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Modular Design of Coiled Coils to Target Bzip Transcription Factors

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Modular Design of Coiled Coils to Target bZIP Transcription Factors by Jenifer Kaplan B.S. Molecular and Cellular Biology B.A. Mathematics Johns Hopkins University, Baltimore, MD, 2008 SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY FEBRUARY 2014 Massachusetts Institute of Technology. All rights reserved. Signature of Author: _________________________________________________ Department of Biology February 3, 2014 Certified by:______________________________________________________________ Amy Keating Associate Professor of Biology Thesis Supervisor Accepted by:_____________________________________________________________ Stephen Bell Professor of Biology Co-Chair, Biology Graduate Committee 1 2 Modular Design of Coiled Coils to Target bZIP Transcription Factors by Jenifer Kaplan Submitted to the Department of Biology On February 3, 2014 in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology at the Massachusetts Institute of Technology Abstract Basic leucine-zipper (bZIP) transcription factors regulate many important cellular processes including tissue differentiation, stress responses and the unfolded protein response, the cell cycle, and apoptosis. Understanding the genes and processes regulated by bZIPs is imperative for understanding different diseases including diabetes and cancer, but there is still much unknown about how certain bZIPs function. Reagents capable of studying bZIP-regulated processes are therefore needed to specifically target the proteins under study. Recent work suggests that previous reagents, including siRNAs and dominant-negative bZIP mutants, may not have been as specific for the target bZIP as intended. In an effort to develop new reagents capable of specifically interacting with target bZIPs, I tested two protein design methods to determine whether they could successfully generate tight and specific binders of the leucine-zipper coiled-coil domain. Both methods tested take advantage of the structure and biophysical properties of a coiled coil. In the coiled-coil dimer, two helices wrap around each other into a super helix. At the sequence level, coiled coils have a repeating heptad sequence with seven positions denoted (a-b-c-d-e-f-g) and typically a hydrophobic residue at a and d positions. Due to a buried hydrophobic interface between the helices, individual coiled coils are unfolded in solution and only fold upon binding to a partner. The first method used to design peptides that would bind tightly and specifically to bZIPs depended on the coupled binding and folding within coiled coils. In a previous study, core positions of the peptide pointing inward to the coiled-coil interface were optimized for stably and specifically binding to the target, as these positions have been shown to be primarily responsible for specificity and affinity of interactions. The solvent-exposed positions were designed to complement the core positions. I measured the affinitiy and specificity of some of these designed peptides for their bZIP target. I then redesigned the solvent-exposed positions to include more helix-promoting residues that would increase the affinity of the interaction between the designed peptide and target bZIP. Using a solution FRET assay to test both the original and redesigned peptide’s affinity for the target and 30 off-target bZIPs, I showed that redesigning the solvent-exposed positions did stabilize the design-target interaction from 3-fold to 90-fold but the redesign process also changed the specificity of the peptide. The second design method reduced the full coiled-coil interaction into interactions between individual heptads. Each heptad in the designed peptide was predicted to bind tightly and specifically to the corresponding heptad in the target bZIP. Using this design method, tight and very specific peptides were generated targeting different bZIPs and shown to be potent inhibitors. Finally, I proposed how these two methods can be combined to generate more tight and specific binders of bZIPs that can be used to reveal new insights into genes and cellular processes regulated by bZIPs. Thesis Supervisor: Amy Keating Title: Associate Professor of Biology 3 Acknowledgements I would first like to think my advisor, Amy Keating, for everything. There are too many things to list, but thank you for your constant enthusiasm, guidance, support, advice, efforts in turning me into a better scientific communicator, and wonderful lab environment. I have truly enjoyed my time in the lab and I hope future scientific experiences are just as enjoyable. I would also like to thank Bob Sauer and Thomas Schwartz for having served on my committee since my second year. Both were very generous with comments and technical advice during my committee meetings, and it was all much appreciated. Thank you also to Krishna Kumar for agreeing to serve at my defense. To the members past and present of the Keating lab: Raheleh Rezaei Araghi, Orr Ashenberg, Judy Baek, Scott Chen, Jeremy Curuksu, Joe DeBartolo, Sanjib Dutta, Emiko Fire, Glenna Foight, Karl Gutwin, Seungsoo Hahn, Karl Hauschild, Justin Jenson, Yong Ho Kim, Christos Kougentakis, Chris Negron, Vladimir Potapov, Luther Reich, Aaron Reinke, Josh Sims, Evan Thompson, Vincent Xue, and Nora Zizlsperger. Thank you all for making the lab a truly awesome place to work. I’ve enjoyed being with you all early in the morning to late nights on weekends and in between. Both inside and outside the lab, you’ve all been great companions. I would especially like to thank Orr and Aaron. Both have been incredibly helpful and freely giving of knowledge, technical expertise, and reagents, and I would likely not have reached this point without them. I’d also like to thank the Baker, Bell, Walker, Sauer, and Laub labs for use of equipment. To Nathalia and Ala in the Biopolymers Facility, thank you for all the peptides and MALDI analysis. To Debby Pheasant in the BIF, thank you for all the assistance with cleaning and building AUC cells. To my friends outside of lab and Biograd 2008, thank you for pulling me away from lab and reminding me there’s more to life than science. The fun times we shared will not be forgotten. And finally, I’d like to thank my family: my parents for always encouraging me to be myself and pursue my interests, and my sisters for encouraging me to play with my beakers. I did it Merm! 4 5 Table of Contents List of Figures……………………………………………………………………………………..9 List of Tables………………………………………………………………………………….....11 Chapter 1 Introduction…………………………………………………………………………...13 Repeat proteins…………………………………………………………………………...16 Ankyrin repeats…………………………………………………………………..16 Armadillo repeats………………………………………………………………...17 Tetratricopeptide repeat……………………………………………………….....19 Coiled-coil domain……………………………………………………………….21 Basic leucine-zipper transcription factors………………………………………………..23 FOS and JUN…………………………………………………………………….26 Activating Transcription Factor (ATF) 4 and ATF5…………………………….27 The large MAF family: MAF and MAFB……………………………………….29 X-box Binding Protein 1 (XBP1), ATF6, and CREBZF………………………...31 Targeting bZIPs………………………………………………………………………….33 Gene knockouts and knockdowns………………………………………………..33 Small-molecule inihibitors……………………………………………………….34 Rational design of dominant-negative bZIP mutants…………………………….36 Library selection of designed coiled coils……………………………………….38 Computational design of coiled coils…………………………………………….39 Experimental methods to measure coiled-coil interactions……………………………...41 Circular Dichroism (CD) spectroscopy………………………………………….41 Calorimetry………………………………………………………………………42 6 Electrophoresis mobility-shift assay (EMSA) ...………………………………...43 Coiled-coil arrays………………………………………………………………...43 References………………………………………………………………………………..46 Chapter 2 Increasing the affinity of selective bZIP-binding peptides through surface residue redesign…………………………………………………………………………………………..55 Introduction………………………………………………………………………………56 Results……………………………………………………………………………………61 Solution characterization of original designed anti-bZIP peptides………………61 Design and testing of surface-redesigned anti-bZIP peptides……………………66 Peptides designed to bind to XBP1………………………………………………67 Peptides designed to bind to ATF6 and FOS…………………………………….73 Discussion………………………………………………………………………………..76 Additional tables…………………………………………………………………………80 Methods…………………………………………………………………………………..82 References………………………………………………………………………………..89 Chapter 3 Data-driven prediction and design of bZIP coiled-coil interactions………………….93 Introduction………………………………………………………………………………94 Results……………………………………………………………………………………96 Model benchmarking………………………………………………………….....96 Designing specific binders…………………………………………………….....97 Discussion………………………………………………………………………………106 Additional tables………………………………………………………………………..109 Methods…………………………………………………………………………………112 7 References………………………………………………………………………………119 Chapter 4 Conclusions and future directions…………………………………………………...121 Summary of design methods……………………………………………………………122 Surface-core modularity………………………………………………………...122 Heptad assembly………………………………………………………………..124 Combining heptad assembly and surface-core modularity……………………………..125 Anti-bZIPs as reagents for understanding bZIP function………………………………129 References………………………………………………………………………………132 Appendix A Characterization of original designed anti-bZIP peptides anti-LMAF, anti-LMAF-3, anti-JUN, anti-CREB-3, and anti-CREBZF-2………………………………………………….134 Characterization of the
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