Small Ankyrin-1 and Obscurin as a Model for Ankyrin Binding Motifs Item Type dissertation Authors Willis, Chris Publication Date 2011 Abstract Small ankyrin-1 binds with high affinity to two regions within obscurin A. This interaction provides a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscle. Here, I show that four hydrophobic residues within the hydro... Keywords ankyrin binding motifs; hydrophobic interactions; obscurin; protein-protein interaction; small ankyrin-1; Ankyrins; Hydrophobic and Hydrophilic Interactions Download date 03/10/2021 19:07:22 Item License https://creativecommons.org/licenses/by-nc-nd/4.0/ Link to Item http://hdl.handle.net/10713/683 Abstract Title of Dissertation: Small Ankyrin-1 and Obscurin as a Model for Ankyrin Binding Motifs Chris D. Willis, Doctor of Philosophy, 2011 Dissertation Directed by: Robert J. Bloch, Ph.D., Professor, Department of Physiology Small ankyrin-1 binds with high affinity to two regions within obscurin A. This interaction provides a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscle. Here, I show that four hydrophobic residues within the hydrophobic “hotspot” of the ankyrin-like repeats of sAnk1 are involved in binding obscurin. Alanine scanning mutagenesis of each of the four residues inhibits binding to the high affinity binding site, Obsc6316-6345, whereas two of the mutations had no effect on binding to the lower affinity site, Obsc6231-6260. Mutagenesis identified three central residues within Obsc6316-6345 that are critical for binding sAnk1, but no single residue within Obsc6231-6260 that is essential. Instead, only a triple mutant of neighboring residues of Obsc6231-6260 decreases binding. This suggests the positional importance of the hydrophobic residues within the central region of Obsc6316-6345 that have a direct role in docking to sAnk1, in contrast to a more general, hydrophobic contribution of the hydrophobic residues of Obsc6231-6260. Modeling of sAnk1 and Obsc6316-6345 are consistent with the idea that specific hydrophobic residues interact in the docked complex. Using the identified hydrophobic and previously reported charged amino acids of obscurin involved in binding, I identify two classes of ankyrin binding motifs (ABMs) The first, type 1, contains ~35% α-helix, between two less structured regions. The second, type 2, contains~17% α-helix at its amino-terminal, flanked by a disordered carboxy-terminal sequence. Using a custom matrix, I classify several known ABMs as type 1 or 2 by structural comparison and sequence alignment within the ABM-specific matrix. I use this matrix to predict ABMs in proteins that bind ankyrins. This method identified a type 1 ABM in histone deacetylase-4 and in retinoblastoma protein that bind their ligands, RFXAnk and gankyrin, respectively. I confirm this binding with in vitro assays and the ~30% helical content of retinoblastoma by circular dichroism. The method also identified a type 2 ABM in the retinoblastoma protein that binds gankyrin in vitro and has ~15% helical content. I conclude that my methods can be used to predict ABMs from primary sequence which will be applicable to the prediction of other protein-protein interactions. Small Ankyrin-1 and Obscurin as a Model for Ankyrin Binding Motifs by Chris D. Willis Dissertation submitted to the faculty of the Graduate School of the University of Maryland, Baltimore in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2011 ACKNOWLEDGMENTS First and foremost, I must state how lucky I have been to be surrounded by such a supportive laboratory. This stems from the leadership at the top, and my mentor Dr. Robert Bloch. I am both honored and humbled that such an accomplished scientist was willing to mentor a graduate student who had never taken a physiology course. With his guidance, I have learned more than I ever thought possible about the skeletal muscle field as well as the invaluable tool of what productive research entails. Any of my future success will be a direct result of the solid foundation Dr. Bloch has instilled in me. During my career in the Bloch laboratory, I have been around amazing scientists who have always been there to help me and my research. I cannot express my gratitude toward every past and current member who has assisted me but in particular I would like to thank Dr. Ben Busby who has been a both a great laboratory partner and friend. The projects we have been involved in together have contributed to my skill set and professional accomplishments, which I am very proud to present on my CV. The biggest supporting cast in my graduate career must be my family and friends. My parents have been so generous to allow me to receive such a fine education which paved the way for continued success in graduate school. Hearing how proud the completion of my dissertation makes them is as rewarding as it is personally. Part of my rationale for selecting UMB was to be close to my best friend, my little brother while he was at Towson. Thanks to him, I had some of my best times while in Baltimore when he would come downtown to visit. Lastly, my girlfriend Jenn who I have been with through this whole experience deserves all the credit in the world for making it work 100 miles away. iii Table of Contents I. Introduction………………………………………………………………………................1 a. Skeletal Muscle Biology………………………………………………………..1 1. Physiological Overview………………………………………………...1 2. The Sarcomere…………………………………………………………..2 3. Molecular Mechanism of Muscle Contraction………………………….5 4. Myofibrillogenesis and Development…………………………………...7 b. Obscurin………………………………………………………………………..11 1. Gene Product…………………………………………………………….11 2. Protein Domains…………………………………………………………12 3. Localization in striated muscle…………………………………………..14 4. Functional Roles………………………………………………………....16 5. Obscurin Binding Partners…………………………………………….…18 i. The interaction with Small Ankyrin-1………………..………...…….23 c. The Ankyrin and Ankyrin-Like Repeat Superfamily…………………………..29 1. Consensus Ankyrin Repeats……………………………………………...29 2. Role in Striated Muscle (isoforms, cytoskeletal linkage, regulation of membrane systems) ……………………………………………………...36 3. Muscle Specific Ankyrin Isoforms…………………………………….…37 i. Small Ankyrin-1…………………………………………….………..38 4. Gankyrin …………………………………………………………………43 5. RFXANK………………………………………………………………...45 6. DARPins as Therapeutics………………………………………………...47 d. Ankyrin Binding Proteins………………………………………………………47 1. Co-crystal structures……………………………………………………...48 2. Nuclear Factor Kappa-Light-Chain-Enhancer……………………………49 3. Retinoblastoma…………………………………………………………...49 4. Histone Deacetylase 4…………………………………………………….50 e. Hypotheses and Significance of this Project……………………………………51 II. Hydrophobic Interactions Mediate Binding of Obscurin and Small Ankyrin 1………...54 iv a. Abstract…………………………………………………………………………..54 b. Introduction……………………………………………………………………...55 c. Materials and Methods…………………………………………………………..57 d. Results…………………………………………………………………………...65 e. Discussion………………………………………………………………………..79 f. Supplemental Addendum………………………………………………………..85 III. Two Types of Ankyrin Binding Motifs Dictate Interaction with Ankyrin Repeat Proteins…………………………………………………………………………………….95 a. Abstract………………………………………………………………………….95 b. Introduction……………………………………………………………………..96 c. Materials and Methods………………………………………………………….98 d. Results…………………………………………………………………………..102 e. Discussion……………………………………………………………………….115 IV. Future Directions and Conclusions………………………………………………………...123 a. Physiological Significance of Two Obscurin Binding Sites for sAnk1……….123 b. Attempts to Obtain Structural Information on sAnk1 by NMR……………….125 c. Role of Divalent Cations………………………………………………………128 d. A Single ABM Can Bind to Different AR Proteins…………………………...132 e. Ankyrin-Like Repeats…………………………………………………………138 f. Concluding Remarks…………………………………………………………..140 Bibliography…………………………………………………………………………………...143 v List of Figures and Tables: Figure 1.1. Distinct compartments and myofilaments which make up the sarcomere…………..2 Figure 1.2. Identified protein binding partners of titin………………………………..................4 Figure 1.3. The premyofibril model of myofibrillogenesis……………………………………...9 Figure 1.4. Identified isoforms of obscurin……………………………………………………..13 Figure 1.5. Localization pattern of obscurin A in skeletal muscle……………………………...15 Figure 1.6. Primary sequence analysis of ankyrin isoforms including sAnk1………………….22 Figure 1.7. Kinetics of binding of GST-Obsc6231-6260 and GST-Obsc6316-6345 to MBP-sAnk129- 155………………………………………………………………………………………………...25 Figure 1.8. Docked model of Obsc6322-6339 and sAnk157-122…………………………………….26 Figure 1.9. Alpha helical content of peptides for Obsc6316-6345 versus Obsc6231-6260…………….28 Figure 1.10. The primary sequence and secondary structure of a consensus ankyrin repeat…...30 Figure 1.11. The secondary structure of Notch1 with the consensus ankyrin repeats…………..33 Figure 1.12. Sequence alignment of the amino acids of tankyrase C-terminal to the twelfth consensus ankyrin repeat………………………………………………………………………...34 Figure 1.13. p53bp2 protein isoforms secondary structure in complex with p53………………36 Figure 1.14. The effect of siRNA on network SR proteins……………………………………..40 Figure 1.15. Refined model of sAnk1 ALRs following molecular dynamics simulations……...42 Figure 1.16. The secondary structure of the ARD protein gankyrin……………………………44 Figure 1.17. The predicted secondary structure N-terminal to the ARs of RFXANK…………46 Figure 2.1. Images of sAnk-1 alone and the sAnk-157-122:Obsc6322-6339