DYNAMIC STUDIES OF TYPE I AND TYPE II CADHERINS EC DOMAINS WU FEI (B.Sc., University of Science and Technology of China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSICS NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. WU FEI 2014-01-22 Acknowledgements Foremost, I wish to express my sincere gratitude to my supervisor Professor Liu Xiangyang and former supervisor Dr. Liu Ruchuan, for their invaluable advices, patience, kindness and encouragement throughout my Ph.D. candidature. Professor Liu Xiangyang provided me a global insight and guided the direction of my research. Dr. Liu Ruchuan took care of the details of my research works. His valuable experiences and suggestions have made me through all the difficulties during the experiment. I would like to acknowledge Professor Jean Paul Thiery. This thesis would not have been completed without his kind support and guidance, for which I am always grateful. I am also indebted to the people in his group for being friendly and helpful. Dr. Prashant Kumar, Dr. Shen Shuo, Ms Ahmed El Marjou and Ms Nandi Sayantani provided me the protein sample which is critical for my research project. I also thank Professor Lim Chwee Teck and his group members for their help with instruments. Especially I want to thank Dr. Kong Fang and Dr. Zhong Shaoping for their kind advices with AFM experiments set up. I enjoyed the discussions with them. Meanwhile, I would like to thank my colleagues, Ms Liu Min, Mr Lu Chen, Dr. Manoj Kumar Manna, Dr. Deng Qinqiu, Mr Qiu Wu and Mr Thuan Beng Saw for their help during my research life. i Special thank you to my girlfriend Guo Xixian for her consistent encouragement, help, and love during my struggling with the research project. Last and the most important, thank my parents who give me unconditional love and support. They are always there to encourage me whenever I met with difficulties. As their only son, I regret being so far away from them and could not be with them when they needed me. ii Table of Content Summary . v List of tables . vi List of Figures . vii Publications ix Chapter Introduction 1.1 Cadherin mediated cell adhesion and cell sorting . 1.2 Cadherin molecular structure and homophilic interaction between EC domains 1.2.1 Type I and Type II cadherins share similar molecular structure . 1.2.2 Type I and Type II cadherins show distinct interaction mechanisms. 1.3 The role of dynamic force in cadherins physiology function. . 11 1.4 Question addressed in this thesis . 15 Chapter Experimental technologies and theories . 18 2.1 Protein expression and sample preparation . 20 2.1.1 Protein expression and purification 20 2.1.2 Sample preparation and surface chemistry . 22 2.2 Atomic Force Microscopy . 29 2.2.1 Instrumentation 29 2.2.2 Measurement procedure . 33 2.2.3 Data Analysis . 36 2.3 Magnetic tweezers . 38 2.3.1 Instrumentation 38 2.3.2 Measurement procedure and data analysis . 47 2.4 SMD simulation 49 2.4.1 Steered Molecular Dynamics . 49 2.4.2 SMD simulation of cadherin EC domains 53 2.5 Forced bond dissociation . 58 2.5.1 Physical description of bond dissociation 58 2.5.2 Bond dissociation under force 59 Chapter Dynamic measurements on homophilic interaction between cadherin EC domains 63 3.1 Strand-swap dimer unbinding of Type I and Type II cadherins in SMD simulations . 63 3.2 Dimer unbinding of Type I and Type II cadherins in AFM experiments 66 3.2.1 Control experiments . 66 3.2.2 Type I and Type II cadherins show distinct unbinding behavior in AFM experiments . 69 3.3 Mechanical properties of Type I and Type II cadherins homophilic interaction pairs 75 3.3.1 SMD simulation results partly account for different adhesivity between Type I and Type II cadherins . 75 3.3.2 Mechanical properties of cadherins homophilic interaction pairs in AFM experiments . 76 Chapter Partial unfolding of cadherin EC domains 80 4.1 Forced unfolding of cadherins EC domains in AFM experiments 80 4.1.1 AFM unfolding control experiments 80 iii 4.1.2 Forced cadherin EC domains unfolding in AFM experiments . 83 4.1.3 Comparison of unfolding and unbinding force in AFM experiments 84 4.2 Forced cadherin EC domains unfolding in magnetic tweezers experiments . 87 4.3 Forced cadherin EC domains unfolding in SMD simulations . 89 4.3.1 Force-extension unfolding trajectories in SMD simulations 89 4.3.2 Comparison between unfolding pathways of Type I and Type II cadherins . 91 4.3.3 The role of Ca2+ ions in cadherin unfolding pathway . 92 4.4 Partial unfolding of EC domains may be involved in cadherin physiology function 95 4.4.1 Partial unfolding of cadherins EC domains 95 4.4.2 Partial unfolding of cadherins EC domains may exist in vivo 96 4.4.3 Possible role of partial unfolding of cadherins EC domains in vivo 97 Chapter Conclusion 100 References .104 Appendix .110 Appendix I AFM data analysis program .110 Appendix II Magnetic tweezers data analysis program 112 Appendix III Ca2+ Bridge rupture in SMD simulation 114 iv Summary Cadherins are a class of protein that dominate cell-cell adhesion in most tissues. Their dysfunction correlates with diseases such as breast cancer, tumor progression and neuropsychiatric disorders. A better understanding of their adhesion mechanism is thus vital for assailing their role in these disease processes. Although extensive studies have been performed, the adhesion mechanism of cadherins has not been fully understood yet. Particularly, distinct adhesion mechanisms between Type I and Type II cadherins and the role of dynamic force in the adhesion process are still being elucidated. In the present study, by utilizing Atomic Force Microscopy (AFM), magnetic tweezers as well as Steered Molecular Dynamics (SMD) simulations, homophilic interactions and mechanical stability of classical Type I and Type II cadherins extracellular (EC) domains were investigated at the single molecule level. The results show that the unbinding force of Type I cadherins homophilic interaction pairs are stronger than that of Type II cadherins. In addition, unbinding forces of the homophilic interaction pairs for both cadherins show overlap with unfolding forces of their monomers. This phenomenon indicates that partial unfolding/deformation of the cadherin monomers may take place before the rupture of their homophilic interactions in vivo. This possible conformational change may expose new interaction interfaces or trigger cortical actin cytoskeletal remodeling in strengthening cadherin-mediated adhesion. Furthermore, it may also contribute to the significant adhesive strength difference between Type I and Type II cadherins. v List of tables Table 2.1 Parameters of SMD simulations. . 57 Table 3.1 Binding probabilities in different conditions of AFM unbinding experiments. Numbers in parentheses indicate the number of curves with unfolding events divided by the total number of curves achieved in the corresponding experiment. 68 Table 4.1 Pick up rate in the AFM unfolding control and unfolding experiments. . 82 Table 4.2 Multiple peaks ratio in AFM unbinding experiments. . 86 vi List of Figures Figure 1.1 Architecture of classical cadherins. . Figure 1.2 Multiple-protein complex interact with cadherin cytoplasmic region. Figure 1.3 Crystallographic structure of C-cadherin extracellular region. Figure 1.4 Two-step binding model of classical cadherins. Figure 1.5 The structure of artificial E-cadherin junction. 10 Figure 1.6 Molecular basis of mechanical sensing of cadherins complex. . 13 Figure 2.1 The photo of SDS-PAGE. 22 Figure 2.2 Chemical modification method for AFM unbinding experiments. 25 Figure 2.3 Preparation for magnetic tweezers sample. . 28 Figure 2.4 Schematic of AFM. 30 Figure 2.5 Working principle of PZT scanner. 31 Figure 2.6 Force probe of AFM 32 Figure 2.7 Spring constant calibration of AFM cantilever. . 33 Figure 2.8 Schemes of AFM unbinding experiments. . 34 Figure 2.9 Schemes of AFM unfolding experiments. . 35 Figure 2.10 WLC fitting of unfolding force-extension curve. 37 Figure 2.11 Schematic of Magnetic tweezers/evanescent nanometry system. 40 Figure 2.12 Force calibration of magnetic tweezers. 41 Figure 2.13 Force versus distance curve in force calibration. . 42 Figure 2.14 Preparation process of fluorescent bead modified cantilever. 45 Figure 2.15 TIRF depth calibration. 47 Figure 2.16 A typical curve in magnetic tweezers experiments. . 48 Figure 2.17 Energy barrier of protein unfolding/unbinding. . 59 Figure 2.18 Lower energy barrier caused by a constant force. . 61 Figure 3.1 Force-extension curves of strand-swap dimers dissociation in SMD simulations. 65 Figure 3.2 Evaluating protein density on the slide. . 69 Figure 3.3 Unbinding forces of cadherin homophilic interaction pairs . 71 Figure 3.4 Unbinding forces of curves with single force peak. 73 Figure 4.1 Unfolding force of EC domains by AFM. . 82 Figure 4.2 Forced unfolding of cadherins EC domains by AFM. . 84 Figure 4.3 Indication of unfolding happens prior to unbinding. . 86 Figure 4.4 Forced unfolding of E-cadherin EC domains by magnetic tweezers. 88 Figure 4.5 Unfolding trajectories of EC12 domains. 90 Figure 4.6 Distinct unfolding pathways of E-cadherin and cadherin EC12 domains. . 92 Figure 4.7 Unfolding force-extension curves of E-cadherin EC12. 94 vii Figure A.1 The program for AFM results analysis. .111 Figure A.2 The program for magnetic tweezers results analysis .113 Figure A.3 The GUI of Ca2+ bridges information representation program. 117 viii to that borne by the protein molecules in vivo. These results indicate that the partial unfolding of cadherins EC domains may happen in vivo during the turnover of cadherins molecules at the adhesion junction. Finally, SMD simulations results indicate the different unfolding pathways between Type I and Type II cadherins. This may contribute to their distinct adhesion strength in vivo. Based on all these findings, we proposed two possible strengthening mechanisms of cadherin-mediated adhesion which involves partial unfolding or deformation of EC domains. One is exposing new binding interface and the other is conformational signal which can trigger a series of processes in cytoskeleton. Furthermore, These two possible mechanisms may also account for the diversity between the adhesion strength of Type I and Type II cadherins. The weaker homophilic interactions between the EC domains of Type II cadherins may limit the mechanical signal it can transmit. Also, the unfolding pathways of Type II cadherins EC domains may not favour a strong adhesion as that of Type I cadherins. After decades of study, the mechanisms of cadherin mediated cell-cell adhesion are still being elucidated. The present study directly compared the mechanical property of Type I and Type II cadherins monomers as well as dimers. Also, we propose that force could account for the strengthening effect in cadherins adhesion and contribute to the distinct adhesion strength between Type I and Type II cadherins. This is the first step towards uncovering the role of force in cadherin adhesion process in vivo. However, there are still questions remain to be addressed:  Although the present study suggests that the partial unfolding may happen prior 102 to the unbinding, the evidence is indirect. A magnetic tweezers experiment may provide a solid evidence. If the fluorescent magnetic bead linked to the slide via cadherins homophilic interaction pairs, before the bead pulled away by the magnetic tweezers, an unfolding step should be observed by the TIRF microscopy.  In the present study, isolated EC domains were utilized. Previous studies show that the distinct adhesion strength between Type I and Type II cadherins is probably due to this region (10). Also, this is a widely used method for in vitro studies to simplify the experiments (33, 41, 101, 102). However, cadherin cytoplasmic domain, together with other adhesion molecules in cytoskeleton can remodel the conformation of homophilic interaction pairs and the junction of cadherins. Therefore, further dynamic studies using full length cadherins expressed on living cells should be conducted to investigate the junction remodeling in vivo.  The unfolding pathways of cadherins EC-domains were investigated by SMD simulation in the present study. 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During the fitting, after choosing a force peak by mouse, the program can fit this peak with a WLC curve whose persistence length and contour length are adjustable. The fitting is implemented by Levenberg–Marquardt algorithm. Then by clicking Save button, the program can save the information including rupture extension, contour length, persistence length, rupture force, fitting residual and the curve sequence. These data will be represented into histograms by a Labview program. Below is the list of main buttons and textboxes: Load: Load the folder of raw .txt data of AFM. Mouse mode: Including “zoom in” mode in which dragging mouse can choose a region of curve to zoom in and “fit” mode in which dragging mouse can choose a segment of curve to fit. The two modes can be switched between each other by double clicking the mouse. 110 Path: The path of folder which containing the raw .txt data of AFM. ResultPath: The path of file which record the target information extracting from force-extension curves. Progress: Show the current curve sequence in the folder. Save: Record the target information into the file Prior/next: Move to the prior or next curve in the folder -/+: Adjust the force baseline of the curve in case the baseline found by the program is not accurate. KAdj: The spring constant of the cantilever which can be used to adjust the extension of protein molecule by removing the deflection of the cantilever, i.e. the force divided by the spring constant. Figure A.1 The program for AFM results analysis. 111 Appendix II Magnetic tweezers data analysis program The AFM data analysis program is also wrote by me independently in C#. It main function is finding out the unfolding steps in each magnetic tweezers unfolding curve. The algorithm is based a previous publication (82) and its GUI is shown in Figure A.2. During the fitting, firstly the program read in the extension-time curve from the raw data file. Then it can fit the curve with given step number and calculate the step indicator which corresponds to the quality of this step number. By trying different step number and find out the one with the maximum step indicator, the optimized fitting can be found. Then by clicking the Save button the fitting results can be saved to the target file. The results will be represented into histograms by a Labview program, Below is the list of main buttons and textboxes: File path: The path of the txt file which containing the extension-time unfolding curve. LoadData: Read in the data. StepNum: Expected step number, will be evaluated by Stepindicator. ChiFit: Use the algorithm in this publication (82) to fit. HMMInitial, HMMFit, PrintHMM: these three buttons are for fitting the curve by Hidden Markov model, however, because this fitting algorithm always give the similar results as the ChiFit and is more time consuming, thus was not utilized for data analysis in the present study. StepIndicator: This value is the evaluation of the StepNum, can be used to adjust how many steps are there in the curve (82). 112 SAVE: Save the fitting results to target txt file. SavePath: Path of the target txt file which store the fitting results. In the chart, X-axis is time and Y-axis is extension. Blue curve shows the unfolding curve, red curve shows the fitting curve. Figure A.2 The program for magnetic tweezers results analysis 113 Appendix III Ca2+ Bridge rupture in SMD simulation Previous crystallographic data have shown that Ca2+ ions form bridges with highly conserved residues in cadherins (9, 14, 37). In the SMD simulations of cadherin EC12 domains unfolding, rupture of theses Ca2+ bridges occurred sequentially as unfolding progressed. The Ca2+ bridges which involves the three Ca2+ ions at the linker between EC1 and EC2 domains are listed as below: Cad8: CA1_GLU11_OE1, CA1_ASP101_OD2, CA1_GLU12_OE2, CA1_ASP64_OD2, CA1_GLU66_OE1, CA1_ASP101_OD1, CA2_GLU11_OE2, CA2_GLU66_OE2, CA2_ASP98_OD1, CA2_ILE99_O, CA2_ASP101_OD1, CA2_GLU66_OE1, CA2_ASP134_OD1, CA2_GLU11_OE1, CA3_ASN100_OD1, CA3_ASN102_O, CA3_ASP132_OD2, CA3_ASP132_OD1, CA3_ASP134_OD2, CA3_SER141_O, CA3_ASP187_OD2, CA3_ASP187_OD1. Ca11: CA1_GLU11_OE2, CA1_GLU12_OE2, CA1_ASP64_OD2, CA1_GLU66_OE2, CA1_ASP101_OD1, CA2_GLU11_OE1, CA2_ASP101_OD2, CA2_ASP134_OD1, CA2_GLU66_OE1, CA2_ASP98_OD1, CA2_ILE99_O, CA3_ASN100_OD1, CA3_ASN102_O, CA3_ASP132_OD2, CA3_ASP132_OD1, CA3_ASP134_OD2, CA3_ASP187_OD2, CA3_ASP187_OD1 Ecad: CA1_ASP103_OD1, CA1_ASP103_OD2, CA1_GLU69_OE1, CA1_ASP67_OD2, CA1_GLU11_OE1, CA1_ASP67_OD1, CA2_GLU11_OE2, CA2_ASP100_OD1, 114 CA2_GLN101_O, CA2_GLU69_OE1, CA2_ASP103_OD1, CA2_GLU69_OE2, CA2_ASP136_OD1, CA3_ASP134_OD1, CA3_ASP195_OD2, CA3_ASN104_O, CA3_ASN102_OD1, CA3_ASP136_OD2, CA3_ASN143_O, CA3_ASP134_OD2 Ccad: CA1_ASN104_O, CA1_ASN102_OD1, CA1_ASP195_OD1, CA1_ASP136_OD2, CA1_ASP134_OD1, CA1_ASP134_OD2, CA1_ASP195_OD2, CA2_GLU69_OE1, CA2_GLU69_OE2, CA2_ASP100_OD2, CA2_ASP136_OD1, CA2_GLN101_O, CA2_ASP103_OD2, CA2_GLU11_OE1, CA3_GLU69_OE1, CA3_ASP103_OD2, CA3_ASP103_OD1, CA3_GLU11_OE2, CA3_ASP67_OD2, CA3_ASP67_OD1 Ncad: CA1_ASN142_O, CA1_ASN102_OD1, CA1_ASP194_OD2, CA1_ASP136_OD2, CA1_ASN104_O, CA1_ASP134_OD2, CA1_ASP134_OD1, CA2_ASP136_OD1, CA2_MET101_O, CA2_GLU11_OE2, CA2_ASP100_OD2, CA2_ASP100_OD1, CA2_GLU69_OE2, CA2_GLU69_OE1, CA2_ASP103_OD1, CA3_ASP103_OD1, CA3_ASP103_OD2, CA3_GLU11_OE2, CA3_GLU11_OE1, CA3_GLU69_OE1, CA3_ASP67_OD1, CA3_ASP67_OD2 In the names of Ca2+ bridges in the above list, the first part, i.e. CA1, CA2, CA3 refers to the three different Ca2+ ions and the rest of the name, e.g. ASP67_OD2 refers to the atom in the protein structure which forms bridge with the corresponding Ca 2+ ion. The rupture information of the Ca2+ bridges was extracted from the simulation results by a tk/tcl script wrote by me for analysis. Because there are around twenty targeted Ca2+ bridges in each cadherin structure, it would be quite messy to plot all 115 their extension curves together with the corresponding unfolding force-extension curve. I wrote a program in c# to represent this information and the GUI is shown in Figure A.3. Below is the list of main buttons and textboxes: Curve: The name of the file which containing the target force-time curve. As in Figure A.3, it is Ecad0.txt which refers to the first trial of E-cadherin. Plot Ruptured: Plot the extension-time curves of all Ca2+ bridges which ruptured during simulation. Plot All: Plot the extension-time curves of all Ca2+ bridges. Reset view: Reset the view of curves after zoom in/out. Clear All: Remove all the plotted extension-time curves of Ca2+ bridges. In the table at left: Bridge: The bridge name, e.g. CA1_ASP103_O refers to the bridges between the first Ca2+ ion and the atom ASP103_O in the protein molecule. Rupture Frame: The rupture time of the bridge, the unit is ps. -1 refers to no rupture during the simulation. Avg b4 rup: The average extension of the bridge before its rupture, the unit is Å. Sdv b4 rup: The standard deviation of the bridge extension before its rupture, the unit is Å. In the chart at right: The X-axis is the time and the unit is ps. The left Y-axis is the extension of Ca2+ bridges with the unit Å and the right Y-axis is the tension force of the protein molecule with the unit pN. The black curve is the protein unfolding force-time curve. 116 By clicking the bridge name in the left table, the program can add its corresponding extension-time curve into the chart, as the red curve in Figure A.3. The green dots on the unfolding force-time curve correspond to the rupture events of Ca2+ bridges. Figure A.3 The GUI of Ca2+ bridges information representation program. 117 [...]... cadherins (8) and are divided into 5 distinct families: classical Type I cadherins, classical Type II cadherins, desmosomal cadherins, protocadherins and seven-pass transmembrane cadherins (9, 10) Among them, classical Type I and Type II cadherins are the best understood families in both structure and physiological function so far 1 The sequence characteristics of Type I and Type II cadherins result in... between Type I and Type II cadherins Among total ~550 amino acids in the extracellular region, 21 out of 180 conserved residues of Type II cadherins are not found in Type I cadherins In contrast, in the cytoplasmic region, this number is 3 out of 52 (13) Studies have shown that the distinct adhesive strength and binding specificity between Type I and Type II cadherins probably are governed by this region... strand-swap dimers is due to the lacking of glycosylation since the EC domains used in these experiments are bacterially expressed Their experiment (40) exclude this trimeric interaction by utilizing mammalian-expressed EC domains of Type II cadherins However, even though the clustering mechanism of Type II cadherins is still unsolved, it probably is different from that of Type I cadherins This is because... because Type II cadherins lack the pseudo-β helix region which is indispensable to the lateral clustering of Type I cadherins (37) In addition, the lateral clustering structure has not been observed in the Type II cadherins artificial junction under the similar condition (40) The different clustering mechanism of Type I and Type II cadherins may partly account for their distinct 9 adhesion strength in vivo... I and Type II cadherins, the EC domains explored in the present study should be enough for the comparison between Type I and Type II cadherins Also, the experimental results in this study do not unveil the detailed unfolding pathway of Type I and Type II cadherins EC domains Although this information is important, it is difficult to 16 achieve regarding nowadays technology limitation Therefore, this... neuropsychiatric disorders (6) Understanding the adhesion mechanism of cadherins is thus vital for assailing their role in these disease processes In 1991, Suzuki et al first proposed grouping all 11 types of classical cadherins identified by that time into two families, Type I and Type II cadherins, based on their overall similarities in sequence (7) To date, cadherins super-family comprises over 80 types of cadherins. .. II cadherins, Type I cadherins expressed cells show stronger separation force between each other In addition, the homophilic adhesion of Type I cadherins are more rapid (10) EC2 EC1 EC2 EC1 EC2 EC1 EC1 EC2 EC1 EC2 EC2 EC1 Figure 1.4 Two-step binding model of classical cadherins Cadherin monomers dimerize via the interaction between EC1 2 domains Two monomers first associate to form X-dimer, then this... their distinct behaviour in vivo Type I cadherins, including E-cadherin, N-cadherin and C-cadherin etc., show stronger and more rapid adhesion than Type II cadherins and are found primarily in tissues where the requirement for integrity is high In contrast, Type II cadherins such as cadherin 7, cadherin 8 and cadherin 11 are highly related and expressed in cells with more mobility and more temporary intercellular... with these studies, dissociation constants kd of different cadherins EC domains measured by ultracentrifugation experiments (35) also show that the 7 strand-swap dimer of Type II cadherins have higher binding energy than that of Type I cadherins However, the stronger strand-swap dimer of Type II cadherins does not result in a stronger adhesion junction in vivo In cell based studies, comparing with Type. .. and B When cadherins EC domains subject external force, the cadherin-catenin complex is stretched between the cadherins EC domains and the actin in the presence of Myosin II Then the vinculin binding site in α-catenin exposes to recruit vinculin, as shown in Figure 1.6C and D This process may trigger junction remodeling ii) Besides the direct force transmission, the conformational change of cadherin . 3.2.2 Type I and Type II cadherins show distinct unbinding behavior in AFM experiments 69 3.3 Mechanical properties of Type I and Type II cadherins homophilic interaction pairs 75 3.3.1 SMD simulation. 1.2.1 Type I and Type II cadherins share similar molecular structure 3 1.2.2 Type I and Type II cadherins show distinct interaction mechanisms. 6 1.3 The role of dynamic force in cadherins physiology. 3.1 Strand-swap dimer unbinding of Type I and Type II cadherins in SMD simulations 63 3.2 Dimer unbinding of Type I and Type II cadherins in AFM experiments 66 3.2.1 Control experiments 66