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

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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]

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©Copyright by Sanjana Saravanan March 11, 2019

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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

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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.

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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

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Coiled Coil Propensity 0 478 680

p150ABC

p150A

p150B

p150C

p150AB

p150BC p150486-645 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

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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. ITC experiments tell us if and how strongly two proteins bind, as well as the thermodynamic parameters of the interaction. The secondary structure and changes that occur upon binding were captured using CD spectra. Finally, NMR was able to verify the secondary structure of CT IC and elucidate and map the sites of interactions between IC and p150.

Methods

Sequence Selection for Agadir Predictions

Sequences for dynein intermediate chain from a range of species were obtained from the UniProt protein database (Apweiler et al. 2004). Species with available biophysical data were automatically selected, while other species were included to fill in the phylogenetic spectrum. IC from three species have been used in previous studies: Rattus norvegicus (rat), Drosophila melanogaster (fruit fly) and Saccharomyces cerevisiae (yeast). Six other species used were: Homo sapiens (human), Danio rerio (zebrafish), Callorhinchus milii (Australian ghostshark), Octopus bimaculoides (Californian two-spot octopus), Caenorhabditis elegans (nematode) and Chaetomium thermophilum (thermophilic fungus). The first one hundred amino acids of IC from each species were scored via the Agadir algorithm, which outputs a prediction for percent helicity at the residue level (Lacroix et al., 1998; Muñoz et al., 1994a-c, 1997).

Protein Expression and Purification

The CT IC88, IC35, p150ABC (res. 478-680), p150486-645, p150AB (res. 478-622), p150BC (res. 542- 680), p150A (res. 478-541), p150B (res. 542-622) and p150C (res. 623-680) constructs were prepared by PCR and cloned into a pET24d vector with an N terminus His6 tag using the Gibson Assembly protocol (Gibson et al., 2009, 2010). An internal TEV cleavage site is used to remove the His6 tag, leaving a GAHM sequence on the N-terminus of the protein. DNA sequences were verified by Sanger sequencing. The recombinant plasmids were transformed into E. coli (BL21 DE3 Rosetta) cell lines for protein expression.

Bacterial cultures for expression of unlabeled proteins were grown in lysogeny broth (LB) at 37o C to an optical density (A600) of 0.6-0.8. Protein synthesis was then induced with 0.4 mM isopropyl-B-D-thiogalactopyranoside (IPTG), and expression proceeded overnight at 26o C. Labeled proteins were grown in minimal MJ9 media under half the concentration of antibiotic at o 37 C to an A600 of 0.8 and expressed as described above. Cells were harvested from the cultures by centrifugation at 4000 RPM, lysed by sonification, and centrifuged a second time at 15000 RPM, to remove all cell debris. The supernatant was purified using TALON Metal Affinity Resin and the His6 tag was cleaved using tobacco etch virus (TEV) protease. Proteins were further purified using a Superdex 75 (GE Healthcare) size-exclusion chromatography column. Protein concentrations were determined from absorbance at 280 nm for proteins with high molar extinction coefficient values. Molar extinction coefficients measured at 280 nm were computed with the ProtParam tool on the ExPASy website (Gasteiger et al. 2005). The concentrations of proteins that lack aromatic residues were determined using a Bradford assay (Bio-Rad), absorbance at 205 nm and by comparing their band size on SDS polyacrylamide gels against

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predetermined standards. Molar extinction coefficients measured at 205 nm were computed with a Protein Calculator tool (Anthis and Clore 2013). All purified proteins were stored at 4o C with a protease inhibitor mixture of pepstatin A and phenylmethanesulfonyl fluoride.

CD

Spectra were collected on a JASCO 720 spectropolarimeter using a 1 mm cell, with protein concentrations of 10-20 µM in 10 mM sodium phosphate, pH 7.5. All experiments were done using a 300 µL cuvette with a path length of 0.1 cm. The data shown are the average of three scans from wavelengths of 195-250 nm. The thermal unfolding data were collected at temperatures between 5o- 50o C in increments of 5o C.

ITC

ITC experiments were conducted using a MicroCal VP-ITC microcalorimeter. All experiments were performed at 25o C in a 50 mM sodium phosphate, 50 mM sodium chloride, 1 mM sodium azide, pH 7.5 buffer. Samples were degassed at 25o C for 15 minutes prior to loading. Each experiment was started with a 2 µL injection and followed by 27-33 10µL injections. In every experiment, IC at a concentration of 200-300 µM was titrated into p150 at a concentration of 20- 30 µM.

NMR Measurements and Analysis

NMR samples were buffer exchanged into a 10 mM sodium phosphate, 50 mM sodium chloride, 1 mM sodium azide, pH 6.0 buffer with 5% D2O, 0.2 mM 2,2-dimethylsilapentane-5-sulfonic acid (DSS) and a Roche protease inhibitor cocktail. Experiments and data analysis were done by Nathan Jespersen, a graduate student in the Barbar Lab, and Dr. Nikolaus Loening, a collaborator from the department of chemistry at Lewis and Clark College.

Results

Folding of H2 varies among species

NMR data on IC from rat, Drosophila and yeast have shown that both the helicity and location of H2 vary across species (Figure 1A and 2B). While H2 is a strong helix in rat, it has been relegated to a nascent helix in Drosophila and yeast. Additionally, comparative ITC studies between rat and yeast IC have shown that the binding affinity to p150 increases with a weaker H2 helix (Jie et al. 2017, 2015). In order to understand how helicity varies with species complexity, we used the Agadir program, which uses a sequence-based algorithm to predict helicity at the residue level. Agadir predictions do not account for long-range or tertiary interactions. It is therefore most useful for predicting the helicity of short sequences, or intrinsically disordered regions such as the N-terminus of IC. Predictions for the selected species illustrate a highly conserved SAH, as well as a secondary helix, H2, which decreases in helicity with decreasing species complexity (Figure 2A and 2B). Human and zebrafish IC are predicted to have an H2 that is equal in strength to rat, while the H2 in the shark species selected is predicted to be of intermediate strength. The predicted H2 in IC from an octopus species, C. elegans, and C. thermophilum are weak, similar to the weak H2 predictions in Drosophila and yeast. NMR secondary chemical shift data for rat, Drosophila and yeast are consistent with their

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Agadir predictions (Figure 2B), suggesting Agadir accurately predicts both strong and nascent helices.

The N-terminal region of IC is highly conserved in chordates, correlating with the high conservation of secondary structure (Figure 2C). On the other hand, IC from non-chordates shows little conservation, in either the sequence or position of H2 (Figure 2C). Interestingly, despite the lack of conservation in sequence, the nascent helix structure is well conserved in non- chordates, suggesting that the weak characteristic of H2 is necessary or functionally important. A H. sapiens D. rerio C. milii 100 100 100 Strong Helix

50 50 50 Transient/Weak Helix 0 0 0 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 Disordered Linker O. bimaculoides C. elegans C. thermophilum % Helicity 100 5 50 50 5

50 25 25 0 0 44 54 42 62 0 0 0 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 B R. norvegicus D. melanogaster S. cerevisiae 100 100 5 60 5

50 50 30 0 0 48 58 63 73

% Helicity 0 0 0 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 4 4 4

β 3 3 3 C 2 2 2 Δ

– 1 1 1 ! 0 0 0 C

Δ -1 -1 -1 0 25 50 75 0 25 50 75 100 0 25 50 75 Residue C Experimentally Verified H2 Agadir Predicted H2

H. sapiens MSDK--SDLKAELERKKQRLAQIREEKKRKEEERKKKEADMQQKKE-PVQDDSDLDRKRRETEALLQSIGISPEPPLVPTPMSPSSKSVSTPSEAGSQDSGDL 100 R. norvegicus MSDK--SELKAELERKKQRLAQIREEKKRKEEERKKKETDQKKEAAVSVQEESDLEKKRREAEALLQSMGLTTDSPIVPPPMSPSSKSVSTPSEAGSQDSGD- 100 D. rerio MADR--SDLKAELERKKQRLAQIREEKKRKEEERKKKESEMLQRPE-NVTEDSDLDRKRRETEALLQSIGISPEPPLVPTPMSPSSQSVSPPSETGSQESIDG 100 C. milii MSDKDKSELKAELERKKQRLAQIREEKKRKEEERKRKEADQKKEVV--QNEELDIEKKRREADALLQSMGITSEPSIVP-PTSPSSKSVSTPSEAGSQDSADG 100 *:*: *:***************************:**:: :. :: *:::****::*****:*::.:..:** * ****:***.***:***:* *

O. bimaculoides MSD-RKAELERKKARLEQMRKERQEKEQRRK------LKDNEEVKPGKPPQDLRAETDALLQDLGIPPNENSGRSGSISPTVQDTEQTNVVISGGSTRPKVK 95 D. melanogaster -MD-RKAELERKKAKLAALREEKDRRRREKE--IKDMEEAAGRIGGGAGIDKDQRKDLDEMLSSLGVAP------VSEVLSSLSSVNSMTSDNSNTQTPD 90 C. elegans MSELRKLELERKKQKLAELKSQRKREEDARVQNLLRSTNENGTTQNGTSRQTLSSNEVEDILRQVGIST---EPTVREESPAPPPGSHSDNHVDPSASHPRIS 100 C. thermophilum -MQARREELLAKKARLAEIKRQRE----LRAQQAAGRSITPSELVSPTPSRANSRREIESLIDSILSSS------AGANSPRRGSRPNSVISTGELSTDNA 90 S. cerevisiae -ME-RLKQLEEKRRQLKELRERR------KQASLFPGSETMGHHPTEVHAKATMVSVSVQTDMEEGSKIQEPQSAYLRRKEVITYDKGIQTDQ 85 : * :* *: :* :: .: : . : . . . Figure 2. IC secondary structure conservation (A) Percent helicity predictions by residue for the first 100 amino acids of IC from Homo sapiens (human), Danio rerio (zebrafish), Callorhinchus milii (Australian ghostshark), Octopus bimaculoides (Californian two-spot octopus), Caenorhabditis elegans (nematode) and Chaetomium thermophilum (thermophilic fungus). Predictions were generated using the algorithm provided by Agadir. The insets for octopus and CT show detail for the region predicted to be H2. (B) The top graphs show the percent helicity prediction by residue for the first 100 amino acids of IC from Rattus norvegicus (rat), Drosophila melanogaster (fruit fly) and Saccharomyces cerevisiae (yeast). The insets for Drosophila and yeast show detail for the region predicted to be H2. The bottom graphs show experimentally obtained secondary structural data for constructs of rat, Drosophila and yeast IC used in previous studies. The experimental secondary structure was determined using the differences in the chemical shifts of Ca and Cb from random coil values. (C) Sequence alignment of IC from chordates (human, rat, zebrafish and shark) versus non-chordates (octopus, fruit fly, C. elegans, CT and yeast) using the MAFFT alignment program (Katoh and Standley 2013; Li et al. 2015; McWilliam et al. 2013). Highlighted residues show predicted versus experimentally determined H2 regions. Identical (asterisk), strongly similar (colon) and weakly similar (period) residues are shown.

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IC and p150 from Chaetomium thermophilum

IC from Chaetomium thermophilum (CT IC) is predicted to have the weakest H2 among the organisms selected for predictions. Therefore, it is the ideal system to determine the importance of H2 helicity in IC-p150 interactions. Constructs were designed to include either the SAH domain (IC35, res. 1-35) or the SAH domain plus the potential H2 region (IC88, res. 1-88) (Figure 1B).

CD experiments on IC35 indicate that the construct is completely helical, characterized by the double minima at 222 nm and 208 nm (Wei et al., 2014; Figure 4C). IC88, however, is a mixture of helical and disordered regions, based on the shift of the first minima to 205 nm (Wei et al. 2014; Figure 4B). These spectra are consistent with the Agadir prediction for CT IC.

Studies on mammalian p150 have shown that the IC binding site is located within the CC1B domain (Siglin et al. 2013; Figure 1C). Alignment of human, Drosophila, yeast and CT p150 sequences was used to identify the CC1B region in CT, resulting in the generation of the p150ABC constructs (Figure 1D). Given that the p150 binding region in IC is less than 80 amino acids, it is possible that the IC binding site in p150 can be more discretely defined within the CC1B domain. Analysis of p150ABC using the COILS Server (Lupas et al. 1991) revealed that the coiled coil contains two areas of decreased structure, suggesting the presence of short linkers between coiled coils (Figure 1D). This prediction was used to further separate the p150ABC construct into smaller fragments p150AB, p150BC, p150A, p150B, and p150C (Figure 1D). The presence of linkers provides an option to safely cut the coiled coil structure of CC1B without risk of completely losing structure. Another construct, p150486-645, was created in an effort to design a more concise binding region, as well as to determine the necessity of the C-terminal region of p150ABC (Figure 1D).

CD data indicate that CT p150ABC, p150AB and p150A are fully helical, p150BC is a mixture of helix and disorder, and p150B and p150C are mostly disordered (Figure 5A). It seems that p150A is largely stabilizing the rest of the coiled-coil and separating the structure causes the individual fragments to unravel. p150C is predicted to have decreased coiled-coil structure (Figure 1D) and CD indicates that it does not have any helical structure either (Figure 5A). p150B and p150C are disordered on their own, but those regions together seem to be regaining some structure (Figure 5A).

CT IC and p150ABC form a tight, stable complex, with contributions from H2

Binding between CT IC and p150 was verified using Isothermal Titration Calorimetry (ITC). The ITC thermogram revealed a two-step binding event where there is a tight initial exothermic reaction, followed by a weaker, endothermic reaction. While ITC on rat IC showed binding on the order of 10 µM (Jie et al. 2017), the first step in the IC88/p150ABC binding had a dissociation constant (Kd) of 2 nM and the second step had a Kd of 500 nM (Figure 3A). This binding is substantially stronger than the mammalian system.

In comparison, thermograms for IC35 binding to p150ABC showed that removing H2 causes a decrease in binding affinity to p150 (Figure 3B). Furthermore, binding between IC35 and p150ABC showed a drastically diminished second step and was unable to be fit to either a one- or two-step binding curve. The decrease in overall affinity and reduction of the second binding step

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indicates that the H2 region in CT IC is important for strong interactions with p150 and promotes a more complex binding behavior.

Time˚(min) Time (min) Time˚(min) 0 30 60 90 120 150 0 30 60 90 120 A 0.1 B 0.0 0.0 c c e e /sec -0.1 s -0.2 s ˚ ˚ / / l l a a c c cal µ µ

µ -0.2 -0.4

-0.3 2 t t 0 n n a a t t c 0 c e e j j n n i i

f f o -2 o

-2 e e l l o o cal/mole m m / / k l

-4 l a a c c k k -6 -4 0 1 0 1 2 3 Molar˚Ratio Molar Ratio Molar˚Ratio

Figure 3. Interactions of CT IC constructs with p150ABC Representative thermograms (top) and binding isotherms (bottom) from ITC titrations of (A) IC88 and (B) IC35 with o p150ABC collected at 25 C, pH 7.5.

Denaturing curves for the various IC/p150 complexes, generated using CD data, reveal that the most stable complex contains the H2 region of IC as well as the C-terminal portion of p150ABC (Figure 4E). In other words, the IC88/p150ABC complex is more stable at high temperatures than the IC35/p150486-645 complex (Figure 4E and 4I). A comparison of the signal at 220 nm for the complexes and free proteins clearly illustrates the greater thermostability of the IC88/p150ABC complex (Figure 4A). On its own, p150ABC is structurally unstable at high temperatures, and the addition of IC, with or without H2, dramatically stabilizes the structure (Figure 4D, 4E and 4F). However, the presence of H2 provides more structural stability to the complex than just the SAH region, demonstrating that H2 is necessary for a tight and stable IC/p150 complex.

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A B C -5 10 10 IC88 IC35 p150ABC

-10 0 5 IC35/p150486-645

IC88 -15 -10 0

IC88/p150ABC -20 -20 -5 CD at 220 nm -25 -30 -10 5 10 15 20 25 30 35 40 45 50 195 205 215 225 235 245 255 195 205 215 225 235 245 255 Temperature (°C) D E F 60 80 75 p150ABC IC88/p150ABC IC35/p150ABC 60 40 50 40 25 20 20

0 0 0 -20

CD (mdeg) -20 -25 -40

-50 -40 -60 195 205 215 225 235 245 255 195 205 215 225 235 245 255 195 205 215 225 235 245 255 G H I 45 20 75 p150486-645 IC88/p150486-645 IC35/p150486-645 30 0 50

15 -20 25

0 -40 0

-15 -60 -25

-30 -80 -50 195 205 215 225 235 245 255 195 205 215 225 235 245 255 195 205 215 225 235 245 255

Wavelength (nm)

5 oC 50 oC

Figure 4. Complex dependent secondary structure stability detected using CD (A) CD signal (mdeg) as temperature increases at 220 nm shown for free IC88 and p150ABC and bound IC88/p150ABC and IC35/p150486-645. (B-I) Thermal unfolding CD spectra of IC88 (B), IC35 (C), p150ABC (D), IC88/p150ABC (E), IC35/p150ABC (F), p150486- 645 (G), IC88/p150465-465 (H), IC35/p150486-645 (I) collected at increasing temperatures between 5°-50° C.

Mapping sites of interactions

ITC was used to broadly determine the necessary regions for binding between IC88 and the fragments of p150ABC (Figure 5B). p150BC is the only construct that bound as tightly and in a similar two-step manner as p150ABC (Figure 5B). Alternatively, p150AB, p150A and p150B all bound weakly, and p150C did not bind under our experimental conditions (Figure 5B). NMR titrations of p150 constructs into labeled CT IC were used to map exact binding regions on IC88 (Figure 5C). While ITC showed no measurable interactions between p150C and IC88, NMR revealed that p150C interacts directly with, and exclusively to H2. In fact, all constructs of CT

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p150, including p150ABC, caused the peaks in and around the H2 region to disappear. These data indicate that while the “A” and “B” segments, on their own and together, are forming cursory interactions with IC, the “C” region is crucial for complete and tight binding. It is apparent that a mixture of the helical and disordered regions of CC1B are necessary to replicate the strong, two- step binding shown by p150ABC, since neither p150B or p150C behaved similarly to p150BC (Figure 5B).

While previous work has indicated that H2 is somehow important for regulating the interactions between IC and p150, it was believed that direct interactions only occurred between the SAH domain of IC and p150. Here we provide the first example of direct binding between p150 and H2. ABC AB BC A B C 478 680 478 622 542 680 478 541 542 622 623 680

10 0 0 0 0 0 A 0 -10 -1 -5 -10 -2 -10 -20 -20 -3 -20 -30 -40 -10 -20 -4 -30 -40 -5

CD (mdeg) -40 -60 -50 -15 -30 -6 200 210 220 230 240 250 200 210 220 230 240 250 200 210 220 230 240 250 200 210 220 230 240 250 200 210 220 230 240 250 200 210 220 230 240 250 Wavelength (nm) B µcal/sec kcal/mole

Molar Ratio C 1.2 1.2 1.2 1.2 1.2 1.2

0.8 0.8 0.8 0.8 0.8 0.8

0.4 0.4 0.4 0.4 0.4 0.4

0 0 0 0 0 0 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 Peak VolumeRatio IC88 Residue Number

Figure 5. Comparison of CD, ITC and NMR titrations for p150ABC and sub-constructs (A) CD spectra at 25° C for p150ABC, p150AB, p150BC, p150A, p150B, and p150C. (B) ITC data showing respective thermograms (top) and binding isotherms (bottom) from titrations of p150ABC, p150AB, p150BC, p150A, p150B, and p150C with IC88. (C) HSQC peak volume ratios from titrations of 15N-labeled IC88 with unlabeled p150ABC, p150AB, p150BC, p150A, p150B, and p150C. All data collected at 800 MHz, pH 6.0 and 15° C. Samples for p150ABC, p150A, and p150C are at an IC:ligand molar ratio of ~1:1. Samples for p150AB, p150BC, and p150B are at an IC:ligand molar ratio of 1:1.2.

Discussion

H2 contributions to tight binding

Evidence that a nascent H2 is essential for IC/p150 binding is in its conservation in less complex organisms. Although the location, sequence, and size were not similar in the non-chordate species selected in this study, their structural predictions all indicate the presence of a secondary helix. NMR secondary chemical shift data show that SAH is similar in strength in rat,

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Drosophila, yeast, and CT (Figure 2B; Figure 6B), which suggests that differences in p150 binding must be caused by other differences in IC sequence. As species complexity increases, H2 becomes a strong helix. Interestingly, binding to p150 is the weakest in rat IC, the most complex organism in this study. Binding affinity for rat IC and p150 is 12.6 µM (Jie et al. 2017), while the Kd value was 3.6 µM for Drosophila (Morgan et al. 2011) and 5.6 µM for yeast (Jie et al. 2015). Additionally, binding to p150 drastically weakens in Drosophila and yeast when H2 is removed, but the strength of binding in rat is preserved when H2 is removed. A similar effect is seen in CT IC; binding to p150 is significantly stronger, with an apparent Kd of less than 1 µM, and was remarkably weakened with the removal of H2 (Figure 3A and 3B). These data suggest that a weak H2 is necessary for tight binding, and a strong H2 somehow inhibits overall binding strength. A possible explanation for a nascent helix being more favorable for strong binding is that it provides more flexibility for the disordered regions of IC and p150 to interact. A stronger H2 may inhibit IC from folding in a way that allows that region to come into contact with p150 and form the additional bonds that make binding tight. There is a possibility that the disordered linkers between the two helices may have an impact on binding, and other studies have investigated the role of phosphorylation sites distant from SAH on p150 binding (Jie et al. 2017). However, it seems that H2 has a substantial impact on the strength of interactions between IC and p150. A B

C. thermophilum 50 5

25 ) 0 42 62 % Helicity ppm ( 0 N

15 0 25 50 75 100 4

3

β 2 C Δ

– 1 ! C

Δ 0

-1 0 25 50 75 Residue 1H (ppm)

Figure 6. IC88 Structure 15 1 (A) An [ N, H] – TROSY HSQC spectrum of CT IC88 with backbone NH assignments. Spectra were collected at pH 6.0 and 15° C. (B) The experimentally obtained secondary structure obtained using the differences in chemical shifts of Ca and Cb from random coil values. The data show a strong SAH and a weak H2 at residues 50-60, matching the Agadir prediction.

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Further investigation into the exact effect of a strong versus weak H2 can be done by replacing the weak H2 in CT IC with the strong H2 from rat IC. After confirming that the residues replacing the wild type H2 form a strong helix in the new sequence, the same experiments used in this study should determine if H2 folding is correlated with strong binding. The presence of the strong H2 would be expected to weaken binding to p150 compared to wild type CT IC. The intermediate helical propensity predicted for the H2 in the shark species (Figure 1A) is a good candidate for filling in the range of H2 folding.

A C. thermophilum p150FL H. sapiens p150FL CC1B CC1B 100 100

75 75

50 50

25 25

0 0 1 201 401 601 801 1001 1201 1 201 401 601 801 1001 1201

B C. thermophilum p150ABC H. sapiens p150* 100 100

75 75

50 50

25 25

0 0 478 528 578 628 678 382 412 442 472 502 Figure 7. Coiled-coil predictions for p150 from C. thermophilum and H. sapiens (A) COILS Server (Lupas et al. 1991) predictions for full length p150 from C. thermophilum and H. sapiens. Comparison of the full-length structures shows that the CC1B region in p150 from CT has a portion that is predicted to be greatly decreased in coiled-coil structure, while the mammalian p150 does not. This region of lower structure corresponds to the p150C construct. (B) COILS Server (Lupas et al. 1991) predictions for the p150ABC and p150* constructs. These predictions more clearly show how the mammalian p150 construct lacks the region of diminished structure that CT p150 has. This is the region that is shown to interact with H2 by NMR. Although the p150* construct is around 50 residues shorter than the p150ABC construct, including the rest of the CC1B domain into the construct would not introduce a disordered region.

Disordered regions of p150 are interacting with H2

Our data affirm direct interactions between H2 and p150. All constructs of CT p150 caused peaks corresponding to and surrounding H2 to disappear when titrated into IC88 (Figure 5C). In particular, NMR shows that p150C, which is also the most disordered piece of p150ABC, only affects the peaks corresponding to H2. This interaction was too weak to be measured by ITC. The only construct that exhibited similar binding characteristics as p150ABC was p150BC. Since p150B does not have the same two-step binding to IC88, p150C must be necessary for tight

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binding. Furthermore, both p150C and H2 must be present, as those two regions interact with each other exclusively. In the two-step binding event, it is possible that the second step describes interactions between H2 and the disordered region of p150, which is why it is not present in constructs without p150C or H2. The thermal unfolding curves from the CD data further corroborates this by demonstrating that there is only one complex, IC88/p150ABC, that was completely resistant to denaturation at high temperatures (Figure 4E). Free p150ABC denatured sooner and to a greater degree than p150486-645, confirming that the C-terminal region is less structured and is being stabilized by IC. Although both IC88 and IC35 stabilize the secondary structure of p150ABC, the missing second step in the ITC of p150ABC into IC35 further indicates the involvement of H2.

The IC binding site on p150 is unlikely to include the entire CC1B region, but evidence points to a majority of the domain being required for strong binding. The “BC” region was sufficient to replicate the binding of the full construct, but p150A and p150AB also showed weak binding under ITC and NMR conditions. There may be a number of non-specific interactions that occur along the entire regions of IC and p150 in these constructs. While we may not be able to refine the structure down to a single binding motif, we have uncovered several key factors contributing to tight complex formation.

The disordered region in CC1B is unique to p150 from CT (Figure 7A). Comparison of the domain between CT and mammalian p150 shows that the decreased structure we see in p150C is not present in mammalian p150 (Figure 7A and 7B). This may be an attribute of the evolution of IC and p150 in less complex organisms, or organisms that have an IC with a weak H2. It seems that the folding of H2 correlates with the C-terminal portion of CC1B in p150. A weaker H2 is able to interact with that region when it is also less structured, and a stronger H2 is unable to interact with p150 due to its folding. Therefore, the respective p150 has a more structured CC1B and the dynein/dynactin system relies on other methods of regulation, such as phosphorylation sites downstream from the binding region.

References

Anthis, N.J., Clore, G.M. (2013) Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm. Protein Science. 22, 851-858.

Apweiler, R., Bairoch, A., Wu, C.H., Barker, W.C., Boeckmann, B., Ferro, S., Gasteiger, E., Huang, H., Lopez, R., Magrane, M., Martin, M.J., Natale, D.A., O'Donovan, C., Redaschi, N., Yeh, L.L. (2004) Uniprot: the universal protein knowledgebase. Nucleic Acid Res. 32, 115D-119.

Benison, G., Nyarko, A., and Barbar, E. (2006) Heteronuclear NMR identifies a nascent helix in intrinsically disordered dynein intermediate chain: implications for folding and dimerization. J. Mol. Biol. 362, 1082–1093.

Clark, S.A., Jespersen, N., Woodward, C., and Barbar, E. (2015) Multivalent IDP assemblies: unique properties of LC8-associated, IDP duplex scaffolds. FEBS Lett. 589, 2543–2551.

Eschbach, J. and Dupuis, L. (2011) Cytoplasmic dynein in neurodegeneration. Pharm. Thera. 130(11), 348-363.

18

Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D., Bairoch, A. (2005). Protein Identification and Analysis Tools on the ExPASy Server, (In) John. M. Walker (ed): The Proteomics Protocols Handbook, Humana Press. 571-607.

Gibson, D.G., et al. (2009) Nat. Methods. 6, 343-345.

Gibson, D.G., et al. (2010) Science. 329, 52-56

Jie, J., Löhr, F., Barbar, E. (2015) Interactions of Yeast Dynein with Dynein Light Chain and Dynactin. J. Biol. Chem. 290, 23863-23874.

Jie, J., Löhr, F., Barbar, E. (2017) Dynein binding of competitive regulators Dynactin and NudE involves novel interplay between phosphorylation site and disordered spliced linkers. Structure. 25, 421-433.

Kardon, J.R., and Vale, R.D. (2009) Regulators of the cytoplasmic dynein motor. Nat. Rev. Mol. Cell Biol. 10, 854–865.

Katoh, K., and Standley, D.M. (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution. 4, 772-780.

Kellner, N., Schwarz, J., Sturm, M., Fernandez-Martinez, J., Griesel, S., Zhang, W., Chait, B.T., Rout, M.P., Kück, U., Hurt, E. (2016) Developing genetic tools to exploit Chaetomium thermophilum for biochemical analyses of eukaryotic macromolecular assemblies. Scientific Reports. 6, 20937

Kini, A. R., and Collins, C. A. (2001) Modulation of cytoplasmic dynein ATPase activity by the accessory subunits. Cell Motil. Cytoskeleton. 48, 52–60.

Lacroix, E., Viguera AR, Serrano, L. (1998) Elucidating the folding problem of a-helices: Local motifs, long-range electrostatics, ionic strength dependence and prediction of NMR parameters. J. Mol. Biol. 284, 173-191.

Li, W., Cowley, A., Uludag, M., Gur, T., McWilliam, H., Squizzato, S., Park, Y.M., Buso, N., Lopez, R. (2015) The EMBL-EBI bioinformatics web and programmatic tools framework. Nucleic Acids Research. 43(W1), W580-4.

Lupas, A., Van Dyke, M., and Stock, J. (1991) Predicting Coiled Coils from Protein Sequences. Science. 252, 1162-1164.

Makokha, M., Hare, M., Li, M., Hays, T., and Barbar, E. (2002) Interactions of cytoplasmic dynein light chains Tctex-1 and LC8 with the intermediate chain IC74. Biochemistry (Mosc.) 41, 4302–4311.

McWilliam, H., Li, W., Uludag, M., Squizzato, S., Park, W.M., Buso, N., Cowley, A.P., Lopez, R. (2013) Analysis Tool Web Services from the EMBL-EBI. Nucleic Acids Research. 41(Web Server issue), W597-600.

19

Moore, J.K., Sept, D., Cooper, J.A. (2009) Neurodegeneration mutations in dynactin impair dynein-dependent nuclear migration. PNAS. 106(13), 5147-5152.

Morgan, J.L., Song, Y., and Barbar, E. (2011) Structural dynamics and multi-region interactions in dynein-dynactin recognition. J. Biol. Chem. 286, 39349–39359.

Muñoz, V. & Serrano, L. (1994a) Elucidating the folding problem of helical peptides using empirical parameters. Nature: Struct. Biol. 1, 399-409.

Muñoz, V. & Serrano, L. (1994b) Elucidating the folding problem of a-helical peptides using empirical parameters, II. Helix macrodipole effects and rational modification of the helical content of natural peptides. J. Mol. Biol. 245, 275-296.

Muñoz, V. & Serrano, L. (1994c) Elucidating the folding problem of a-helical peptides using empirical parameters III: Temperature and pH dependence. J. Mol. Biol. 245, 297-308.

Muñoz, V. & Serrano, L. (1997) Development of the Multiple Sequence Approximation within the Agadir Model of a-Helix Formation: comparison with Zimm-Bragg and Lifson-Roig Formalisms. Biopolymers. 41, 495-509.

Siglin, A.E., Sun, S., Moore, J.K., Tan, S., Poenie, M., Lear, J.D., Polenova, T., Cooper, J.A., Williams, J.C. (2013) Dynein and dynactin leverage their bivalent character to form a high- affinity interaction. PLoS One. 8(4), e59453.

Tompa, P., and Fuxreiter, M. (2008) Fuzzy complexes: polymorphism and structural disorder in protein-protein interactions. Trends Biochem. Sci. 33, 2–8.

Urnavicius, L., Zhang, K., Diamant, A.G., Motz, C., Schlager, M.A., Yu, M., Patel, N.A., Robinson, C.V., and Carter, A.P. (2015) The structure of the dynactin complex and its interaction with dynein. Science. 347, 1441–1446.

Vallee, R.B., McKenney, R.J., and Ori-McKenney, K.M. (2012) Multiple modes of cytoplasmic dynein regulation. Nat. Cell Biol. 14, 224–230.

Vallee, R.B., Williams, J.C., Varma, D., and Barnhart, L.E. (2004) Dynein: an ancient motor protein involved in multiple modes of transport. J. Neurobiol. 58, 189–200.

Wei, Y., Thyparambil, A.A., Latour, R.A. (2014) Protein helical structure determination using CD spectroscopy for solutions with strong background absorbance from 190-230 nm. Biochim Biophys Acta. 1844(12), 2331-2337.

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