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Unveiling the Molecular Mechanisms Regulating the Activation of the Erbb Family Receptors at Atomic Resolution Through Molecular Modeling and Simulations

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Unveiling the Molecular Mechanisms Regulating the Activation of the Erbb Family Receptors at Atomic Resolution Through Molecular Modeling and Simulations University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Spring 2011 Unveiling the Molecular Mechanisms Regulating the Activation of the ErbB Family Receptors at Atomic Resolution through Molecular Modeling and Simulations Andrew Shih University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biophysics Commons, Other Biomedical Engineering and Bioengineering Commons, and the Structural Biology Commons Recommended Citation Shih, Andrew, "Unveiling the Molecular Mechanisms Regulating the Activation of the ErbB Family Receptors at Atomic Resolution through Molecular Modeling and Simulations" (2011). Publicly Accessible Penn Dissertations. 302. https://repository.upenn.edu/edissertations/302 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/302 For more information, please contact [email protected]. Unveiling the Molecular Mechanisms Regulating the Activation of the ErbB Family Receptors at Atomic Resolution through Molecular Modeling and Simulations Abstract The EGFR/ErbB/HER family of kinases contains four homologous receptor tyrosine kinases that are important regulatory elements in key signaling pathways. To elucidate the atomistic mechanisms of dimerization-dependent activation in the ErbB family, we have performed molecular dynamics simulations of the intracellular kinase domains of the four members of the ErbB family (those with known kinase activity), namely EGFR, ErbB2 (HER2) and ErbB4 (HER4) as well as ErbB3 (HER3), an assumed pseudokinase, in different molecular contexts: monomer vs. dimer, wildtype vs. mutant. Using bioinformatics and fluctuation analyses of the molecular dynamics trajectories, we relate sequence similarities to correspondence of specific bond-interaction networks and collective dynamical modes. We find that in the active conformation of the ErbB kinases (except ErbB3), key subdomain motions are coordinated through conserved hydrophilic interactions: activating bond-networks consisting of hydrogen bonds and salt bridges. The inactive conformations also demonstrate conserved bonding patterns (albeit less extensive) that sequester key residues and disrupt the activating bond network. Both conformational states have distinct hydrophobic advantages through context-specific hydrophobic interactions. The inactive ErbB3 kinase domain also shows coordinated motions similar to the active conformations, in line with recent evidence that ErbB3 is a weakly active kinase, though the coordination seems to arise from hydrophobic interactions rather than hydrophilic ones. We show that the functional (activating) asymmetric kinase dimer interface forces a corresponding change in the hydrophobic and hydrophilic interactions that characterize the inactivating interaction network, resulting in motion of the αC-helix through allostery. Several of the clinically identified activating kinase mutations of EGFR act in a similar fashion to disrupt the inactivating interaction network. Our molecular dynamics study reveals the asymmetric dimer interface helps progress the ErbB family through the activation pathway using both hydrophilic and hydrophobic interaction. There is a fundamental difference in the sequence of events in EGFR activation compared with that described for the Src kinase Hck. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Bioengineering First Advisor Ravi Radhakrishnan Keywords ErbB family, kinase activation, molecular dynamics, hydrophilic and hydrophobic interactions Subject Categories Biophysics | Other Biomedical Engineering and Bioengineering | Structural Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/302 UNVEILING THE MOLECULAR MECHANISMS REGULATING THE ACTIVATION OF THE ERBB FAMILY RECEPTORS AT ATOMIC RESOLUTION THROUGH MOLECULAR MODELING AND SIMULATIONS Andrew J. Shih A DISSERTATION in Bioengineering Presented to the Faculties of the University of Pennsylvania In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2011 Supervisor of Dissertation Dr. Ravi Radhakrishnan, PhD, Associate Professor of Bioengineering Graduate Group Chair Dr. Christopher S. Chen, MD, PhD, Professor of Bioengineering Dissertation Committee Dr. Casim A. Sarkar, PhD, Assistant Professor of Bioengineering Dr. Mark A. Lemmon, PhD, Professor of Biochemistry and Biophysics Dr. Jeffery G. Saven, PhD, Associate Professor of Chemistry Acknowledgements First and foremost my sincerest gratitude to my advisor, Professor Ravi Radhakrishnan, for guiding me and staying with me through the many twists and turns (and sometimes dead ends) in exploring the EGFR kinase that eventually expanded into the other ErbB members. His passion and creativity made it a joy delving deeper into the activation mechanisms of kinases. A special thanks to Professor Casim Sarkar, Professor Mark Lemmon and Professor Jeff Saven, as my thesis committee members and advisors: for being understanding during my first forays into molecular biology as well as for advice inside and outside my field of expertise, giving me the breadth and scope of knowledge for proper perspective. I wish to thank the Sung Hee Choi and Fumin Shi of the Lemmon Lab for all the insights into the ErbB family kinases. Also, I greatly appreciate all the lab members of the Radhakrishnan lab for an enjoyable research environment and knowledgeable discussions. In particular, I would like to thank Shannon Telesco and Yingting Liu for help with new programs and processing developments as we intensified our studies of EGFR and other members of the ErbB family. Beyond the lab, this work could not have possible without Alice Chau and Yuuai, for unquestioned support, keeping me grounded and serving as sounding board to my (sometimes crazy) ideas. Finally, I want to thank my family for everything they have done to make me who I am. ii Abstract Unveiling the Molecular Mechanisms Regulating the Activation of the ErbB Family Receptors at Atomic Resolution through Molecular Modeling and Simulations Andrew Shih Advisor: Ravi Radhakrishnan The EGFR/ErbB/HER family of kinases contains four homologous receptor tyrosine kinases that are important regulatory elements in key signaling pathways. To elucidate the atomistic mechanisms of dimerization-dependent activation in the ErbB family, we have performed molecular dynamics simulations of the intracellular kinase domains of the four members of the ErbB family (those with known kinase activity), namely EGFR, ErbB2 (HER2) and ErbB4 (HER4) as well as ErbB3 (HER3), an assumed pseudokinase, in different molecular contexts: monomer vs. dimer, wildtype vs. mutant. Using bioinformatics and fluctuation analyses of the molecular dynamics trajectories, we relate sequence similarities to correspondence of specific bond-interaction networks and collective dynamical modes. We find that in the active conformation of the ErbB kinases (except ErbB3), key subdomain motions are coordinated through conserved hydrophilic interactions: activating bond-networks consisting of hydrogen bonds and salt bridges. The inactive conformations also demonstrate conserved bonding patterns (albeit less extensive) that sequester key residues and disrupt the activating bond network. Both conformational states have distinct hydrophobic advantages through context-specific hydrophobic interactions. The inactive ErbB3 kinase domain also shows coordinated iii motions similar to the active conformations, in line with recent evidence that ErbB3 is a weakly active kinase, though the coordination seems to arise from hydrophobic interactions rather than hydrophilic ones. We show that the functional (activating) asymmetric kinase dimer interface forces a corresponding change in the hydrophobic and hydrophilic interactions that characterize the inactivating interaction network, resulting in motion of the αC-helix through allostery. Several of the clinically identified activating kinase mutations of EGFR act in a similar fashion to disrupt the inactivating interaction network. Our molecular dynamics study reveals the asymmetric dimer interface helps progress the ErbB family through the activation pathway using both hydrophilic and hydrophobic interaction. There is a fundamental difference in the sequence of events in EGFR activation compared with that described for the Src kinase Hck. iv Table of Contents List of Tables .................................................................................................................. viii List of Figures ................................................................................................................... ix Chapter 1.) Regulation of ErbB Receptor Tyrosine Kinases ........................................ 1 1.1) RTK Structure and Signaling ................................................................................ 1 1.2) Regulation of RTK signaling duration at the cell surface ..................................... 3 1.3) Regulation, Structure and Auto-inhibition of RTKs ............................................. 5 1.4) Kinase Domain Mutations in Cancer and their Therapeutic Importance .............. 8 Chapter 2.) Computational Methods Related to Reaction Chemistry ....................... 15 2.1) Introduction ......................................................................................................... 15 2.2) Computational Methods .....................................................................................
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