EFFECTS OF PROTONATION STATES OF CATALYTICALLY IMPORTANT RESIDUES AND ACTIVE SITE WATER MOLECULES ON PTP1B CONFORMATION

by Ahmet zcan B.S., Chemical Engineering, Yeditepe University, 2008

Submitted to the Institute for Graduate Studies in Science and Engineering in partial fulfillment of the requirements for the degree of Master of Science

Graduate Program in Computational Science and Engineering Boazii University 2011

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EFFECTS OF PROTONATION STATES OF CATALYTICALLY IMPORTANT RESIDUES AND ACTIVE SITE WATER MOLECULES ON PTP1B CONFORMATION

APPROVED BY:

Assist. Prof. Elif zkrml lmez (Thesis Supervisor) Assist. Prof. Burak Alakent (Thesis Co-Supervisor) Prof. Viktorya Aviyente Assist. Prof. Blent Balta Prof. Pemra Doruker Turgut

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DATE OF APPROVAL: ../../

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ACKNOWLEDGEMENTS

It was a pleasure for me to work on such a project. Firstly I would like to give special thanks to my thesis supervisor and co-supervisor, Assist. Prof. Elif zkrml lmez and Assist. Prof. Burak Alakent, for their helps, suggestions and supports during my research. I would like to thank my thesis committee members Prof. Pemra Doruker Turgut, Prof. Viktorya Aviyente and Assist. Prof. Blent Balta for their participations in the project and for their evaluations. First of all, I am very grateful to Pnar Kanlkler and Burcu zkaral for their helps and for enlightening me before starting the project. I am also grateful to my lab-partners namely, hsan mr Akda, Ezgi Akkaya, Nilay Bdeyri, Celal Ceylan, Aslgl Doan, Dilek Eren, Yasemen Gngrmez, Deniz Menekeda, Begm Alaybeyolu and Simay Yalaz for their helps and encouragements, for sharing all the good and bad times. My sincere thanks go to my friends zer zcan and Tark Can, for their supports, helps and friendships. My special thanks go to Glah Tokgz for her motivation and support. Finally, the biggest thanks go to my mother Muazzez zcan and my father Mithat zcan for their ever-lasting encouragement and support. TUBITAK Project No. 107T863 and BAP Project No. 5101 are gratefully acknowledged for the funding.

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ABSTRACT

EFFECTS OF PROTONATION STATES OF CATALYTICALLY IMPORTANT RESIDUES AND ACTIVE SITE WATER MOLECULES ON PTP1B CONFORMATION

Protein Tyrosine Phosphatases 1B (PTP1B) play a critical role in normal cell proliferation, differentiation, and metabolism, by removing the phosphate group from phosphotyrosine. Unliganded and liganded states of PTP1B are mostly associated with the open state of WPD loop (WPDopen) and the open state of WPD loop (WPDclosed) conformations in Protein Tyrosine Phosphatase (PTP) family, but in some cases WPD loop has been observed to adopt the closed conformation in the unliganded structures, and open conformation in the liganded structures. Position of the water molecules in the active site are suggested to help stabilize the closed conformation in PTP1B, and prevent the loop closure and maintain an atypical WPD loop conformation in STEP, which is another member of PTP family. Another controversial issue that may be related with the active site conformation and dynamics is the unusual protonation states of the active site residues Asp181 and Cys215: Asp181 is suggested to be protonated and Cys215 sidechain is suggested to be a thiolate. In this thesis, molecular dynamic (MD) simulations were used to study the effects of protonation states of Asp181 and Cys215, and the active site waters on the WPD loop conformations. MD simulations were performed on the unliganded PTP1B in WPDopen and WPDclosed, and the liganded PTP1B in WPDclosed conformations. In the WPDopen unliganded state, protonation state of Asp181 did not have a significant effect on the conformation or dynamics of the catalytically important loop regions in the vicinity of Asp181. The WPDclosed crystal structure conformation was maintained only in the MD simulations with a protonated Asp181 and with the initial positioning of crystal structure waters. With the active site waters missing, WPD loop moves from the closed to an intermediate conformation. Crystal structure conformations of the Michaelis complex were maintained only in the MD simulations with the protonated Asp181, protonated Cys215, and initialized with the single crystal structure water at the active site. When Cys215 was

v deprotonated, either the peptide or WPD loop moved out of the pocket. These results bring doubt on the controversial assumption that Cys215 should be in thiolate form in the Michaelis complex. In the absence of the active site water, Asp181 moves toward the position preoccupied by the water, and pTyr is slightly displaced. As a result, one may say that proper protonation states of active site residues and proper positioning of water molecules play important roles for the WPD loop activation/inactivation.

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ZET

KATALTK AIDAN NEML KALINTILARIN PROTONASYON DURUMLARININ VE AKTF BLGE SU MOLEKLLERNN PTP1B KONFORMASYONUNA ETKS

Tirosin Fosfataz 1B (PTP1B) enzimi, fosfotirozinden fosfat grubunu kopararak, normal hcrenin oalmasnda, farkllamasnda ve metabolizmasnda kritik bir rol oynar. PTP1Bnin substrat bal ve bal olmayan yaplar ounlukla Protein Trozin Fosfataz (PTP) ailesindeki WPD dngs konformasyonlarnn ak hali (WPDak) ve kapal hali (WPDkapal) ile ilikilendirilir ama baz durumlarda WPD dngsnn substrat bal deil iken kapal ve substrat bal iken ak olduu gzlemlenmitir. Aktif blgedeki sularn substratsz PTP1Bdeki kapal yapnn dengede durmasna yardm ettii, ve dngnn kapanmasn engelledii ve PTP ailesinin bir baka yesi olan STEPteki WPD dngs konformasyonun atipik durumda kalmasn salad ne srlyor. Aktif blge konformasyonu ve dinamii ile ilgili olabilecek bir baka tartmaya yol aan konu ise aktif blgedeki kalntlardan Asp181in ve Cys215in allmadk protonasyon durumlar: Asp181in protone edilmesi ve Cys215 yan zincirinin tiyolat olmas ileri srlyor. Bu tezde deiik WPD dngs konformasyonlarnda Asp181in ve Cys215in iyonizasyon durumlarnn ve aktif blge sularnn etkilerini incelemek Molekler Dinamik (MD) simlasyonlar kullanld. Substratsz PTP1Bnin WPDak ve WPDkapal, ve substratl PTP1Bnin WPDkapal konformasyonlarna MD simlasyonlar koturuldu. WPDak substratsz durumda, Asp181nin protonasyon durumun Asp181 komu olan katalitik adan nemli olan blgelerin konformasyonda ve dinamiinde nemli bir etkisi yoktu. Sadece protone edilmi Asp181 ve kristal yapdaki sularn bata konumlandrlmas ile WPDkapal kristal yaps korunabildi. Aktif blgedeki sularn yokluunda WPD dngs kapal konformasyondan ara konformasyona doru hareket etti. Michaelis kompleksin kristal yap konformasyonu ise sadece protone edilmi Asp181li ve Cys215li, ve aktif blgedeki kristal sular ile balatlm MD simlasyonlarnda korundu. Ne zaman Cys215 deprotone edilse, ya peptit ya da WPD dngs aktif blgeden uzaklat. Bu sonular

vii Cys215in Michaelis komplekste tiyolat olmas gerektiren tartmaya ak varsaymlara phe getirdi. Aktif blgedeki suyun yokluunda ise, Asp181 kristaldeki suyun kaplad pozisyona doru yneldi ve pTyr ok az yerini deitirdi. Sonu olarak aktif blgedeki kalntlarn protonasyon durumlarnn ve su molekllerinin uygun konumlandrlmasnn WPD dngs aktivasyon/inaktivasyonu iin nemi roller oynayabilirler.

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TABLE OF CONTENTS ACKNOWLEDGEMENTS .................................................................................. ABSTRACT .......................................................................................................... ZET ..................................................................................................................... LIST OF FIGURES ............................................................................................... LIST OF TABLES ................................................................................................ iii iv vi xi xx

LIST OF SYMBOLS............................................................................................. xxii LIST OF ACCRONYMS / ABBREVIATIONS ................................................... xxiiv 1. INTRODUCTION ............................................................................................. 2. PROTEIN TYROSINE PHOSPHATASE ........................................................ 2.1. Protein Tyrosine Phosphatase 1B .............................................................. 2.1.1. Biological Significance of PTP1B ................................................... 2.1.2. Tertiary Structure and Catalytic Mechanism of PTP1B .................. 2.1.3. PTP Signature Motif (P-Loop) ....................................................... 2.1.4. WPD loop ........................................................................................ 2.2. Effect of Water Molecules on the PTP1B Conformation .......................... 2.3. Conformational Activation of WPD Loop in Other Members of PTP family ......................................................................................................... 2.4. Ionization States of the Catalytically Important Residues in the Active Site ............................................................................................................. 3. METHODS ........................................................................................................ 3.1. Molecular Dynamics Simulations ............................................................. 3.1.1. CHARMM Force Field.................................................................... 3.1.2. NAMD ............................................................................................. 3.1.3. MD Simulation Structures and Parameters ..................................... 13 16 16 17 18 19 11 1 3 3 4 5 6 7 9

ix 3.2. Trajectory Analysis.................................................................................... 3.2.1. Root Mean Square Deviations (RMSD) .......................................... 3.2.2. Mean Square Fluctuations (MSF).................................................... 3.2.3. Hydrogen Bond Criteria .................................................................. 3.2.4. Probability Distribution ................................................................... 4. RESULTS AND DISCUSSION ....................................................................... 4.1. PTP1B in the unliganded WPDopen state.................................................... 4.1.1. Structural Analysis of the WPDopen Simulations ............................. 4.1.2. Analysis of the Residue Fluctuations in the WPDopenSimulations .. 4.1.3. Polar Interactions between WPD loop and R-Loop ........................ 4.2. PTP1B in the unliganded WPD loop closed (WPDclosed) state .................. 4.2.1. Structural Analysis of the WPDclosed Simulations ........................... 4.2.2. Analysis of the Dynamics of the WPDclosed Simulations ................. 4.2.3. Atomistic Analysis of the Active Site Residues in the WPDclosed Simulations....................................................................................... 4.2.4. Analysis of the Active Site Water Molecules in WPDclosed Simulations....................................................................................... 4.3. PTP1B in the Liganded WPDclosed State .................................................... 4.3.1. MD Simulations with Asp181 and Cys215 both in Protonated States and Initialized with Crystal Waters ....................................... 4.3.2. MD Simulations with Asp181 Protonated, Cys215 Negatively Charged and Initialized with Crystal Waters ................................... 4.3.3. Analysis of MD Simulations with Asp181 in Protonated State and Initialized with Random Waters....................................................... 4.3.4. Analysis of MD Simulations with Asp181 in Negatively Charged State and Initialized with Crystal Waters ......................................... 5. CONCLUSIONS AND RECOMMENDATIONS............................................ 5.1. Conclusions ............................................................................................... 80 84 84 74 68 63 44 62 39 20 20 20 21 21 22 22 23 25 27 31 31 35

x APPENDIX A: Similarity of the Structures and the Dynamics of MDc3 and MDc4 ..................................................................................................................... APPENDIX B: VMD commands for the selection of active site waters .............. APPENDIX C: Additional analyses for liganded WPDclosed simulations ............. APPENDIX D: Scripts Used in the Thesis ........................................................... D.1. Distance Measurement.............................................................................. D.2. Angle Measurement between Three Atoms ............................................. D.3. Dihedral Angle Measurement ................................................................... D.4. RMSD Measurement ................................................................................ D.5. MSF Measurement.................................................................................... D.6. Energy Measurement ................................................................................ D.7. Hydrogen Bond Measurement .................................................................. D.8. Solvent Accessible Surface Area Measurement ....................................... D.9. Water Counting ......................................................................................... 89 91 93 97 97 97 97 98 98 98 99 99 99

D.10. Probability Distribution .......................................................................... 100 REFERENCES ...................................................................................................... 101

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LIST OF FIGURES

Figure 2.1.

The superfamily of protein tyrosine phosphatases and dualspecificity phosphatases: (A) Classical protein tyrosine 4 phosphatases. (B) Some of the dual-specificity phosphatases ..........

Figure 2.2.

Tertiary Structure of liganded PTP1B (PDB code: 1PTU). The helices and 12 strands are labeled. WPD and P-loop are shown as blue and red, respectively. pTyr sidechain is shown ......................... 5 6 9

Figure 2.3. Figure 2.4. Figure 2.5.

Schematic representation of the catalytic mechanism of PTP1B ...... Structures of open and closed conformations of PTP1B ................... Interactions of WPD loop in unliganded open structure of PTP1B with the water molecules in the active site.........................................

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Figure 2.6.

Interactions of WPD loop in closed structure of PTP1B with the active site waters ................................................................................ 11

Figure 2.7.

Interaction network between the PTP1B active site residues, the inhibitor and water molecules in the open conformation of the WPD loop. .......................................................................................... 11

Figure 2.8.

Interaction between the ligand and the water molecule in the active site of apo-Yersinia PTPase in WPD loop open conformation, and WPD loop closed conformation in sulfate bound Yersinia PTPase ... 12 13

Figure 2.9.

Atypical state of WPD loop in two members in PTP families ..........

xii Figure 4.1. RMSD of the C atoms of the MD simulation structures from the crystal structure (PDB ID: 2F6F): MDo1 (red), MDo2 (black), and MDo3 (blue) ....................................................................................... Figure 4.2. RMSD of WPD loop relative to: (A) WPDopen and (B) WPDclosed crystal structures: MDo1 (red), MDo2 (black), and MDo3 (blue) ....... Figure 4.3. RMSD of the R- loop relative to the crystal structure: MDo1 (red), MDo2 (black), and MDo3 (blue)......................................................... Figure 4.4. R-loop conformation of the crystal structure (cyan) and average conformation between 25-40 ns: MDo1 (red), MDo2 (black), and MDo3 (blue) ....................................................................................... Figure 4.5. MSF of the residues in MDo1 (red), MDo2 (black), and MDo3 (blue)simulations ................................................................................ Figure 4.6. Figure 4.7. MSF of WPD loop in MDo1 (red), MDo2 (black), and MDo3 (blue). MSF of R-loop (113-120. residues) in MDo1 (red), MDo2 (black), and MDo3 (blue) ................................................................................. Figure 4.8. (A) Nonbonded energies between WPD lop and R-Loop (B) Probability distribution of the nonbonded energies. In both figures, MDo1 (red), MDo2 (black), and MDo3 (blue)are shown. ................... Figure 4.9. (A) Polar interactions between Arg112 (purple) and Asp181 (green) in MDo2 and MDo3, (B) Polar interaction between Arg112 (purple) and, Pro180 (red) and Trp179 (blue) in MDo1 ..................... Figure 4.10. Arg112 distance to important residues ............................................... 29 30 28 27 26 27 25 25 24 23

xiii Figure 4.11. RMSD of the C atoms of the MD simulation structures from the crystal structure (PDB ID: 1SUG) in MDc1 (black), MDc2 (blue), MDc3 (red) and MDc4 (green)............................................................ Figure 4.12. RMSD of WPD loop relative to (A) WPDclosed and (B) WPDopen crystal structures in MDc1 (black), MDc2 (blue) and MDc3 (red). .... Figure 4.13. Simulation averages of the C trace of the WPDclosed (blue) and WPDopen (red) crystal structure with WPD loop (cyan) of the simulations in (A) MDc1, (B) MDc2 and MDc3 ................................. Figure 4.14. RMSD of the C-terminus of WPD loop to (A) WPDclosed and (B) WPDopen crystal structures in MDc1 (black), MDc2 (blue) and MDc3 (red). ........................................................................................ Figure 4.15. Figure 4.16. MSF in MDc1 (black), MDc2 (blue) and MDc3 (red)......................... MSF of the WPD loop in MDc1 (black), MDc2 (blue) and MDc3 (red). ................................................................................................... Figure 4.17. MSF of the S-loop (A) between 10-40 ns and (B) between 10-27 ns of MDc1 (black), MDc2 (blue) and MDc3 (red). ................................ Figure 4.18. The average conformations of the S-loop before (blue) and after (red) the conformational transition at 27 ns in MDc1 ........................ Figure 4.19. Backbone dihedral (A) , and (B) angles of Glu200 on the N terminus of the S-loop ........................................................................ Figure 4.20. Correlation map of the residues 160-210 in (A) MDc1, (B) MDc2 and (C) MDc3. .................................................................................... 40 39 38 38 37 35 36 35 33 32

xiv Figure 4.21. Correlation map of the residues 160-210 in MDc1 sampled before 27 ns ................................................................................................... Figure 4.22. (A) C C distance between the residues Asp181 in WPD loop and Cys215 in P-loop in MDc1, MDc2 and MDc3. (B) Sidechain (1) dihedral angle of Asp181 in MDc1, MDc2 and MDc3 . ............ Figure 4.23. Polar interaction of Asp181 with the surrounding residues in representative conformations from (A) MDc1 sampled at 30 ns, (B) MDc2 sampled at 30 ns, and (C) MDc3 sampled at 30 ns. Dashed lines show the polar interactions. ....................................................... Figure 4.24. Figure 4.25. Asp181 distances to important residues ............................................. The minimum distance between sidechain O atoms of Asp181 and sidechain N atoms of Arg221: MDc3 (black) and MDc4 (red)........... Figure 4.26. (A) The waters in the active site in the WPDclosed crystal structure (1SUG) (B) Potential hydrogen bonds (