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Thesis Draft Sanjana Saravanan A Nascent Helix in Dynein Intermediate Chain is Essential for Regulating Dynein/Dynactin Complexes by Sanjana Saravanan A THESIS submitted to Oregon State University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in Bioengineering (Honors Scholar) Presented March 11, 2019 Commencement June 2019 1 2 AN ABSTRACT OF THE THESIS OF Sanjana Saravanan for the degree of Honors Baccalaureate of Science in Bioengineering presented on March 11, 2019. Title: A Nascent Helix in Dynein Intermediate Chain is Essential for Regulating Dynein/Dynactin Complexes Abstract approved: _____________________________________________________ Elisar Barbar Cytoplasmic dynein is a motor protein complex found in eukaryotes that is essential to many cellular processes. With dynein being involved in mitosis, axonal transport and organelle transport, disruption of dynein function can lead to neurodegeneration and other diseases. The main function of dynein is transportation of cargo through the attachment of a cofactor or regulatory protein, like dynactin. Many of dynein’s functions are regulated by binding of dynein intermediate chain (IC) to dynactin p150. This interaction involves a single alpha helix (SAH) at the N-terminus of IC and the coiled-coil region (CC1B) on p150. Previous studies in the Barbar lab have shown that there is a secondary helix (H2) downstream of SAH that is structurally varied among the species of IC. This study looks closely at IC from Chaetomium thermophilum (CT), and experiments reveal that it has a weak H2 and substantially stronger binding to p150. The data from this study describe the role of H2 in binding and is the first to show direct interactions between H2 and p150. A combination of results from isothermal titration calorimetry (ITC), circular dichroism (CD) and nuclear magnetic resonance (NMR) are used to map the binding site of CT IC on p150. Key Words: Cytoplasmic dynein, dynein intermediate chain, dynactin p150, secondary helix, intermediate chain and p150 binding Corresponding e-mail address: [email protected] 3 ©Copyright by Sanjana Saravanan March 11, 2019 4 A Nascent Helix in Dynein Intermediate Chain is Essential for Regulating Dynein/Dynactin Complexes by Sanjana Saravanan A THESIS submitted to Oregon State University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in Bioengineering (Honors Scholar) Presented March 11, 2019 Commencement June 2019 5 Honors Baccalaureate of Science in Bioengineering project of Sanjana Saravanan presented on March 11, 2019. APPROVED: Elisar Barbar, Mentor, representing Department of Biochemistry and Biophysics Afua Nyarko, Committee Member, representing Department of Biochemistry and Biophysics Patrick Reardon, Committee Member, representing OSU NMR Facility _____________________________________________________________________ Toni Doolen, Dean, Oregon State University Honors College I understand that my project will become part of the permanent collection of Oregon State University, Honors College. My signature below authorizes release of my project to any reader upon request. _____________________________________________________________________ Sanjana Saravanan, Author 6 Introduction Cytoplasmic dynein is a 1.6 MDa motor complex found along the surface of microtubules inside cells in eukaryotes (Kardon and Vale 2009; Vallee et al. 2004, 2012). Dynein is responsible for the retrograde transport of organelles and other cellular cargo and is essential for a number of cellular processes (Kardon and Vale 2009; Vallee et al. 2004, 2012). These processes include centrosome separation during mitosis, mitotic spindle assembly, and axonogenesis (Kardon and Vale 2009; Vallee et al. 2004). The complex is composed of multiple subunits that form the motor domain and the cargo attachment domain (Jie et al. 2015). The motor domain is made of the heavy chain subunit that is activated by ATP to generate dynein movement (Jie et al. 2015). The cargo attachment domain is used to load cargo, maintain stability and modulate dynein activity (Kini and Collins 2001). Multiple chains assemble to form the cargo attachment domain, one of which is the intermediate chain (IC). This subunit is one of many key components in dynein assembly and function (Makokha et al. 2002). Dynein carries out its functions by binding to various cofactors and regulatory proteins (Vallee et al. 2012), including dynactin, a 1.0 MDa protein complex composed of more than 20 subunits (Urnavicius et al. 2015). Binding of dynein to dynactin involves interactions of IC and p150, the largest subunit of dynactin (Jie et al. 2015). With dynein being involved in numerous critical cellular processes, inhibition of dynein severely disrupts normal function of cells. Failed interaction between dynein and dynactin has been shown to cause motor neuron degeneration resembling ALS (Moore et al. 2009). Axonal transport, autophagy and the clearance of aggregated proteins are also affected by defective dynein regulation, leading to several different neurodegenerative diseases, such as Huntington’s, Parkinson’s and Alzheimer’s diseases (Eschbach and Dupuis 2011). Interaction between dynein and p150 occurs at the intrinsically disordered N-terminal region of IC. Intrinsic disorder is a unique property of a class of proteins that are flexible, multivalent, and lack a fixed 3D structure (Clark et al. 2015). Intrinsically disordered proteins (IDPs) like IC maintain partial disorder even when interacting with partners, providing flexibly and functional versatility (Benison et al. 2006; Clark et al. 2015; Tompa and Fuxreiter 2008). Previous studies have shown that binding to p150 involves a single a-helix (SAH) at the N-terminus in IC from rat, Drosophila, and yeast (Jie et al. 2017, 2015). This structure is highly conserved among IC homologs (Jie et al. 2015). There is, however, a second helix present in each sequence of IC, indicated by NMR data (Jie et al. 2015; Morgan et al. 2011). This helix is nascent in Drosophila and yeast IC, and strong in rat IC (Figure 1A). Isothermal titration calorimetry (ITC) was used to examine the role of helix 2 (H2) in binding to p150 by removing it, which showed that it had no effect in rat and caused weakened binding in yeast (Jie et al. 2017, 2015). It seems that a strong H2 does not contribute to binding of p150 at SAH, while a weak H2 does play a role. Additionally, yeast IC binds more tightly to p150 than rat (Jie et al. 2017, 2015). There is no evidence that H2 in any of these species directly interacts with p150, but it may have a role in regulating interactions with SAH. 7 A 1 38 52 66 Tctex/Lc8 LC7 259 612 IC2C WD 40 Light Chain Binding Site 1 41 49 63 289 642 Predicted Light Chain Binding Site Dros IC WD 40 Alpha Helix 1 24 66 73 385 533 Pac11 WD 40 Transient/Weak Alpha Helix Dyn2 Dyn2 B 1 30 50 60 323 707 CTIC WD 40 IC88 IC35 C Cap-Gly CC1A CC1B CC2 1 48 90 217 350 548 926 1049 1278 p150Glued ICD p150* D 1 23 65 343 478 715 1071 1215 1400 CT p150 ICD CT p150ABC 100 50 Coiled Coil Propensity 0 478 680 p150ABC p150A p150B p150C p150AB p150BC p150 486-645 Figure 1. Dynein Intermediate Chain and Dynactin p150 Domain Architecture (A) IC from rat (IC2C), Drosophila (Dros IC), and yeast (IC homolog Pac 11) have an N-terminus single a-helix (SAH) as well as a second helix (H2) (Jie et al. 2015). H2 is either strong (black helix) or nascent (grey helix). Rat and Drosophila IC have binding sites for three light chains: Tctex, LC8, and LC7, while yeast IC has binding site for two LC8 homolog copies (DYN2). The C-terminal is predicted to be a WD repeat domain (WD40). (B) IC from Chaetomium thermophilum (CT IC) is predicted to have a SAH and a nascent H2. Based on sequence motifs, the Tctex, LC8 and LC7 binding sites are predicted to be in regions similar to Dros IC. IC88 and IC35 show the constructs used in this paper. (C) Sequence domains in mammalian p150 (p150Glued) show a Cap-Gly domain at the N-terminus. Two coiled-coil regions, CC1 and CC2, occur further along in the protein and are separated by an intercoiled domain (ICD). CC1 is further divided into two domains called CC1A and CC1B. Previous studies have shown that the IC binding site is located within the CC1B region. The p150* construct was used in previous studies investigating the binding of mammalian IC and p150. (D) CT p150 is predicted to have similar sequence domains to mammalian p150. The COILS Server prediction tool (Lupas et al. 1991) was used to predict the coiled-coil propensity of each residue in p150ABC, one of the constructs used in this study. This prediction was used to create the smaller constructs. Comparing the secondary structures of a selection of ICs from a range of species revealed that H2 is a conserved structural feature. The secondary structure prediction algorithm from Agadir (Lacroix et al. 1998; Muñoz et al. 1994a-c, 1997) allowed us to map the size and location of H2 in species of varying complexity. The species Chaetomium thermophilum (CT), a thermophilic fungus, stood out as a model organism based on a very weak H2 prediction (Figure 2A). CT has been used in studies involving thermostable proteins due to its ability to grow at temperatures up to 60° C (Kellner et al. 2016). In this study we investigate the function and contributions of H2 in binding to p150, using IC from Chaetomium thermophilum. The biophysical techniques used 8 in this study answer two major questions: (1) How does H2 folding correlate with species complexity? (2) If and how is H2 involved in regulating interactions with p150? ITC, together with circular dichroism (CD) and nuclear magnetic resonance (NMR), were used to provide insight to the residue level interactions between various constructs of CT IC and p150.
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