RAP1-TRIGGERED PATHWAYS FOR TALIN-MEDIATED INTEGRIN ACTIVATION by LIANG ZHU Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Jun Qin Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY January, 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Liang Zhu candidate for the degree of Doctor of Philosophy *. Committee Chair Vivien Yee, Ph.D. Committee Member Jun Qin, Ph.D. Committee Member Edward F. Plow, Ph.D. Committee Member Focco van den Akker, Ph.D. Committee Member Sadashiva S. Karnik, Ph.D. Date of Defense November 28, 2017 *We also certify that written approval has been obtained for any proprietary material contained therein Table of Contents List of Tables ..................................................................................................................... 1 List of Figures .................................................................................................................... 2 Acknowledgement ............................................................................................................. 4 List of Abbreviations ......................................................................................................... 6 Abstract .............................................................................................................................. 9 Chapter 1 Background and Significance ......................................................................... 11 Background ................................................................................................................ 11 Integrin ................................................................................................................. 11 Talin ..................................................................................................................... 15 Rap1 ..................................................................................................................... 18 RIAM ................................................................................................................... 19 PIPKI ................................................................................................................... 22 Significance ............................................................................................................... 24 Chapter 2 Conformational Activation of Talin by RIAM Triggers Integrin-mediated Cell Adhesion .................................................................................................................. 26 Abstract ...................................................................................................................... 27 Introduction ................................................................................................................ 28 Results........................................................................................................................ 31 I A novel RIAM-binding site on talin-F3 .............................................................. 31 RIAM-N binds next to integrin site in talin-F3 to occlude talin-R9 .................... 34 RIAM promotes talin unmasking to activate integrin ......................................... 38 Discussion .................................................................................................................. 42 Materials and Methods .............................................................................................. 44 Chapter 3 Structure of Rap1b Bound to Talin Reveals a Pathway for Triggering Integrin Activation ........................................................................................................................ 48 Abstract ...................................................................................................................... 49 Introduction ................................................................................................................ 50 Results........................................................................................................................ 52 Rap1 recognizes talin F0 domain in a GTP-dependent manner .......................... 52 Rap1b/talin interface exhibits distinct features .................................................... 63 Rap1b binding to talin is crucial for integrin activation ...................................... 75 Membrane anchored Rap1b shows enhanced binding to talin ............................ 82 Discussion .................................................................................................................. 94 Materials and Methods .............................................................................................. 97 Chapter 4 Summary and Future direction ...................................................................... 114 Summary .................................................................................................................. 114 Future direction ........................................................................................................ 117 Reference ....................................................................................................................... 123 II List of Tables Table 3.1 Structural Statistics of the Rap1b/talin-F0 complex ........................................ 69 Table 3.2 Comparison of binding interface between Rap1 and its effector proteins ...... 71 1 List of Figures Figure 1.1 The structural model of integrin activation .................................................... 13 Figure 1.2 The classification of integrins ........................................................................ 14 Figure 1.3 Domain organization and a linear structural model of talin ........................... 17 Figure 1.4 The mechanism of Rap1 activation ................................................................ 20 Figure 1.5 Domain organization of RIAM ...................................................................... 21 Figure 1.6 PIPKIγ peptide and integrin β-tail bind to talin-F3 in a similar mode ........... 23 Figure 2.1 RIAM interacts with talin-F2F3 but not talin-F0F1 ....................................... 32 Figure 2.2 RIAM specifically interacts with talin-H F3 domain ..................................... 33 Figure 2.3 The interface between talin-F3 and RIAM-N ................................................ 35 Figure 2.4 RIAM-N occludes talin-R from binding to talin-F3 and may otherwise promote integrin binding to talin-F3 ................................................................................ 36 Figure 2.5 RIAM promotes talin unmasking to activate integrin .................................... 40 Figure 3.1 Rap1b induces residue-specific chemical shift changes of 15N-labeled talin-F0 ......................................................................................................................................... 54 Figure 3.2 Rap1b/talin-F0 interaction is GTP dependent but of weak affinity ............... 55 Figure 3.3 H-Ras does not interact with talin-F0 ............................................................ 56 Figure 3.4 Kindlin2-F0 does not interact with Rap1b ..................................................... 58 Figure 3.5 Talin-F1F2 does not interact with Rap1b ....................................................... 59 Figure 3.6 Talin-F0 induced residue-specific chemical shift changes of 15N-labeled Rap1b ............................................................................................................................... 60 Figure 3.7 Rap1b recognizes F0 domain of talin in a GTP-dependent manner .............. 62 2 Figure 3.8 Representative intermolecular NOEs for the structure calculation of Rap1b/talin-F0 complex .................................................................................................. 65 Figure 3.9 Solution structure of Rap1b/talin-F0 complex by NMR ................................ 67 Figure 3.10 Detailed binding interface between Rap1b and talin-F0 .............................. 70 Figure 3.11 Rap1b/talin-F0 interaction is modest but highly specific ............................. 72 Figure 3.12 Rap1/talin interaction is evolutionally conserved ........................................ 74 Figure 3.13 Rap1b/talin interaction is drastically reduced by double mutations (K15A, R35A) in talin-F0 domain ................................................................................................ 77 Figure 3.14 Impaired Rap1/talin interaction results in defective integrin activation ...... 78 Figure 3.15 The Rap1b binding to talin is crucial for cell adhesion and spreading ........ 80 Figure 3.16 The mechanism of talin recruitment by Rap1 .............................................. 85 Figure 3.17 Membrane anchored Rap1b robustly enhances its binding to talin-H ......... 86 Figure 3.18 Binding affinity between membrane-anchored Rap1b and talin-H measured by isothermal titration calorimetry (ITC) ........................................................................ 88 Figure 3.19 Membrane anchored Rap1b interacts with talin in a GTP dependent manner ......................................................................................................................................... 91 Figure 3.20 Membrane anchored Rap1b promotes talin unmasking ............................... 93 Figure 3.21 The anchorage of Rap1b to membrane vesicles ........................................