non-specific bindings. Additionally, the SPR studies of ligand bindings of low molecular weight usually do not have a good sensitivity (93-95). Thus we argue that the confirmation of a specific and direct interaction between the extracellular IgV domain and phosphoantigens by SPR lacks credibility.

Do Phosphoantigens Bind to the Extracellular IgV Domain of BTN3A1?

In summary, the extracellular binding presented by De Libero and colleagues is unclear as all of three main biophysical experiments had noticeable flaws. Almost at the same time,

Adams group and our group published two independent studies. Both studies undoubtedly suggest an intracellular binding of BTN3A1 to phosphoantigens. Confirmed by other following independent studies (96), the intracellular binding model of BTN3A1 to phosphoantigens has been widely acknowledged. A better understanding of the interaction between BTN3A1 and phosphoantigens sets the right course for future studies on phosphoantigens in Vγ9Vδ2 T cell activation, and lays the groundwork for development of next-generation Vγ9Vδ2 T cell-based immunotherapies.

Intracellular Binding of BTN3A1 to Phosphoantigens

Chapter 7 Intracellular Binding of BTN3A1 to Phosphoantigens

Based on Hsiao, C.H.C., Lin, X., Barney, R.J., Shippy, R.R., Li, J., Vinogradova, O., Wiemer, D.F. and Wiemer, A.J., 2014. Synthesis of a phosphoantigen prodrug that potently activates Vγ9Vδ2 T-lymphocytes. Chemistry & biology, 21(8), pp.945-954.

A POM-protected Phosphoantigen is Highly Potent in Vγ9Vδ2 Stimulation.

Drugs of phosphates and phosphonates are being explored exceedingly in anticancer and antiviral therapy. Prodrugs of phosphates and phosphonates have significantly improved bioavailability, membrane permeability and plasma stability (97). Small and highly charged molecules (such as phosphoantigens) exhibit low cellular concentration due to the need for endocytic uptake. Prodrugs (such as POM-protected molecules) mask the negative charged groups ensuring a passive diffusion across the cell membrane. After the cellular entry, the

POM-protecting groups are removed by cellular esterases generating the functional phosphoantigens.

Compound 1a, a modest phosphoantigen (98), was chosen for our prodrug strategy due to its better chemical stability against phosphatase metabolism. A series of protected compounds (Figure 32) were synthesized and tested in human peripheral blood Vγ9Vδ2 T cell stimulation and the cell-based assay provided very exciting results (Table 7).

Intracellular Binding of BTN3A1 to Phosphoantigens

Figure 32. Chemical structures of phosphoantigens used in the study

Fold Fold IC50 Selectivity Compound EC50 (μM) difference difference (μM) index from HMBPP from 1a HMBPP 0.00051 1.0 NA 0.50 980 IPP 36 71000 NA ND ND Risedronate 1.6 3100 NA 61 38 1a 4.0 7800 1 ND ND 1b 0.0054 10 740 0.60 110 1c 0.50 980 8 8.6 17 1d > 10 > 19000 NA ND ND NA, not applicable; ND, not determined. Table 7. Activity of compounds for expansion of Vγ9Vδ2 T Cells from human peripheral blood mononuclear cells and inhibition of cell viability

Compound 1a showed a moderate potency in Vγ9Vδ2 T cell stimulation (Table 7). The dimethyl phosphonate, compound 1d, was predicted to be inactive for its metabolism stability

(99) and indeed was not able to stimulate Vγ9Vδ2 T cell (Table 7). Compound 1c is compound

1d with a labile POM group replacing one of the two methyl groups (Figure 32). The introduction of a POM group was able to restore the stimulatory effect and was more potent than compound 1a (Table 7). The double POM-protected or bis-POM phosphoantigen,

Intracellular Binding of BTN3A1 to Phosphoantigens

compound 1d, exhibited the highest potency among compounds 1a-1d and provided an EC50 of 0.0054 µM (Table 7). The results proved the concept of using a bis-POM phosphoantigen to achieve Vγ9Vδ2 T cell stimulation.

HMBPP, a potent direct phosphoantigen, showed an EC50 of 0.00051 µM which is the strongest in all the phosphoantigens tested. However, due to the highly-charged negative groups, HMBPP suffers from poor pharmacokinetic properties, rapid plasma clearance and low membrane permeability (100). Compared to HMBPP, our bis-POM phosphoantigens, compound 1b, showed a much higher potency in our 2-hr Vγ9Vδ2 T cell stimulation (Figure

33D). The fast stimulatory effect is believed to be a result from the enhanced uptake of compound 1b. Although less potent than HMBPP, compound 1b was still able to stimulate

Vγ9Vδ2 T cell at low nanomolar concentrations.

Figure 33. Bis-POM prodrug compound 1b functions as a direct phosphoantigen with elevated potency relative to compound 1a and HMBPP. * indicates statistical significance.

Risedronate and other indirect phosphoantigens are known to activate Vγ9Vδ2 T cell by inhibiting farnesyl diphosphate synthase in the human isoprenoid metabolism (Figure 25).

The resulted cellular accumulation of IPP, the natural direct antigen, induces stimulation of

Intracellular Binding of BTN3A1 to Phosphoantigens

Vγ9Vδ2 T cells. To prove that our bis-POM compound 1b works as a direct phosphoantigen, compound 1b was compared with Risedronate in Vγ9Vδ2 T cell stimulation with treatment of lovastatin (Figure 33C). Lovastatin inhibits 3-hydroxy-3-methylglutaryl-coenzyme A reductase upstream of farnesyl diphosphate synthase, and counters the induced accumulation of IPP by indirect phosphoantigens such as Risedronate. The stimulated Vγ9Vδ2 T cell proliferation by compound 1b was not decreased by treatment of lovastatin, which notably abated the stimulatory effect of Risedronate. This study, and the biophysical evidence for a direct intracellular between BTN3A1 and compound 1a, prove our hypothesis that our bis-POM compound 1b is a direct-acting phosphoantigen.

The BTN3A1 PRY/SPRY (B30.2) Domain Directly Binds Phosphoantigens.

It recently was reported that BTN3A1 is required for phosphoantigen detection by

Vγ9Vδ2 T cells (88, 101, 102). However, the exact region of BTN3A1 where phosphoantigens bind is a matter of debate (88, 101). Based on the 740x increase in activity of the bis-POM prodrug, we hypothesized that intracellular binding occurs, which is in contrast to the model of extracellular binding to the immunoglobulin-like V (IgV) (88). To determine whether the

BTN3A1-antigen complex forms intracellularly or extracellularly, we expressed the recombinant extracellular IgV domain, the intracellular PRY/SPRY (B30.2) domain, and the full intracellular domain (BFI) and purified the soluble proteins. First, we verified the conformations of the constructs by NMR, and, according to the nicely dispersed 1H-15NHeteronuclear Single

Quantum Correlation (HSQC) spectra, all the recombinant proteins are well folded and remain in monomeric state. Most of the amide resonance frequencies for PRY/SPRY domain overlapped with the peaks in the spectra for BFI (presented overlaid in Figure 34). This suggests the structure of PRY/SPRY domain in isolated form and in the context of the full-

Intracellular Binding of BTN3A1 to Phosphoantigens

length protein remains the same and that the 68-residue membrane proximal region does not interact with the PRY/SPRY domain intramolecularly.

Figure 34. Evidence for the absence of major 18 intramolecular interactions between the 68-residue membrane-proximal region and PRY/SPRY domain: HSQC spectra of BFI (black) and PRY/SPRY (gray)

We performed ITC to measure the interactions between these three BTN3A1 constructs and HMBPP (Figure 35A). While no binding was observed with the extracellular

BTN3A1 IgV domain, both the intracellular PRY/SPRY (B30.2) domain and the BFI domain showed strong binding with dissociation constants (Kd) of 1.61 μM and 1.54 μM, respectively.

The interaction of HMBPP with BTN3A1 was enthalpy-driven with an unfavorable entropy change. HMBPP binding to the full intracellular domain (ΔH = -51.9 kJ/mol and TΔS = -18.7 kJ/mol) and the PRY/SPRY domain (ΔH = -49.4 kJ/mol and TΔS = -16.4 kJ/mol) shares very similar enthalpy and entropy terms, which reveals the binding site on BTN3A1 for phosphoantigens is enclosed in the PRY/SPRY domain (Table 8). 1H-15N HSQC titrations of

Intracellular Binding of BTN3A1 to Phosphoantigens

HMBPP agreed with the ITC results. While there were no chemical shift perturbations in the spectrum for the IgV domain upon HMBPP titration (Figure 36) the effects from HMBPP on the spectra of BFI and PRY/SPRY were very pronounced. HMBPP-concentration-dependent chemical shift perturbations were observed in the spectra for both BFI (Figure 37A) and the

PRY/SPRY domain (Figure 37B) and reached saturation at an HMBPP:protein ratio of 3:1.

Combining ITC and NMR data, we have demonstrated that HMBPP interacts with BTN3A1 at its intracellular domain at 1:1 ratio and is likely capable of inducing significant conformational rearrangements in the intracellular domain of BTN3A1.

Figure 35. Binding of HMBPP and Compound 1a to the Intracellular Domain of BTN3A1. (A) Isothermal titration calorimetry plots for the interaction of HMBPP with the extracellular IgV domain (gray line/dots), the full intracellular domain (BFI) (black line/dots), or the isolated PRY/SPRY (B30.2) domain (red line/dots). (B) Isothermal titration calorimetry plots for the interaction of the full intracellular domain (BFI) of BTN3A1 with HMBPP in the absence of compound 1a (red line/dots), or the presence of 10x compound 1a (gray line/dots), or 100x compound 1a (blue line/dots).

Intracellular Binding of BTN3A1 to Phosphoantigens

K ΔH TΔS ΔG Protein or protein-ligand complex Ligand d µM kJ/mol kJ/mol kJ/mol IgV No binding

PRY/SPRY HMBPP 1.61 -49.4 -16.4 -33.0

BFI 1.54 -51.9 -18.7 -33.2

1:10 1.70 -49.1 -16.2 -32.9 BFI + Compound 1a HMBPP 1:100 1.26 -28.3 5.33 -33.6

1:10 1.15 -46.6 -12.7 -33.9 BFI + IPP HMBPP 1:100 2.55 -38.7 -6.78 -31.9

BFI C-HDMAPP 1.95 -47.1 -14.5 -32.6 Table 8. Summary of the thermodynamic data derived from the ITC measurements using different ligands

Figure 36. Evidence for the absence of interaction between HMBPP and the extracellular IgV domain of BTN3A1. HSQC of spectra of free IgV domain (black), IgV + HMBPP at 1:1 protein-to-compound ratio (blue) and IgV + HMBPP at 1:3 protein-to-compound ratio (red). The three spectra overlap perfectly indicating no interaction between HMBPP and the IgV domain.

Intracellular Binding of BTN3A1 to Phosphoantigens

Figure 37. 1H-15N HSQC titration of HMBPP into the full-length intracellular domain and the PRY/SPRY domain of BTN3A1. Compared to the PRY/SPRY domain, the full-length intracellular domain of BTN3A1 (BFI) includes an additional membrane-proximal region of 68 residues at the N-terminus, which is demonstrated to be involved in the interaction with HMBPP and to undergo significant structural changes upon binding. (A) Chemical shift perturbations

Intracellular Binding of BTN3A1 to Phosphoantigens

for the interaction of HMBPP and BFI at ratios of 0:1 (black), 1:1 (blue), 3:1 (red). (B) Chemical shift perturbations for the interaction of HMBPP and the PRY/SPRY domain at ratios of 0:1 (gray), 1:1 (orange), 3:1 (green). (C) Evidence for the involvement of the 68-residues membrane-proximal region in interaction with HMBPP: HSQC spectra of the free BFI (black), BFI + HMBPP at 1:3 ratio (red) and PRY/SPRY + HMBPP at 1:3 ratio (green). Peaks which belong to the 68-residue membrane-proximal region are absent in the spectrum of PRY/SPRY domain. Arrows indicate the peaks corresponding to the amides from this region that have showed remarkable shifts upon binding to HMBPP, thus suggesting significant structural rearrangements.

To determine whether binding was specific to HMBPP or a general property of phosphoantigens, we also tested whether another potent phosphoantigen, C-HDMAPP, could bind to internal domain (Figure 38). C-HDMAPP is a phosphonate analog of HBMPP (98).

Interestingly, C-HDMAPP exhibited similar energetics relative to HMBPP when binding to the

PRY/SPRY domain (Kd = 1.95 μM, ΔH = -47.1 kJ/mol, TΔS = -14.5 kJ/mol). The data reveals that both HMBPP and C-HDMAPP bind with high affinity to the intracellular PRY/SPRY (B30.2) domain but not the extracellular V domain. C-HDMAPP has a slightly weaker interaction which is consistent with its activity in cellular assays (98). Therefore, we have demonstrated that two different phosphoantigens bind strongly to the intracellular domain of BTN3A1.

Intracellular Binding of BTN3A1 to Phosphoantigens

Figure 38. Binding of C-HDMAPP to the BTN3A1 full intracellular domain

Because IPP also acts as a direct phosphoantigen in cellular assays, we tested its ability to interact with BTN3A1. However, we were unable to measure directly the binding of

IPP to BTN3A1 by ITC. This is perhaps not surprising, given that the activity in cellular assays is approximately 71,000 fold less for IPP relative to HMBPP (Table 8) making the projected dissociation constant well outside of the detection limit of ITC. Therefore, we performed a competition ITC study to determine whether increasing concentrations of IPP would affect the binding of HMBPP to BTN3A1 (Figure 39). IPP dose-dependently decreased the favorable enthalpy of HMBPP binding from a ΔH of -51.9 kJ/mol to -47.7 kJ/mol and -38.7 kJ/mol at

IPP:BTN3A1 ratios of 10:1 and 100:1, respectively (Table 8). IPP also dose-dependently made more favorable the TΔS term of HMBPP binding, changing it from -18.7 kJ/mol to -12.7 kJ/mol and -6.78 kJ/mol at IPP:BTN3A1 ratios of 10:1 and 100:1 respectively. Taken together, the data show that IPP binds to BTN3A1 with a much weaker affinity compared to HMBPP and that both compounds compete for the same binding site within the PRY/SPRY domain.

Intracellular Binding of BTN3A1 to Phosphoantigens

Figure 39. Binding of HMBPP to the BTN3A1 full intracellular domain in the presence of IPP (no IPP, red line; IPP:BTN3A1=10:1, gray line; IPP:BTN3A1=100:1, blue line)

Because compound 1b is expected to undergo metabolic cleavage, resulting in accumulation of compound 1a, we examined the binding affinity between compound 1a and the BTN3A1 PRY/SPRY domain. Like IPP, the binding affinity of 1a was undetectable in ITC when using compound 1a as the sole ligand, as we had purposely chosen a weak phosphoantigen for the prodrug studies. However, as expected, compound 1a had a markedly stronger effect on the HMBPP binding to the PRY/SPRY domain relative to IPP (Figure 35).

Like IPP, compound 1a dose-dependently decreased the favorable enthalpy of HMBPP binding to BTN3A1 (Table 8). These effects also were measured at 1a:BTN3A1 ratios of

0:1(ΔH of -51.9 kJ/mol), 10:1 (-49.1 kJ/mol) and 100:1 (-28.5 kJ/mol). Compound 1a also dose- dependently made more favorable the TΔS term of HMBPP binding at IPP:BTN3A1 ratios of

10:1 (-16.2 kJ/mol) and 100:1 (+5.19 kJ/mol). These data fit well with the cellular data in that

1a is a more potent phosphoantigen relative to IPP and would be expected to have a stronger effect. Perhaps surprisingly, the entropy term became positive at the highest concentration of

1a. This finding demonstrates that the prenyl alcohol and/or the olefin position play a more important role in the phosphoantigen-BTN3A1 interaction as compared to the distal phosphate group. Thus, according to ITC data, compound 1a may induce conformational changes in the intracellular domain of BTN3A1 similar to the ones induced by the stronger interaction with

HMBPP and absent in the interface with IPP.

To confirm that IPP and compound 1a do have direct interactions with the intracellular domain of BTN3A1, 1H-15N HSQC titrations of IPP and compound 1a into BFI were performed at ligand:protein molar ratios of 0:1, 10:1 and 100:1. Both IPP (Figure 40A) and compound 1a

(Figure 40B) showed concentration-dependent perturbations in the spectra. These two sets of titration data demonstrate that both IPP and compound 1a bind to BFI at a similar binding site with only a limited number of distinct differences in amide chemical shift perturbations between

Intracellular Binding of BTN3A1 to Phosphoantigens

the two. In addition, the spectrum of compound 1a, but not IPP, shows several chemical shift perturbations identical to the ones observed in the titration of HMBPP (marked by arrows on

Figure 40C). Thus our NMR and ITC results are mutually consistent and suggest that binding sites for IPP and compound 1a on the PRY/SPRY domain are quite similar, although each compound is also involved in unique interactions with BTN3A1.

Intracellular Binding of BTN3A1 to Phosphoantigens

Figure 40. 1H-15N HSQC titration of IPP and compound 1a into the full-length intracellular domain of BTN3A1 (BFI). (A) Chemical shift perturbations for the interaction of IPP and BFI at ratios of 0:1 (black), 10:1 (blue), 100:1 (red). (B) Chemical shift perturbations for the interaction of compound 1a and BFI at ratios of 0:1 (gray), 10:1 (orange), 100:1 (green). (C) Comparison of BFI chemical shifts and in the presence of IPP (red) and compound 1a (green) at 100:1 compound-to-protein ratio (the spectrum of BFI in free form shown in black). Arrows indicate chemical shift perturbations found for compound 1a, but not IPP, which are identical to the ones observed in the titration of HMBPP.

The Intracellular Domain of BTN3A1 Undergoes Conformational Changes after

Binding to Phosphoantigens.

Adams group was able to determine the structures of BTN3A1 B30.2 domain in free and bound forms with a resolution of 1.4 Å (89). The structures revealed a basic pocket for phosphoantigen binding and mutations of key residues from positive to negative completely ablated phosphoantigen binding. The phosphoantigen used for crystallization structural determination was cHDMAPP, which showed a similar thermodynamic profile as HMBPP

(Table 8) in our ITC experiments, which is also consistent with their results.

Surprisingly, the free B30.2 domain and the B30.2-cHDMAPP complex are almost identical structurally as the phosphoantigen was not able to induce any noticeable conformational changes in BTN3A1 (Figure 41). This leads to a question: what is the molecular mechanism connecting the phosphoantigen binding to the downstream signaling then to the eventual activation of Vγ9Vδ2 T cells?

Intracellular Binding of BTN3A1 to Phosphoantigens

Figure 41. Structural comparison between free BTN3A1 B30.2 domain (gray, PDB entry: 4N7I) and B30.2-cHDMAPP complex (green, PDB entry: 4N7U)

The unobserved structural changes in B30.2 domain may be explained by the BTN3A1 construct used in the study. Compared to our BFI construct, the B30.2 domain is truncated at the N-terminus and lacks the 68-residue juxtamembrane (JM) region. Our 1H-15N HSQC titration data indicate the involvement of the 68-residue JM region in interactions with phosphoantigens (Figure 37). This evidence indirectly proves the significance of the JM region in the probable structural rearrangements of intracellular BTN3A1. As the JM region may be the harbor for cellular binding partners such as periplakin (96), biologically and functionally, it is reasonable to speculate the JM region can be switched from “inactive” form to “active” form upon binding of phosphoantigens. Another possibility is that the JM region interacts with the cell membrane, which enables BTN3A1 to have an “inside-out” signaling like integrins. In this scenario, the intracellular binding of BTN3A1 to phosphoantigens causes a change in binding

Intracellular Binding of BTN3A1 to Phosphoantigens

affinity of the extracellular domain to its unknown counter-partners. It is plausible since the ectodomain of BTN3A1 seems to dimerize in two different modes, IgC-to-IgC and IgV-to-IgC

(103).

To further investigate the potential structural changes in intracellular BTN3A1 especially the JM region, we performed SAXS analysis on BFI in absence and presence of

HMBPP. Our initial results did suggest changes in the overall shape of BFI. The shape of BFI can be viewed as a body part and a head part connected by a short neck region. We suspect the body part encompasses the B30.2 domain due to size similarity in comparison to structure determined by X-ray. (89). We also believe the JM region forms the head and neck and extends to the body part. The most pronounced change in shape upon HMBPP binding occurs in the neck region, where a narrowing is observed. In addition, there are other distinctions between the free and bound structures, which are difficult to describe at the moment due to the low- resolution of the models. Overall, the SAXS results are consistent with our hypothesis of

BTN3A1 intracellular conformational changes and warrant further structural investigations of intracellular BTN3A1, especially the JM region.

Intracellular Binding of BTN3A1 to Phosphoantigens

Figure 42. Structures of BFI with (green) and without (gray) HMBPP by SAXS

Discussion

During the course of these studies, two groups reported binding of phosphoantigens to

BTN3A1 via conflicting mechanisms (88, 89). Initially, di Libero and colleagues demonstrated extracellular binding whereas a recent paper by Adams clearly showed intracellular binding.

Our studies are in strong agreement with those from the Adams lab. In addition to the calorimetry assay, we also see strong interactions as assessed by NMR spectroscopy. Our results also demonstrate for the first time the relative contribution of the prenyl alcohol group and/or olefin position is more important that the second phosphate group, because compound

1a affects the binding of HMBPP to BTN3A1 to a much greater extent than IPP, which lacks the alcohol functional group. Additionally, we have compared the effects of different antigen binding on the full intracellular domain and the PRY/SPRY domain by NMR. Our results indicate that although the binding site for phosphoantigens is localized in PRY/SPRY domain, the 68-residue membrane proximal region, preceding PRY/SPRY, also undergoes major structural rearrangement upon binding (Figure 37C). These findings clearly promote a model in which intracellular binding of phosphoantigens to BTN3A1 induces a conformational change, which either propagates across the membrane to the extracellular region or promotes an interaction with another intracellular protein thus allowing new transmembrane complex formation necessary for recognition by the Vγ9Vδ2 T cell receptor.

Intracellular Binding of BTN3A1 to Phosphoantigens

Research Design and Methods

Cloning and Purification of BTN3A1 Constructs

BTN3A1 constructs were purified based on the method that De Libero and colleagues

(88) used for the IgV domain with some modifications. Amplicons were obtained from THP-1 cDNA using the following primers in Table 9.

Construct Forward Primer Reverse Primer GCT ATC CAT ATG TCA GCT ATC CTC GAG AAC IgV GCT CAG TTT TCT GTG CTT CAG CTC CAC CAG GCT ATC CAT ATG CAA GCT ATC CTC GAG CGC BFI CAG CAG GAG GAA AAA TGG ACA AAT AGT CAG GCT ATC CAT ATG GCG GCT ATC CTC GAG CGC PRY/SPRY GAT GTG ATT CTG GAT TGG ACA AAT AGT CAG Table 9. Primers used for BTN3A1 constructs

Each forward primer contained an NdeI site while each reverse primer contained an

XhoI site. Following amplification and digest, sequences were cloned into the NdeI and XhoI sites in pET21b+, resulting in a 6x His-tagged construct. Each construct was validated by sequencing using the standard T7 forward and T7 reverse primers. The plasmids were transformed into BL21(DE) E. coli. The cells were grown in LB broth until OD600 reached 0.6 at which point the overexpression was induced by 1 mM IPTG. Cells were lysed by French

Press and the cell debris was removed by centrifugation at 18,000 rpm at 4 °C. The supernatant containing the soluble target protein was collected for further purification. The C- terminal His-tag fused protein was purified under native conditions with Ni-NTA resin (QIAGEN) using the standard protocol followed by a buffer exchange to 50 mM Tris, pH 7.5, 100 mM

NaCl, 5 mM β-mercaptoethanol (BME) in a Superdex-75 column (GE Healthcare) where all three recombinant proteins showed a proper folding in monomeric state. The overexpression of the target protein and the purified protein were confirmed by SDS-PAGE and bands of the expected mass were observed.

Intracellular Binding of BTN3A1 to Phosphoantigens

ITC Experiments

Calorimetric measurements of BTN3A1 domains to various compounds were performed on a low volume Nano ITC (TA Instruments). All experiments were carried out in a buffer of 50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM BME at 25 °C with a stirring speed of 200 rpm to minimize the precipitation of the protein. Each titration consisted of twenty 2.5-µl injections with 300 second time intervals. The concentrations of protein samples were determined by UV and the titrant of the compounds were prepared in the same buffer from the dilution of 10 mM or 100 mM stock. The concentrations of the protein ranged from 30 μM to 60

μM. The concentration of protein was examined after each ITC experiment and a good solubility (> 85%) of the protein under this condition was shown. The analysis of the data was done in NanoAnalyze Software (TA Instruments) suite using an “independent” model. In all cases, a stoichiometry of 1 ± 0.1 was revealed.

15N-HSQC Titration by NMR

15N-labelled recombinant proteins were overexpressed in M9 medium supplemented

15 with NH4Cl as the sole nitrogen source. Same purification procedures as described above were followed. NMR samples of 100-200 µM were prepared in a buffer of 50 mM Tris, pH 7.5,

1 15 100 mM NaCl, 5 mM BME, 7% D2O, 1 mM DSS as the internal standard. H- N Heteronuclear

Single Quantum Correlation (HSQC) titration experiments were performed on Varian Inova

600 MHz spectrometer equipped with inverse-triple resonance cyroprobe at 25 °C. All spectra were processed with NMRPipe (104) and analyzed in CcpNmr Analysis software suite (67).

Author Contributions

C-H.C.H. and A.J.W. performed molecular biology and cell-based experiments. R.J.B. and R.R.S. performed the chemical synthesis under supervision of D.F.W. Protein purification,

Intracellular Binding of BTN3A1 to Phosphoantigens

ITC, and HSQC NMR were performed by X.L. under supervision of O.V. Cell viability experiments were performed by J.L. The manuscript was written by C-H.C.H. and A.J.W. with the support of X.L, O.V., and D.F.W.

Conclusions

Chapter 8 Conclusions

In summary, the above studies on p52 Shc and BTN3A1 demonstrate how ITC and

NMR can be utilized to characterize protein interactions.

Chapter 1 and Chapter 2 serve as a brief overview of the two biophysical techniques.

Chapter 3 discusses the significance of p52 Shc in MAPK and PI3K/AKT pathways. In

Chapter 4, we presented a study on phospho-Tyrosine(s) vs. phosphatidylinositol binding in p52 Shc-mediated integrin signaling. p52 Shc has been identified as a primary signaling partner for the tyrosine(s)-phosphorylated cytoplasmic tails of activated β3 integrins. Inspired by our recent structure of the Shc PTB domain in complex with a bi-phosphorylated peptide derived from β3 cytoplasmic tail, we have initiated the investigation of Shc interaction with phospholipids of the membrane. We are particularly focused on PtdIns and their effects on Shc mediated integrin signaling in vitro. Here we present thermodynamic profiles and molecular details of the interactions between Shc, integrin, and PtdIns, all of which have been studied by

ITC and solution NMR methods. A model of p52 Shc interaction with phosphorylated β3 integrin cytoplasmic tail at the cytosolic face of the plasma membrane is proposed based on these data.

Chapter 5 introduces the importance of the Vγ9Vδ2 T cells in human immune system and highlights the significance of BTN3A1 in the phosphoantigen-induced Vγ9Vδ2 T cell activation. Chapter 6 is a discussion why the extracellular binding of BTN3A1 to phosphoantigens is unlikely. In Chapter 7, we demonstrate high affinity interaction between phosphoantigens and the intracellular domain of BTN3A1: while the PRY/SPRY (B30.2) domain forms the binding pocket, perturbations in the JM region were also observed upon this interaction. In addition, our cell-based assay proves a prodrug approach may represent the future direction of phosphoantigen development in clinical Vγ9Vδ2 T cell-based immunotherapies.

References

1. Leavitt S, Freire E. Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr Opin Struct Biol. 2001;11(5):560-6.

2. Freire E, Mayorga OL, Straume M. Isothermal titration calorimetry. Anal Chem. 1990;62(18):950A-9A.

3. Wiseman T, Williston S, Brandts JF, Lin L. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem. 1989;179(1):131-7.

4. Jelesarov I, Bosshard HR. Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition. Journal of molecular recognition. 1999;12(1):3-18.

5. Qin J, Vinogradova O, Gronenborn AM. [18]-Protein–protein interactions probed by nuclear magnetic resonance spectroscopy. Meth Enzymol. 2001;339:377-89.

6. Ni F, Scheraga HA. Use of the transferred nuclear Overhauser effect to determine the conformations of ligands bound to proteins. Acc Chem Res. 1994;27(9):257-64.

7. Brunner E. Residual dipolar couplings in protein NMR. Concepts Magn Reson. 2001;13(4):238-59.

8. Kleckner IR, Foster MP. An introduction to NMR-based approaches for measuring protein dynamics. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics. 2011;1814(8):942- 68.

9. Clore GM, Gronenborn AM. Determining the structures of large proteins and protein complexes by NMR. Trends Biotechnol. 1998;16(1):22-34.

10. Arora A, Abildgaard F, Bushweller JH, Tamm LK. Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nature Structural & Molecular Biology. 2001;8(4):334-8.

11. Williamson M, Asakura T. Empirical comparisons of models for chemical-shift calculation in proteins. Journal of Magnetic Resonance, Series B. 1993;101(1):63-71.

12. Wishart DS, Case DA. Use of chemical shifts in macromolecular structure determination. Meth Enzymol. 2002;338:3-34.

13. Shen Y, Lange O, Delaglio F, Rossi P, Aramini JM, Liu G, et al. Consistent blind protein structure generation from NMR chemical shift data. Proceedings of the National Academy of Sciences. 2008;105(12):4685-90.

14. Shen Y, Vernon R, Baker D, Bax A. De novo protein structure generation from incomplete chemical shift assignments. J Biomol NMR. 2009;43(2):63-78.

References

15. Wuthrich K. NMR of proteins and nucleic acids. Wiley; 1986.

16. Melanie K, Nina J. Teaching an old dogma new tricks: twenty years of Shc adaptor signalling. Biochem J. 2012;447(1):1-16.

17. Ravichandran KS. Signaling via Shc family adapter proteins. Oncogene. 2001 Oct 1;20(44):6322-30.

18. Sweet DT, Tzima E. Spatial signaling networks converge at the adaptor protein Shc. Cell cycle. 2009;8(2):231-5.

19. Lee M, Igawa T, Chen S, Van Bemmel D, Lin JS, Lin F, et al. p66Shc protein is upregulated by steroid hormones in hormone‐sensitive cancer cells and in primary prostate carcinomas. International journal of cancer. 2004;108(5):672-8.

20. Ursini-Siegel J, Muller WJ. The ShcA adaptor protein is a critical regulator of breast cancer progression. Cell Cycle. 2008;7(13):1936-43.

21. Ursini-Siegel J, Hardy W, Zheng Y, Ling C, Zuo D, Zhang C, et al. The ShcA SH2 domain engages a 14-3-3/PI3′ K signaling complex and promotes breast cancer cell survival. Oncogene. 2012;31(48):5038-44.

22. Ursini-Siegel J, Muller WJ. The ShcA adaptor protein is a critical regulator of breast cancer progression. Cell Cycle. 2008;7(13):1936-43.

23. Suen KM, Lin C, George R, Melo FA, Biggs ER, Ahmed Z, et al. Interaction with Shc prevents aberrant Erk activation in the absence of extracellular stimuli. Nature structural & molecular biology. 2013;20(5):620-7.

24. Gu H, Maeda H, Moon JJ, Lord JD, Yoakim M, Nelson BH, et al. New role for Shc in activation of the phosphatidylinositol 3-kinase/Akt pathway. Mol Cell Biol. 2000 Oct;20(19):7109-20.

25. Zhou M, Ravichandran KS, Olejniczak ET, Petros AM, Meadows RP, Sattler M, et al. Structure and ligand recognition of the phosphotyrosine binding domain of Shc. . 1995.

26. Songyang Z, Shoelson SE, McGlade J, Olivier P, Pawson T, Bustelo XR, et al. Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. Mol Cell Biol. 1994 Apr;14(4):2777-85.

27. van der Geer P, Wiley S, Gish GD, Pawson T. The Shc adaptor protein is highly phosphorylated at conserved, twin tyrosine residues (Y239/240) that mediate protein–protein interactions. Current Biology. 1996;6(11):1435-44.

28. Sato K, Gotoh N, Otsuki T, Kakumoto M, Aoto M, Tokmakov AA, et al. Tyrosine residues 239 and 240 of Shc are phosphatidylinositol 4, 5-bisphosphate-dependent phosphorylation sites by c-Src. Biochem Biophys Res Commun. 1997;240(2):399-404.

References

29. Salcini A, McGlade J, Pelicci G, Nicoletti I, Pawson T, Pelicci P. Formation of Shc-Grb2 complexes is necessary to induce neoplastic transformation by overexpression of Shc proteins. . 1994.

30. Pelicci G, Dente L, De Giuseppe A, Verducci-Galletti B, Giuli S, Mele S, et al. A family of Shc related proteins with conserved PTB, CH1 and SH2 regions. Oncogene. 1996 Aug 1;13(3):633-41.

31. Legate KR, Fassler R. Mechanisms that regulate adaptor binding to beta-integrin cytoplasmic tails. J Cell Sci. 2009 Jan 15;122(Pt 2):187-98.

32. Cowan KJ, Law DA, Phillips DR. Identification of shc as the primary protein binding to the tyrosine-phosphorylated beta 3 subunit of alpha IIbbeta 3 during outside-in integrin platelet signaling. J Biol Chem. 2000 Nov 17;275(46):36423-9.

33. Higashi T, Yoshioka A, Shirakawa R, Tabuchi A, Nishioka H, Kita T, et al. Direct demonstration of involvement of the adaptor protein ShcA in the regulation of Ca 2 -induced platelet aggregation. Biochem Biophys Res Commun. 2004;322(2):700-4.

34. Deshmukh L, Gorbatyuk V, Vinogradova O. Integrin {beta}3 phosphorylation dictates its complex with the Shc phosphotyrosine-binding (PTB) domain. J Biol Chem. 2010 Nov 5;285(45):34875-84.

35. Hennessy BT, Smith DL, Ram PT, Lu Y, Mills GB. Exploiting the PI3K/AKT pathway for cancer drug discovery. Nature reviews Drug discovery. 2005;4(12):988-1004.

36. Yaffe MB. How do 14-3-3 proteins work?–Gatekeeper phosphorylation and the molecular anvil hypothesis. FEBS Lett. 2002;513(1):53-7.

37. Barry EF, Felquer FA, Powell JA, Biggs L, Stomski FC, Urbani A, et al. 14-3-3:Shc scaffolds integrate phosphoserine and phosphotyrosine signaling to regulate phosphatidylinositol 3-kinase activation and cell survival. J Biol Chem. 2009 May 1;284(18):12080-90.

38. Neal CL, Xu J, Li P, Mori S, Yang J, Neal NN, et al. Overexpression of 14-3-3ζ in cancer cells activates PI3K via binding the p85 regulatory subunit. Oncogene. 2012;31(7):897-906.

39. Vinogradova O, Velyvis A, Velyviene A, Hu B, Haas TA, Plow EF, et al. A structural mechanism of integrin α IIb β 3 “inside-out” activation as regulated by its cytoplasmic face. Cell. 2002;110(5):587-97.

40. Vinogradova O, Vaynberg J, Kong X, Haas TA, Plow EF, Qin J. Membrane-mediated structural transitions at the cytoplasmic face during integrin activation. Proc Natl Acad Sci U S A. 2004 Mar 23;101(12):4094-9.

41. Deshmukh L, Gorbatyuk V, Vinogradova O. Integrin {beta}3 phosphorylation dictates its complex with the Shc phosphotyrosine-binding (PTB) domain. J Biol Chem. 2010 Nov 5;285(45):34875-84.

References

42. Deshmukh L, Meller N, Alder N, Byzova T, Vinogradova O. Tyrosine phosphorylation as a conformational switch: a case study of integrin beta3 cytoplasmic tail. J Biol Chem. 2011 Nov 25;286(47):40943-53.

43. Schaffner-Reckinger E, Gouon V, Melchior C, Plançon S, Kieffer N. Distinct involvement of β3 integrin cytoplasmic domain tyrosine residues 747 and 759 in integrin-mediated cytoskeletal assembly and phosphotyrosine signaling. J Biol Chem. 1998;273(20):12623-32.

44. Jenkins AL, Nannizzi-Alaimo L, Silver D, Sellers JR, Ginsberg MH, Law DA, et al. Tyrosine phosphorylation of the β3 cytoplasmic domain mediates integrin-cytoskeletal interactions. J Biol Chem. 1998;273(22):13878-85.

45. Phillips DR, Prasad KS, Manganello J, Bao M, Nannizzi-Alaimo L. Integrin tyrosine phosphorylation in platelet signaling. Curr Opin Cell Biol. 2001;13(5):546-54.

46. Law DA, DeGuzman FR, Heiser P, Ministri-Madrid K, Killeen N, Phillips DR. Integrin cytoplasmic tyrosine motif is required for outside-in αIIbβ3 signalling and platelet function. Nature. 1999;401(6755):808-11.

47. Cowan KJ, Law DA, Phillips DR. Identification of shc as the primary protein binding to the tyrosine-phosphorylated beta 3 subunit of alpha IIbbeta 3 during outside-in integrin platelet signaling. J Biol Chem. 2000 Nov 17;275(46):36423-9.

48. Higashi T, Yoshioka A, Shirakawa R, Tabuchi A, Nishioka H, Kita T, et al. Direct demonstration of involvement of the adaptor protein ShcA in the regulation of Ca 2 -induced platelet aggregation. Biochem Biophys Res Commun. 2004;322(2):700-4.

49. Pelicci G, Lanfrancone L, Grignani F, McGlade J, Cavallo F, Forni G, et al. A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell. 1992;70(1):93-104.

50. Ravichandran KS. Signaling via Shc family adapter proteins. Oncogene. 2001;20(44).

51. Rameh LE, Chen C, Cantley LC. Phosphatidylinositol (3, 4, 5) P 3 interacts with SH2 domains and modulates PI 3-kinase association with tyrosine-phosphorylated proteins. Cell. 1995;83(5):821-30.

52. Balla T. Inositol-lipid binding motifs: signal integrators through protein-lipid and protein- protein interactions. J Cell Sci. 2005 May 15;118(Pt 10):2093-104.

53. Zhou M, Ravichandran KS, Olejniczak ET, Petros AM, Meadows RP, Sattler M, et al. Structure and ligand recognition of the phosphotyrosine binding domain of Shc. . 1995.

54. DiNitto JP, Lambright DG. Membrane and juxtamembrane targeting by PH and PTB domains. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids. 2006;1761(8):850-67.

55. Farooq A, Zhou M. PTB or not to be: promiscuous, tolerant and Bizarro domains come of age. IUBMB Life. 2004;56(9):547-57.

References

56. Farooq A, Plotnikova O, Zeng L, Zhou MM. Phosphotyrosine binding domains of Shc and insulin receptor substrate 1 recognize the NPXpY motif in a thermodynamically distinct manner. J Biol Chem. 1999 Mar 5;274(10):6114-21.

57. Zhou MM, Harlan JE, Wade WS, Crosby S, Ravichandran KS, Burakoff SJ, et al. Binding affinities of tyrosine-phosphorylated peptides to the COOH-terminal SH2 and NH2-terminal phosphotyrosine binding domains of Shc. J Biol Chem. 1995 Dec 29;270(52):31119-23.

58. Ravichandran KS, Zhou MM, Pratt JC, Harlan JE, Walk SF, Fesik SW, et al. Evidence for a requirement for both phospholipid and phosphotyrosine binding via the Shc phosphotyrosine-binding domain in vivo. Mol Cell Biol. 1997 Sep;17(9):5540-9.

59. George R, Schuller AC, Harris R, Ladbury JE. A phosphorylation-dependent gating mechanism controls the SH2 domain interactions of the Shc adaptor protein. J Mol Biol. 2008;377(3):740-7.

60. Vinogradova O, Qin J. NMR as a unique tool in assessment and complex determination of weak protein–protein interactions. In: NMR of Proteins and Small Biomolecules. Springer; 2011. p. 35-45.

61. Perozzo R, Folkers G, Scapozza L. Thermodynamics of protein–ligand interactions: history, presence, and future aspects. Journal of Receptors and Signal Transduction. 2004;24(1-2):1-52.

62. Katyal P, Puthenveetil R, Vinogradova O. Structural insights into the recognition of β3 integrin cytoplasmic tail by the SH3 domain of Src kinase. Protein Science. 2013;22(10):1358-65.

63. Ursini-Siegel J, Hardy WR, Zuo D, Lam SH, Sanguin-Gendreau V, Cardiff RD, et al. ShcA signalling is essential for tumour progression in mouse models of human breast cancer. EMBO J. 2008 Mar 19;27(6):910-20.

64. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera—a visualization system for exploratory research and analysis. Journal of computational chemistry. 2004;25(13):1605-12.

65. Gurtovenko AA, Vattulainen I. Lipid transmembrane asymmetry and intrinsic membrane potential: two sides of the same coin. J Am Chem Soc. 2007;129(17):5358-9.

66. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995;6(3):277-93.

67. Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M, et al. The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins: Structure, Function, and Bioinformatics. 2005;59(4):687-96.

68. Parham P. The immune system. Garland Science; 2014.

References

69. Bonneville M, O'Brien RL, Born WK. γδ T cell effector functions: a blend of innate programming and acquired plasticity. Nature Reviews Immunology. 2010;10(7):467-78.

70. Chien Y, Meyer C, Bonneville M. γδ T cells: first line of defense and beyond. Annu Rev Immunol. 2014;32:121-55.

71. Hayday AC. γδ cells: a right time and a right place for a conserved third way of protection. Annu Rev Immunol. 2000;18(1):975-1026.

72. Hayday AC. γδ T cells and the lymphoid stress-surveillance response. Immunity. 2009;31(2):184-96.

73. Wu Y, Ding Y, Tanaka Y, Shen L, Wei C, Minato N, et al. γδ T cells and their potential for immunotherapy. Int J Biol Sci. 2014;10(2):119-35.

74. Bonneville M, O'Brien RL, Born WK. γδ T cell effector functions: a blend of innate programming and acquired plasticity. Nature Reviews Immunology. 2010;10(7):467-78.

75. Kazen AR, Adams EJ. Evolution of the V, D, and J gene segments used in the primate gammadelta T-cell receptor reveals a dichotomy of conservation and diversity. Proc Natl Acad Sci U S A. 2011 Jul 19;108(29):E332-40.

76. Rakasz E, MacDougall AV, Zayas MT, Helgelund JL, Ruckward TJ, Hatfield G, et al. γδ T cell receptor repertoire in blood and colonic mucosa of rhesus macaques. J Med Primatol. 2000;29(6):387-96.

77. Moser B, Eberl M. γδ T-APCs: a novel tool for immunotherapy? Cellular and Molecular Life Sciences. 2011;68(14):2443-52.

78. Allison TJ, Winter CC, Fournié J, Bonneville M, Garboczi DN. Structure of a human γδ T- cell antigen receptor. Nature. 2001;411(6839):820-4.

79. Wiemer DF, Wiemer AJ. Opportunities and challenges in development of phosphoantigens as Vγ9Vδ2 T cell agonists. Biochem Pharmacol. 2014;89(3):301-12.

80. Fournié J, Sicard H, Poupot M, Bezombes C, Blanc A, Romagné F, et al. What lessons can be learned from &ggr; &dgr; T cell-based cancer immunotherapy trials? Cellular & molecular immunology. 2013;10(1):35-41.

81. Kabelitz D. Human gd T lymphocytes for immunotherapeutic strategies against cancer. . 2010.

82. Meraviglia S, Caccamo N, Guggino G, Tolomeo M, Siragusa S, Stassi G, et al. Optimizing Tumor-Reactive γδT Cells for Antibody-Based Cancer Immunotherapy. Curr Mol Med. 2010;10(8):719-26.

83. Kunzmann V, Wilhelm M. Adjuvant zoledronic acid for breast cancer: mechanism of action? The lancet oncology. 2011;12(11):991-2.

References

84. Chlebowski RT, Chen Z, Cauley JA, Anderson G, Rodabough RJ, McTiernan A, et al. Oral bisphosphonate use and breast cancer incidence in postmenopausal women. J Clin Oncol. 2010 Aug 1;28(22):3582-90.

85. Espinosa E, Belmant C, Pont F, Luciani B, Poupot R, Romagne F, et al. Chemical synthesis and biological activity of bromohydrin pyrophosphate, a potent stimulator of human gamma delta T cells. J Biol Chem. 2001 May 25;276(21):18337-44.

86. Arnett HA, Viney JL. Immune modulation by butyrophilins. Nature Reviews Immunology. 2014;14(8):559-69.

87. Franke WW, Heid HW, Grund C, Winter S, Freudenstein C, Schmid E, et al. Antibodies to the major insoluble milk fat globule membrane-associated protein: specific location in apical regions of lactating epithelial cells. J Cell Biol. 1981 Jun;89(3):485-94.

88. Vavassori S, Kumar A, Wan GS, Ramanjaneyulu GS, Cavallari M, El Daker S, et al. Butyrophilin 3A1 binds phosphorylated antigens and stimulates human [gamma][delta] T cells. Nat Immunol. 2013;14(9):908-16.

89. Sandstrom A, Peigné C, Léger A, Crooks JE, Konczak F, Gesnel M, et al. The intracellular B30. 2 domain of butyrophilin 3A1 binds phosphoantigens to mediate activation of human Vγ9Vδ2 T cells. Immunity. 2014;40(4):490-500.

90. Kotlyar SA, Zubatyuk RI, Zhigalko MV, Shishkin OV, Chuprin GN, Kiriyak AV, et al. (cis- syn-cis-Dicyclohexano-18-crown-6) potassium chlorochromate. Acta Crystallographica Section E: Structure Reports Online. 2004;60(12):m1847-8.

91. Julian RR, Beauchamp J. Site specific sequestering and stabilization of charge in peptides by supramolecular adduct formation with 18-crown-6 ether by way of electrospray ionization. International Journal of Mass Spectrometry. 2001;210:613-23.

92. Yin S, Loo JA. Mass spectrometry detection and characterization of noncovalent protein complexes. Mass Spectrometry of Proteins and Peptides: Methods and Protocols. 2009:273- 82.

93. Ahmed FE, Wiley JE, Weidner DA, Bonnerup C, Mota H. Surface plasmon resonance (SPR) spectrometry as a tool to analyze nucleic acid-protein interactions in crude cellular extracts. Cancer Genomics Proteomics. 2010 Nov-Dec;7(6):303-9.

94. Rich RL, Myszka DG. Why you should be using more SPR biosensor technology. Drug Discovery Today: Technologies. 2004;1(3):301-8.

95. Catimel B, Rothacker J, Nice E. The use of biosensors for microaffinity purification: an integrated approach to proteomics. J Biochem Biophys Methods. 2001;49(1):289-312.

96. Rhodes DA, Chen HC, Price AJ, Keeble AH, Davey MS, James LC, et al. Activation of human gammadelta T cells by cytosolic interactions of BTN3A1 with soluble phosphoantigens and the cytoskeletal adaptor periplakin. J Immunol. 2015 Mar 1;194(5):2390-8.

References

97. Hecker SJ, Erion MD. Prodrugs of phosphates and phosphonates. J Med Chem. 2008;51(8):2328-45.

98. Boëdec A, Sicard H, Dessolin J, Herbette G, Ingoure S, Raymond C, et al. Synthesis and biological activity of phosphonate analogues and geometric isomers of the highly potent phosphoantigen (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate. J Med Chem. 2008;51(6):1747-54.

99. Serafinowska HT, Ashton RJ, Bailey S, Harnden MR, Jackson SM, Sutton D. Synthesis and in vivo evaluation of prodrugs of 9-[2-(phosphonomethoxy) ethoxy] adenine. J Med Chem. 1995;38(8):1372-9.

100. Sicard H, Ingoure S, Luciani B, Serraz C, Fournie JJ, Bonneville M, et al. In vivo immunomanipulation of V gamma 9V delta 2 T cells with a synthetic phosphoantigen in a preclinical nonhuman primate model. J Immunol. 2005 Oct 15;175(8):5471-80.

101. Wang H, Henry O, Distefano MD, Wang YC, Raikkonen J, Monkkonen J, et al. Butyrophilin 3A1 plays an essential role in prenyl pyrophosphate stimulation of human Vgamma2Vdelta2 T cells. J Immunol. 2013 Aug 1;191(3):1029-42.

102. Harly C, Guillaume Y, Nedellec S, Peigne CM, Monkkonen H, Monkkonen J, et al. Key implication of CD277/butyrophilin-3 (BTN3A) in cellular stress sensing by a major human gammadelta T-cell subset. Blood. 2012 Sep 13;120(11):2269-79.

103. Palakodeti A, Sandstrom A, Sundaresan L, Harly C, Nedellec S, Olive D, et al. The molecular basis for modulation of human Vgamma9Vdelta2 T cell responses by CD277/butyrophilin-3 (BTN3A)-specific antibodies. J Biol Chem. 2012 Sep 21;287(39):32780-90.

104. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995;6(3):277-93.