Title: Remodeling and activation mechanisms of outer arm dyneins revealed by cryo‐EM
Abstract: Article2 August 2021free access Transparent process Remodeling and activation mechanisms of outer arm dyneins revealed by cryo-EM Shintaroh Kubo Shintaroh Kubo Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada Search for more papers by this author Shun Kai Yang Shun Kai Yang Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada Search for more papers by this author Corbin S Black Corbin S Black Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada Search for more papers by this author Daniel Dai Daniel Dai orcid.org/0000-0002-9973-0446 Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada Search for more papers by this author Melissa Valente-Paterno Melissa Valente-Paterno Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada Search for more papers by this author Jacek Gaertig Jacek Gaertig Department of Cellular Biology, University of Georgia, Athens, GA, USA Search for more papers by this author Muneyoshi Ichikawa Corresponding Author Muneyoshi Ichikawa [email protected] orcid.org/0000-0002-5921-7699 Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma, Japan PRESTO, Japan Science and Technology Agency, Kawaguchi, Japan Search for more papers by this author Khanh Huy Bui Corresponding Author Khanh Huy Bui [email protected] orcid.org/0000-0003-2814-9889 Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada Centre de Recherche en Biologie Structurale, McGill University, Montréal, QC, Canada Search for more papers by this author Shintaroh Kubo Shintaroh Kubo Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada Search for more papers by this author Shun Kai Yang Shun Kai Yang Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada Search for more papers by this author Corbin S Black Corbin S Black Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada Search for more papers by this author Daniel Dai Daniel Dai orcid.org/0000-0002-9973-0446 Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada Search for more papers by this author Melissa Valente-Paterno Melissa Valente-Paterno Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada Search for more papers by this author Jacek Gaertig Jacek Gaertig Department of Cellular Biology, University of Georgia, Athens, GA, USA Search for more papers by this author Muneyoshi Ichikawa Corresponding Author Muneyoshi Ichikawa [email protected] orcid.org/0000-0002-5921-7699 Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma, Japan PRESTO, Japan Science and Technology Agency, Kawaguchi, Japan Search for more papers by this author Khanh Huy Bui Corresponding Author Khanh Huy Bui [email protected] orcid.org/0000-0003-2814-9889 Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada Centre de Recherche en Biologie Structurale, McGill University, Montréal, QC, Canada Search for more papers by this author Author Information Shintaroh Kubo1,†,‡, Shun Kai Yang1,†, Corbin S Black1, Daniel Dai1, Melissa Valente-Paterno1, Jacek Gaertig2, Muneyoshi Ichikawa *,3,4 and Khanh Huy Bui *,1,5 1Department of Anatomy and Cell Biology, McGill University, Montréal, QC, Canada 2Department of Cellular Biology, University of Georgia, Athens, GA, USA 3Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma, Japan 4PRESTO, Japan Science and Technology Agency, Kawaguchi, Japan 5Centre de Recherche en Biologie Structurale, McGill University, Montréal, QC, Canada †These authors contributed equally to this work ‡JSPS Overseas Research Fellow *Corresponding author. Tel: +81-743-72-5575; E-mail: [email protected] *Corresponding author. Tel: +1-514-398-4795; E-mail: [email protected] EMBO Reports (2021)22:e52911https://doi.org/10.15252/embr.202152911 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cilia are thin microtubule-based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs' conformational changes caused by ATP hydrolysis. Here, we report the native ODA complex structure attaching to the doublet microtubule by cryo-electron microscopy. The structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with coarse-grained molecular dynamic simulations, we present a model of how the attachment of the ODA to the doublet microtubule induces remodeling and activation of the ODA complex. Synopsis Cryo-EM structure of the outer dynein arm (ODA) complex attached to the doublet microtubule combined with the coarse-grained molecular dynamics (MD) simulation reveals the active conformation of the ODA and the activation mechanisms of the ODA complex. ODA complex structure natively bound to the doublet microtubule via its tail domain was obtained by cryo-EM. The dynein heavy chain heads of the ODA complex interact with the tail domain of the next ODA complex, forming a row of ODA complexes on the doublet microtubule. Comparison of active and inactive conformation the ODA complex revealed the remodeling of the ODA complex structure, in particular the dynein heavy chain tails. Coarse-grained MD simulation suggests that the attachment of the ODA complex to the doublet microtubule triggers the activation of the complex. Introduction Motion is an important aspect of life. In eukaryotes, cilia and flagella are responsible for cell motility. These microscopic hair-like organelles bend several tens of times per second to generate fluid flows. Cilia in trachea generate the flow of mucus and protect our body from infectious agents such as viruses. There is a canonical axonemal 9 + 2 structure where the central pair microtubules are surrounded by nine doublet microtubules (Fig 1A). The propulsive force generators of cilia and flagella are the axonemal dyneins. As the molecular motors, the axonemal dyneins drive the sliding of doublet microtubules, which is then converted into the bending of the cilium. The axonemal dyneins consist of outer arm dyneins and inner arm dyneins. The outer dynein arm (ODA) regulates the beat frequency while the inner arm dyneins are important for the waveform of the cilium (Brokaw & Kamiya, 1987). Improper assembly of the ODA complex causes ciliopathies in humans (reviewed in Reiter & Leroux, 2017). Figure 1. Cryo-EM structure of the ODA complex on the doublet microtubule A. Schematic diagram of axoneme structure of cilia viewed from the base of the cilia. Doublet microtubule: gray; outer dynein arm: blue; inner dynein arm: red; dynein regulatory complex: green; radial spokes: cyan. Black arrow indicates the view in (B). B. The 24-nm structure of the doublet microtubule from K40R mutant filtered to 18 Å showing the row of the ODA. The green outline indicates the single ODA complex. C. A schematic cartoon of the doublet microtubule and the ODA complex. Arrow indicates the view in (D). D. The focused refined maps of the tail and the heads of the ODA are shown within the map of the entire ODA complex (left). The focused refined maps of Dyh3 and Dyh4 heads and the tail (right). E. Fitting of models into maps (Dyh3, top; DIC3 and Dyh4 tail, bottom). The α-helix part of the DIC3 was more structured in our map compared with that of the Shulin–ODA complex. F, G. The surface render of the model (F) and the schematic cartoon (G) of the ODA with the docking complex. The Dyh5 is too flexible to resolve well by cryo-EM. The Dyh5 head is only shown as the 18-Å resolution surface render, and the Dyh5 tail is drawn as dotted lines. All the components (ICs, LCs, and DCs) are colored and indicated and will be used consistently in all the figures. C-terminal side of the DIC3 has more structured region along the Dyh4 HC tail on the doublet. Download figure Download PowerPoint Unlike cytoplasmic dyneins which walk on microtubules while carrying cargos, axonemal dyneins are anchored firmly on the doublet microtubules (Goodenough & Heuser, 1982; Bui et al, 2008, 2009). The ODA complex is composed of two or three heavy chains (HCs) depending on the species (Diamant & Carter, 2013), two intermediate chains (ICs) and numerous light chains (LCs). For example, human cilia have two-headed ODA (DNAH5 and DNAH9) while ODA of Tetrahymena contains three heavy chains (Dyh3, Dyh4, and Dyh5). Dynein HCs are the most important force-producing components, and each HC has a head domain and a tail domain. The three head domains are coupled together at the level of the tail domain. Within the head domain of each ODA HC, there is an AAA+ ring (composed of AAA1 to AAA6 subdomains) where ATP is hydrolyzed to exert force. The ODA complex is stably attached to the A-tubule of the doublet microtubule and produces force while interacting with the B-tubule of the neighboring doublet microtubule. The docking of the ODA complex to the A-tubule of the doublet is mediated by the HC tail domain. The interaction with B-tubule of the neighboring doublet is mediated by the microtubule-binding domain (MTBD) at the tip of the stalk which extends from the AAA+ ring. ICs and LCs have various regulatory roles (reviewed in King, 2012). Most of the ICs and LCs interact with the tail domain. However, LC1 is bound to the MTBD of the Dyh3 head (Ichikawa et al, 2015; Toda et al, 2020). By conventional quick-frozen deep-etch replicas and cryo-electron tomography (cryo-ET) works, ODAs were shown to form a 24-nm repeating row on the doublet microtubules (Goodenough & Heuser, 1982; Nicastro et al, 2006; Bui et al, 2012; Lin & Nicastro, 2018). The ODA complex is proposed to be attached via the docking complex (DC) (Owa et al, 2014; Oda et al, 2016a). Within each ODA complex, the three heads are aligned parallel to each other so that all three head domains can interact with the adjacent doublet microtubule in a proper orientation. The architecture of ODA complex has been studied by negative staining EM using purified ODA complexes (Ichikawa et al, 2015) or cryo-electron tomography (cryo-ET) of the intact axoneme (Oda et al, 2016b). However, the resolutions were limited in these studies and the modeling of the protein subunits was not possible. Recently, a cryo-EM structure of the inactive Tetrahymena ODA complex (before its incorporation into cilia) was obtained (Mali et al, 2021). In the inactive form, three head domains are packed together by a regulator protein, Shulin. This conformation is markedly different from the active conformation observed in the axoneme where the three heads are in parallel arrangement. The subunit architecture was also revealed in high resolution for the inactive Shulin–ODA complex (Mali et al, 2021). However, to understand how ODA is incorporated into the axoneme structure and activated, it was crucial to obtain a high-resolution structure of the ODA complex in the context of the doublet microtubule. Here, we revealed the Tetrahymena ODA complex on the doublet microtubule at 5.5–7 Å resolutions. Our structure showed in detail how the Tetrahymena ODA complex is attached to the doublet, including its interaction with the DC complex. Very recently, high-resolution structure of Chlamydomonas ODA complex attached to the doublet microtubule was also reported by cryo-EM (Walton et al, 2021). This enabled us to compare the ODA structure from different species. Combined with coarse-grained molecular dynamics (MD) simulations, we have revealed how the ODA complex undergoes an activating rearrangement when it is docked onto the doublet microtubule. Results Cryo-EM structure of the ODA attached to the doublet microtubule In our previous studies of the doublet microtubule structures, the ODA complexes were removed by high-salt treatment from doublets (Ichikawa et al, 2017; Ichikawa et al, 2019; Khalifa et al, 2020). Here, to obtain the native structure of the ODA complex on the doublet, we tried to obtain a wild-type (WT) Tetrahymena doublet microtubule structure without a salt wash (Fig EV1A). The individual doublet microtubules were separated from the rest of the axoneme by induction of microtubule sliding using ATP. However, in the disintegrated WT axonemes, the ODAs tended to detach from the doublet (Fig EV1B) and we were not able to obtain the high-resolution structure of the ODA by single particle analysis (Fig EV1D and E). We noticed that the sliding of doublets in the ATP-reactivated K40R (Gaertig et al, 1995) or MEC17-KO mutant Tetrahymena axonemes (Akella et al, 2010) was less efficient as compared to the WT Tetrahymena axonemes, possibly due to different levels of post-translational modifications of tubulin. This enabled us to obtain cryo-EM images of partially split doublet microtubules with attached ODA complexes (Fig EV1C). Using the cryo-EM images, we first obtained a 24-nm doublet microtubule unit structure as previously described (Ichikawa et al, 2017; Ichikawa et al, 2019; Khalifa et al, 2020) at a 3.9 Å resolution (Fig EV2A). However, the resolution of the ODA part was not high enough due to its flexibility. Therefore, we performed signal subtraction of the doublet and obtained the ODA structure with a 7.8 Å resolution. From here, different regions of the ODA were further revealed by focused refinement. The tail region's resolution was improved to 5.5 Å, the Dyh3 head part to 5.8 Å, and Dyh4 head part to 7 Å (Fig EV2B). The resolution of the Dyh5 head remained at 17 Å since it was furthest from the doublet and more flexible. The head domains were in a parallel configuration (Fig 1B) as previously shown by cryo-ET studies (Bui et al, 2008; Lin & Nicastro, 2018). Our structure fits well with previous cryo-ET structure of the intact axoneme, thereby confirming it is a physiological structure of the ODA complex (Fig EV1F). Click here to expand this figure. Figure EV1. Sample preparation and cryo-EM of the doublet microtubules A. The schematic of our isolation strategy for the intact doublet microtubules. B, C. Micrographs of doublet microtubules from Tetrahymena WT (B) and K40R mutant (C). The red arrowheads indicate the ODA complex falling off from the doublet. The red rectangle indicates the row of intact ODA in the K40R mutant. Scale bars represent 50 nm. D, E. 24 nm structure of doublet from WT and K40R showing the DC is intact in both cases while ODA is clearly present only in K40R. Red arrows indicate the docking complex. Scale bars, 50 nm. F. Fitting of our high-resolution structure into the tomographic map of Tetrahymena showing it is physiological (ΔRib72B mutant rescued with Rib72B-GFP) (EMD-7807, Stoddard et al, 2018). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Cryo-EM processing strategy Alignment strategy for ODA particles. First, we obtained the 24-nm doublet structures. After that, we performed signal subtraction of the doublet microtubule. We centered and boxed out the ODA particles and performed refinement of the entire ODA particles. Focus refinement strategy for different regions of the ODA complex. The Dyh5 seems to be too flexible; therefore, we can only obtain Dyh4 head at 17 Å resolution. Fourier Shell Correlation of the doublet of WT and K40R & MEC17 combined data. Fourier Shell Correlation of the different regions of the ODA complex by focus refinement. Download figure Download PowerPoint Using the recent Shulin–ODA complex structure from Tetrahymena as a starting model, we were able to build in all the components of the ODA complex except for Dyh5's head (Fig 1C–G). Since Dyh5 is not conserved in vertebrates (Lin et al, 2014; Ueno et al, 2014), an atomic model for conserved part of the ODA complex on the doublet was obtained. The ICs and LCs were also assigned to the density map. The majority of LCs were forming the LC tower as earlier observed in the Shulin–ODA complex (discussed below). Compared with the Shulin–ODA complex, there were some parts of ICs which were structured in our map and modeled (Fig 1E–G). There was a density not observed in the Shulin–ODA complex associated with Dyh4, and LC4 was tentatively assigned to this region (Fig 1F and G). Very recently, Tetrahymena ODA model based on cryo-EM structure of reconstituted ODA array on the doublet was reported (preprint: Rao et al, 2020). Our model was similar to obtained reconstituted ODA complex validating our model. When we compare our Tetrahymena ODA structure with recent Chlamydomonas ODA structure attached to the doublet (Walton et al, 2021), both structures appeared strikingly similar in general (Fig EV3). In our structure, there was also an additional density of two segments of coiled coil with a globular domain at the end which is running along the tail domain of the ODA (cyan parts in Fig 1D, F, and G) (discussed later). Click here to expand this figure. Figure EV3. Comparison of the ODA structures obtained by cryo-EM A, B. The model of our Tetrahymena ODA complex (A) and Chlamydomonas ODA complex (PDB ID: 7KZM, Walton et al, 2021) (B) viewed from different sides. The Dyh5 head of Tetrahymena ODA is shown as 18-Å resolution surface rendering. α-Tubulin is shown in green and β-tubulin is shown in blue. The polarities of the doublet microtubules are indicated by + and −. In our Tetrahymena model, there is more modeled region around the linker region. In contrast, there is more modeled region in DC in the Chlamydomonas model from Walton et al (2021), especially the region connecting the DC1/2 on the doublet surface and the extended coiled-coil region associated with the ODA complex (red arrowheads). These differences could relate to different properties of the Tetrahymena and Chlamydomonas ODA or different ways of sample preparation. Download figure Download PowerPoint Attachment of the ODA complex to the doublet via the docking complex The part of DC on the doublet was also visualized in our structure which allowed us to understand how the ODA complex is attached to the doublet (Figs 2 and EV4A). We identified Tetrahymena proteins Q22T00, Q233H6, and I7M2C6 as DC1/2 and 3 following the names of the counterparts of Chlamydomonas (Fig EV4B and C, see Materials & Methods for details). Along the doublet, DC1/2 forms a 24-nm repeating unit, the same periodicity as the repeating unit of the ODA complex. DC3 is associated with the DC1/2 coiled-coil region on the doublet serving as a marker for the ODA attachment (Figs 2C and EV4A) as previously proposed (Ma et al, 2019). The α-helix bundle 3 of Dyh3 HC was interacting with the DC3 (Fig 2C). The tethering of the ODA to the doublet microtubule is mediated by Dyh3 alone, and ICs/LCs are not involved with the microtubule. The DC1/2 also appears to connect to the coiled-coil density with a gap associated with the neighboring ODA tail domain (dashed line in Figs 2A and EV4H). This model is consistent with the coiled-coil prediction of DC1/2 with a gap (Fig EV4B) and the previous tagging results of DC2 of Chlamydomonas reinhardtii by cryo-ET in Oda et al (2016a) (Fig EV4, EV5). Therefore, the density associated with the tail domain corresponds to the C-terminal side of DC1/2 (Fig EV4I and J). This is also consistent with recent Chlamydomonas ODA structure model (Fig EV3). Figure 2. Interactions between the ODA complex and the DC A, B. The stacking of the ODA complex in the axoneme viewed from inside (A) and outside (B) the cilia. All the components belonging to the middle ODA complex are shown in color. The proximal and distal ODA complexes are shown in transparent. C. Model of the DC and the tail of the ODA. D. The interaction of the head of the ODA complex with the tail of the next ODA complex unit. Dyh3 head interacts with the dimerization domain NDD of the proximal Dyh4. The Dyh4 head interacts with the DIC3 WD40 of the proximal ODA complex. Arrows indicate the views in (E) and (F). E. Model view of the interaction between the Dyh3 head with the dimerization domain NDD of the proximal ODA complex. F. Model view of the interaction between the Dyh4 head with the DIC3 WD40 of the proximal ODA complex. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Modeling of the docking complex A. The DC density on the doublet microtubule between PFs-A7 and A8. B. Prediction of coiled coil for DC1 and DC2 using COILS with a window size of 28 (Lupas et al, 1991). C. Sequence alignment of Chlamydomonas DC1 and Tetrahymena CCDC151 homolog (Q22T00). D–G. Structures of Chlamydomonas ODA reconstituted on microtubules with biotin carboxyl carrier protein (BCCP) tagged in different regions of DC2 (residue 76, 276, 412, and 507) from Oda et al (2016a) (EMD-6508, 6509, 6510, 6511). The enhanced signals of BCCP-tag are indicated in colors. H. Slice from a density map showing the docking complex (position indicated in the cartoon). The yellow line indicates one continuous DC1/2. I. The globular density at the end of the coiled coil of DC1/2 (black arrow and dotted line). J. The model of the DC based on our analysis. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Data related to the remodeling of the ODA complex The labeling of helix bundles in the inactive and active Dyh3 and Dyh4. Inactive and active structures are aligned on helix bundle 4 of Dyh4 (residues 414–513). Alignment of the inactive and active Dyh3 at helix bundle 3 (residue 448–536) showing ˜ 90-degree rotation of the head domain (top). Alignment of inactive and active Dyh4 at helix bundle 3 (residue 414–513) showing compressing conformational changes (bottom). Bending conformational change in the LC tower. LC tower from the active ODA complex is in colors, and the LC tower from the Shulin–ODA is shown in transparent. LC towers are aligned based on either Lc8B/10 (left) or Lc8e/f (right) as indicated by green dashed circles. Regions of the ODA that interact with Shulin in the inactive conformation (green regions with green arrowhead) are spread out in the active conformation. The dash arrow indicates the region of Dyh3 head interacting with C3 domain of Shulin, now at the back of the view. Download figure Download PowerPoint The ODA complex is anchored to the doublet by the above-mentioned two sites: DC1/2 and DC3. Over these docking sites, there is a layer composed of Dyh3 HC, DIC2's WD domain, LC tower, and the C-terminal side of DC1/2. Another layer of Dyh4 and DIC3 is further built onto this layer (Fig 2A and B). The three head domains are stacked together in a parallel configuration. The stacking of both the tail and the head domains is important for the proper attachment of the ODA onto the doublet. While the ODA complex is not directly in contact with the doublet microtubule in its native state, ODAs can be reconstituted on preassembled singlet microtubules or doublets lacking DCs with 24-nm periodicity (Ueno et al, 2008; Owa et al, 2014). Our structure suggests that the ODA can be reconstituted on the microtubule without DCs by the interaction of the ODA heads with the proximal neighboring ODA tail domains (Figs 1B and 2D–F). More specifically, the head of Dyh3 seems to interact with the N-terminal dimerization domain (NDD) of the proximal ODA Dyh4 tail while the Dyh4 head interacts with DIC3 WD40 domain of the proximal ODA. These interactions probably enable the spontaneous alignment and stacking of the ODA complexes on the doublet microtubule without the help of the DCs. Similar tail-to-head interaction was recently observed in reconstituted ODA array on the doublet microtubule (preprint: Rao et al, 2020). Since our structure is the native structure of ODA on the doublet, our structure validated their model of the interaction. The interactions between head and tail of neighboring ODA complexes were also observed in Chlamydomonas ODA (Walton et al, 2021). Remodeling of the tail and the head It has been recently proposed that the ODA complex, before its assembly on the doublet, is in an inactive form with the regulatory protein Shulin (Mali et al, 2021). To understand how ODA rearranges into an active form on the doublet, we compared the structures of the Shulin–ODA complex with our ODA complex on the doublet (Figs 3 and EV5). The overall appearance of the active ODA complex on the doublet was quite different from that of the inactive Shulin–ODA complex, which has a more elongated conformation (Fig 3A). The elongated conformation of the inactive Shulin–ODA complex stems from the positions of the motor domains which are fixed in pre-powerstroke conformations (discussed later). The most unchanged region was the base region of the Dyh4 HC up to the bundle 4 region where the DIC3's WD40 domain resides (Figs 3B and EV5A). Dyh3 HC, DIC2, and LC tower were rotating together about ˜ 90 degrees relative to the Dyh4's base region (Fig 3B). With this configuration change, Dyh3 and Dyh4 HCs are no longer crossed to each other and freed. In the active conformation, DIC2's WD40 domain interacts with Dyh4 HC, and the WD40 domain of DIC3 is docked onto Dyh3 HC. Within the HCs, there were also conformational changes. The Dyh3 showed more change in configuration with ˜ 90-degree rotation of the head compared with the inactive form whereas Dyh4 HC showed rather a compressing movement of the head side compared with Shulin–ODA structure (Fig EV5A–C). These conformational changes are mediated by hinge-like motions between the α-helical bundles (Fig 3C). The center region (bundles 4–8) of Dyh3 showed a milder conformational change compared with other regions of the Dyh3 (Fig 3C) since the LC tower is associated with this region. In detail, there is a slight change in conformation of both Dyh3 and LC tower (Figs 3D and EV5D). The conformational change was due to the association of α-helix bundle 8 of the Dyh3 with LC8/8d. This interaction was hindered in the Shulin–ODA complex since Shulin is keeping Dyh3's bundle 8 away from LC8/8d. These tail rearrangements align the head domains to parallel configuration on the doublet (discussed later). Figure 3. Comparison of the active ODA complex structure on the doublet and the inactive Shulin–ODA complex structure The surface rendering of the inactive ODA model (with bound Shulin in green) and the active ODA model. Dyh5 is not shown. Remodeling of Dyh3 and Dyh4 tails and associated IC/LCs. The green circle indicates the region of alignment (Dyh4 residues 414–513, helix bundle 3). Dyh4 tail does not exhibit large conformational change while Dyh3 rotates almost 90 degrees, evident by the rotation of DIC2 WD40 domain. Superimposition of Dyh3 and Dyh4 tails between the inactive (transparent) and the active ODA (Dyh3 is aligned based on res. 535–646, helix bundle 5 while Dyh4 is aligned on res. 414–513, helix bundle 4). The Dyh3 tail exhibits two hinges with larger rotations while the Dyh4 tail also exhibits two hinges with smaller rotations. The changes in interaction between Dyh3 tail and the LC tower due to the remodeling of inactive (with Shulin in green) to active conformations (two structures are aligned based on Lc8b/10). The conformations of Dyh3 bundle 8, Lc8/8d, and Lc2a/9 are significantly different. The release of the Shulin leads to the rotation of the Dyh3 tail, which causes the different interaction pattern between Dyh3 and LC8/8d. Download figure Download PowerPoint Head domain structure of the ODA on the doublet In the Shulin–ODA complex, the heads are fixed in the pre-powerstroke positions (Fig 4A). In our active ODA complex structure, the linkers are in the post-powerstroke configuration both in the Dyh3 and Dyh4 heads. Therefore, the heads are ready to go through transition to next ATPase cycle with Shulin detached. Linker of the Dyh3 head was in a canonical post-powerstroke conformation (Fig 4A). As for the Dyh4 head, LC3 was docked onto the AAA+ ring in the post-powerstroke configuration of the linker. LC3 was interacting with AAA4 of the binding position of the Dyh4 AAA+ ring. However, the binding scheme was different from that of Lis1 which also binds to the AAA3 of cytoplasmic dynein (Toropova et al, 2014) (Fig 4B). LC3 still interacts with AAA4 but it was closer to AAA3. LC1 was previously shown to bind to the MTBD of Dyh3 head (Ichikawa et al, 2015; Toda et al, 2020). LC3 is the second component found to be associated with the head domain of the ODA complex. Since LC3 was interacting with both linker and the AAA+ ring, we carefully examined the linker position of Dyh4. The linker was going at the middle of the apostate and the ADP state and slightly leaning toward AAA4 (Fig 4C). This is a novel configuration of dynein linker. To perform powerstroke, LC3 is thought to be detached from the AAA+ ring (Fig 4D). Figure 4. Structure of the head domain in ODA on the doublet Models of the Dyh3 and Dyh4 heads in post-powerstroke conformations are fitted in the cryo-EM maps of the ODA on the doubl