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The Integrin Equilibrium: Balancing Protein-Protein and Protein-Lipid Interactions" (2012)

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The Integrin Equilibrium: Balancing Protein-Protein and Protein-Lipid Interactions University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2012 The Integrin Equilibrium: Balancing Protein-Protein and Protein- Lipid interactions David Thomas Moore University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, and the Biophysics Commons Recommended Citation Moore, David Thomas, "The Integrin Equilibrium: Balancing Protein-Protein and Protein-Lipid interactions" (2012). Publicly Accessible Penn Dissertations. 553. https://repository.upenn.edu/edissertations/553 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/553 For more information, please contact [email protected]. The Integrin Equilibrium: Balancing Protein-Protein and Protein-Lipid interactions Abstract On circulating platelets, the integrin fibrinogen eceptr or &alphaIIb&beta3 favors inactive conformations. Platelets rapidly activate &alphaIIb&beta3 to bind fibrinogen, mediating a platelet clot. Resting &alphaIIb&beta3 is stabilized by interactions between the &alphaIIb and &beta3 transmembrane domains. Binding of talin-1 and kindlin-3 to the integrin cytoplasmic domain stabilizes separation of the TMs and receptor activation. Src family kinases are needed for transmission of extracellular signals into the cell. We have sought to better understand how signals are transmitted across the &alphaIIb&beta3 TM domain. First we characterized the structure and dynamics of the active and inactive integrin cytoplasmic domain to determine how motifs that bind talin-1 and kindlin-3 are affected by the integrin activation state and the membrane environment. The &alphaIIb&beta3 cytoplasmic domain is disorded, while the &beta3 subunit contains two &alpha-helices, which interact with the phospholipid bilayer. The close proximity of &alphaIIb to &beta3 in the inactive state induces a kink that projects the &beta-chain parallel to the membrane surface. This kink is likely to stabilize interactions between helices in &beta3 with the membrane and possibly compete with binding to talin-1 and kindlin-3. The &beta3 cytoplasmic domain becomes increasingly dynamic further from the membrane, suggesting that distal kindlin-3 binding residues should be more accessible to interact with their binding partners. We also studied the interaction between talin-1 and the &beta3 cytoplasmic domain to determine how the membrane environment affects formation of the talin-1/&beta3 complex. The membrane significantly increases the affinity of talin-1 for &beta3, but that the ternary integrin/talin-1/membrane complex is not significantly more stable than the talin-1/membrane complex alone. We also performed preliminary experiments characterizing the interaction between &alphaIIb&beta3 and both kindlin-3 and c-Src. Unlike talin-1, kindlin-3 does not require the membrane to bind the integrin cytoplasmic domain with high affinity. The interaction between c-Src and the integrin was extremely weak suggesting that it might require colocalization through other interactions. Finally, we developed a simplified model showing how thermodynamic coupling between integrin subunits might allow &alphaIIb&beta3 to adopt multiple activation states. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biochemistry & Molecular Biophysics First Advisor William F. DeGrado Keywords Hemostasis, Integrin, Protein-lipid interactions, Protein-protein interactions, Receptor, Transmembrane Subject Categories Biochemistry | Biophysics This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/553 THE INTEGRIN EQUILIBRIUM: BALANCING PROTEIN-PROTEIN AND PROTEIN-LIPID INTERACTIONS David T. Moore A DISSERTATION in Biochemistry and Molecular Biophysics Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2012 Supervisor of Dissertation William F. DeGrado, Professor of Pharmaceutical Chemistry, University of California, San Francisco Graduate Group Chairperson ___________________________ Kathryn M. Ferguson, Associate Professor of Biochemistry and Biophysics Dissertation Committee Joel S. Bennett, Professor of Medicine Rebecca G. Wells, Associate Professor of Medicine John W. Weisel, Professor of Cell and Developmental Biology Carol J. Deutsch, Professor of Physiology Mark A. Lemmon, Professor of Biochemistry and Biophysics Gregory A. Caputo, Assistant Professor of Chemistry and Biochemistry, Rowan University Dedicated to my family. ii ACKNOWLEDGEMENTS This thesis would not have been completed without the close collaboration with other members of the DeGrado lab. NMR studies described in Chapter 2 were performed by Douglas G. Metcalf (DGM) and previously described in his PhD dissertation. Chapter 2 is adapted from a previous publication (1). SPR experiments in Chapter 3 were performed by or with Patrik Nygren. Chapter 3 is adapted from a previous publication (2). The PIPK-Iγ peptide used in Chapter 3 were prepared by Hyunil Jo. Modeling software used in Chapter 4 was originally written by Gevorg Gregorian and edited by David Moore. Chapter 4 is adapted from a previous publication (3). iii ABSTRACT THE INTEGRIN EQUILIBRIUM: BALANCING PROTEIN-PROTEIN AND PROTEIN-LIPID INTERACTIONS David T. Moore William F. DeGrado On circulating platelets, the integrin fibrinogen receptor αIIb β3 favors inactive conformations. Platelets rapidly activate αIIb β3 to bind fibrinogen, mediating a platelet clot. Resting αIIb β3 is stabilized by interactions between the αIIb and β3 transmembrane domains. Binding of talin-1 and kindlin-3 to the integrin cytoplasmic domain stabilizes separation of the TMs and receptor activation. Src family kinases are needed for transmission of extracellular signals into the cell. We have sought to better understand how signals are transmitted across the αIIb β3 TM domain. First we characterized the structure and dynamics of the active and inactive integrin cytoplasmic domain to determine how motifs that bind talin-1 and kindlin-3 are affected by the integrin activation state and the membrane environment. The αIIb cytoplasmic domain is disorded, while the β3 subunit contains two α-helices, which interact with the iv phospholipid bilayer. The close proximity of αIIb to β3 in the inactive state induces a kink that projects the β-chain parallel to the membrane surface. This kink is likely to stabilize interactions between helices in β3 with the membrane and possibly compete with binding to talin-1 and kindlin-3. The β3 cytoplasmic domain becomes increasingly dynamic further from the membrane, suggesting that distal kindlin-3 binding residues should be more accessible to interact with their binding partners. We also studied the interaction between talin-1 and the β3 cytoplasmic domain to determine how the membrane environment affects formation of the talin- 1/ β3 complex. The membrane siginificantly increases the affinity of talin-1 for β3, but that the ternary integrin/talin-1/membrane complex is not significantly more stable than the talin-1/membrane complex alone. We also performed preliminary experiments characterizing the interaction between αIIb β3 and both kindlin-3 and c-Src. Unlike talin-1, kindlin-3 does not require the membrane to bind the integrin cytoplasmic domain with high affinity. The interaction between c-Src and the integrin was extremely weak suggesting that it might require colocalization through other interactions. Finally, we developed a simplified model showing how thermodynamic coupling between integrin subunits might allow αIIb β3 to adopt multiple activation states. v Table of Contents Chapter 1 Background ............................................................................... 1 I. Introduction ......................................................................................... 1 II. Platelet integrins. ............................................................................... 2 Chapter 2 NMR analysis of the αIIb β3 cytoplasmic interaction suggests a mechanism for integrin regulation. ............................................................. 8 Abstract .................................................................................................. 8 I. Introduction ........................................................................................ 9 II. Results ............................................................................................ 11 Model αIIb β3 cytoplasmic domain constructs. .................................. 11 Association of the αIIb and β3 cytoplasmic domains influences their conformation. .................................................................................... 17 Structure of the β3 cytoplasmic domain crosslinked to the αIIb peptide ......................................................................................................... 21 Conformational flexibility of the αIIb β3 cytoplasmic domains in micelles ............................................................................................ 23 Conformational flexibility of the αIIb β3 cytoplasmic domain in phospholipid bilayers. ....................................................................... 26 III. Discussion ...................................................................................... 31 IV. Methods .......................................................................................... 37 Supplemental ......................................................................................
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