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Short Communication *Corresponding author Esben Lorentzen, Department of Molecular Biology and Genetics, Aarhus University, Gustav WiedsVej 10c, DK- Purification and Structure of 8000 Aarhus C, Denmark; Tel: 45-871-55478; Email:

Submitted: 11 May 2017 Human ODA16: Implications Accepted: 18 July 2017 Published: 21 July 2017 for the Import of Outer Copyright © 2017 Lorentzen et al. Arms into Sperm Flagella by OPEN ACCESS Keywords the • Sperm • • Intraflagellar Transport Machinery • Outer Dynein Arms • ODA16, IFT46 Michael Taschner1, Jerome Basquin2, Sagar Bhogaraju2, Melanie Vetter2, Marie Bech Andersen1, Jiaolong Wang1, Anna Lorentzen1, and Esben Lorentzen1* 1Department of Molecular Biology and Genetics, Aarhus University, Denmark 2Department of Structural Cell Biology, Max-Planck-Institute of Biochemistry, Germany

Abstract A single motile cilium powers the swimming of sperm cells, which is required for fertilization of the egg. At the molecular level, several dyneinmolecular motor complexes including outer dyne in arms (ODAs) are attached periodically to the ciliaryaxoneme where theyhydrolyse ATP to create the force required for bending of the cilium and motility of the sperm cell. ODAs are preassembled in the and subsequently trafficked into the cilium by the intraflagellar transport (IFT) system. In the case of the green alga Chlamydomonasreinhardtii, the adaptor ODA16 binds to ODAs and directly to the IFT complex component IFT46 to facilitate the ciliary import of ODAs. Here, we purified recombinant human IFT46 and ODA16 and determined the high-resolution crystal structure of the ODA16 protein to show that the interaction network resulting in ciliary trafficking of ODAs is different than for the Chlamydomonassystem. Despite similar C-terminal b-propeller domains, the small N-terminal domain that binds IFT46 in Chlamydomonas, is not positioned on top of the b-propeller domain in case of the human ODA16. Consistent with this, we do not observe a direct interaction between human ODA16 and IFT46 suggesting that additional factors may be required for the ciliary import of ODAs in human sperm.

ABBREVIATIONS Cr: Chlamydomonasreinhardtii; Hs: Homo Sapiens; SEC: of the male reproductive system, one long motile cilium powers Size Exclusion Chromatography; ODA: Outer Dynein Arm; IFT: the sperm cell and allows it to swim towards the oocyte for fertilization. Autosomal recessive mutations that hamper with the functionality of the motile cilium thus often result in immotile INTRODUCTIONIntraflagellar Transport; PCD: Primary Cilia Dyskinesia sperm and male infertility [3,5]. signaling, sensory reception, and motility [1]. They probably andThe outer motility dynein of cilia arms relies are on periodically conserved molecular attached [7].structures Outer evolvedCilia toare provide conserved unicellular eukaryotic organisms organelles with that the function ability toin including a (MT)-based onto which inner move in aqueous environments. In humans, motile cilia are lightdynein chains arms that (ODAs) assemble are macro-molecular into ODA complexes motor that complexes associate consisting of several heavy, intermediate, light-intermediate and found in the embryonic node where they establish the left-right asymmetry required for the correct positioning of inner organs, with the MT-doublets of the axoneme. The dynein heavy chains and in the brain where they propel the cerebrospinal fluid flow are 4000-5000 residues long that power the sliding [2,3]. They also propel the extra-cellular mucociliary flow that ciliaryaxonememovement of MT were doublets reported resultingin to have abeating total or of partial the cilium absence [8- clears the airways of inhaled pathogens, and create the fluid-flow 10]. Around 80% of PCD patients with structural defects in the that moves the oocyte through the fallopian tubes to reach the uterus. Mutations in ciliary factors can thuse airways,result in primary and ectopic cilia of dynein arms [3]. Specifically, patient mutations in dynein arm dyskinesia (PCD) with disease phenotypes such as bronchitis heavy chains DNAH11 and DNAH5, intermediate chains DNAI1 and pneumonia due to infections in th and DNAI2 and light-intermediate chain TXNDC3 were reported pregnancy resulting in reduced fertility in females [4-6]. In case to result in PCD [3,5]. Cite this article: Taschner M, Basquin J, Bhogaraju S, Vetter M, Andersen MB, et al. (2017) Purification and Structure of Human ODA16: Implications for the Import of Outer Dynein Arms into Sperm Flagella by the Intraflagellar Transport Machinery. JSM Sexual Med 2(2): 1008. Lorentzen et al. (2017) Email:

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The cell relies on an intracellular transport process known GFP-nanobodycoupled to sepharose beads (‘GFP-binder°C and injectedbeads’). as intraflagellar transport (IFT) to build a cilium and ferry ODAs For the SEC experiment shown in Figure 2A, IFT46 was incubated from their site of assembly in the cytoplasm to their location of with 20% molar excess of HsODA16 for 1h at 4 action in the axoneme [11]. IFT depends on the 22-subunit IFT experimentonto a HiLoadSuperdex shown in Figure 200 2B column was carried in 10mM out Hepes as previously pH 7.5, complexTg737Rpw that mutant serves as mice an adaptor carrying for a ciliary hypermorphic cargo [12-14]. mutation IFT 250mM NaCl, 5% glycerol and 1mM DTT. The affinity pull-down was shown to be essential for spermiogenesis in mice and male Ift88Tg737Rpw described with 150mM NaCl in the binding and washing buffer in the IFT subunit IFT88 are sterile [15].The sperm count in [25,26]. Bound were eluted from the GFP-binder beads Ift88 mice was 350 times lower than in wild-type mice and using 0.1M citric acid. the sperm flagellawere completely immotile [15]. Consistent with this notion, disruption of a different IFT , Ift20, also results in Full-length HsODA16 was crystallized using the hanging drop vapor diffusion method at 18°C by mixing 200nL of purified Additionally,infertile mice [16]. a recently IFT20 is published highly abundant study in demonstrated the testes of mice that and IFT20 expression is upregulated during spermiogenesis[16]. HsODA16 at 26mg/ml with 200nL of the precipitant solution containing50mM Tris pH 8 and 50mM NaOxalate. Crystals were the IFT protein IFT25, which is not required for ciliogenesis in cryo protected by the addition of 30% glycerol and cooled in somatic cells[17], is essential for sperm flagella formation and liquid nitrogen. X-ray diffraction data were collected at 2.8Å fertility in mice, suggesting sperm-specific function of certain resolution at the Swiss Light Source (SLS; Villigen, Switzerland) IFT factors [18]. These studies clearly show that IFT is a required at the PXII beam line on a Pilatus 6M detector and indexed with spermiogenesisprocess for proper but cilium are absent formation in mature in sperm sperm, cells suggestingand for fertility that the XDS package [27] before scaling with Aimless as part of the of mice. Interestingly, IFT proteins are only present during CCP4 package [28]. The structure of HsODA16 was determined by molecular replacement using the CrODA16 structure (pdb IFT is not required for the maintenance of sperm flagella [15]. code 5MZH) as a model and refined in the program Phenix [29]. Chlamydomonasreinhardtii Important work in the green alga model organism The asymmetric unit contained a total of two chains; chain A was (Cr) revealed that the IFT of ODAs not well ordered and had high B-factors whereas chain B was well requires the IFT complex subunit IFT46 as well the cargo adaptor ordered with low B-factors and was consequently used for the structure representation shown in Figure 1 (non crystallographic thisprotein organism, ODA16 [19-21]. demonstrating The zebra evolutionary fish ODA16 conservation ortholog WDR69 [22]. symmetry restraints used in refinement). See Table 1 for data is also required for axonemalCr dynein assembly and motility in collection and refinement statistics. We recently investigated the ODA-ODA16-IFT46 interaction Table 1: HsODA16 (5NNZ) network using purified proteins to show that the CrODA16 X-ray data collection and refinement statistics. C-terminal domain is required for the interaction with ODAs and Wavelength (Å) Resolution range (Å) that the cleft between the N- and C-terminal domainsCr of and CrODA16 Homo 1.000 Space group likely forms the CrIFT46 binding site (Figure 1) [23]. Given that 520 - 2.8 (2.97 - 2.8) Unit cell (a,b,c, α,β,γ) both IFT46 and ODA16 are conserved between P 21 sapiens (Hs) (60% sequence identity over the entire protein Total reflections 52.2 69.5 102.2 90 90.0 90 length), it is a reasonable assumption that the mechanism of Unique reflections 115301 (16503) ciliary ODA trafficking is also conserved. However, as we show Multiplicity 18102 (2798) here, the HsODA16 structure does not have the pronounced Completeness (%) 6.4 (5.9) cleft betweenODA16 domains and does not interact directly Mean I/sigma(I) Cr and 99.5 (96.7) with IFT46 in a high affinity complex. In contrast, the surface of R-merge Hs 5.7 (1.0) the C-terminal domain is completely conserved between CC1/2 0.217 (1.35) ODA16 proteins, suggesting that they interact with ODAs in R-work 0.973 (0.404) ciliarya similar delivery. manner. Our data suggest that additional factors are R-free 0.226 (0.348) required to tether ODA-bound ODA16 to the IFT machinery for Number of non-hydrogen atoms 0.284 (0.469) MATERIALS AND METHODS macromolecules 5248 water 132 5116 Protein residues The coding sequence for human IFT46 ( accession RMS(bonds) 670 code: Q9NQC8-1) and ODA16 (uniprot accession code: Q8N136) RMS(angles) 0.005 were amplified from the Megaman DNA library and cloned with Ramachandran favored (%) 1.08 various N- and C-terminal affinity tags. HsODA16 was expressed Ramachandran outliers (%) 92.9 from recombinant baculovirus in HighFive cells as previously Clashscore described for CrODA16 [23]. Purification of HsODA16 was 0.60 Average B-factor carried out as for CrODA16 but250mM instead of 500mM NaCl 18.2 a cleavable hexahistidine tag in E.coli macromolecules was used in lysis and SEC buffers [23]. HsIFT46 was purified with 29.5 solvent 12.2 and purified as previously 30.0 described for CrIFT46 [24]. Additionally, N-terminally GFP- tagged HsIFT46 was produced in HEK293 cells and purified using Statistics for the highest-resolution shell are shown in parentheses. JSM Sexual Med 2(2): 1008 (2017) 2/5 Lorentzen et al. (2017) Email:

Central Bringing Excellence in Open Access RESULTS AND DISCUSSION not, however, rule out a weak substoichiometric interaction. The elution profiles for both proteins are consistent with To provide insights into the mechanism of IFT of mammalian soluble and folded proteins and no elution peak was observed ODAs, we purified recombinantly expressed full-length HsODA16 at the void volume, demonstrating that any of the proteins was from insect cells and determined the 2.8Å resolution crystal aggregated (Figure 2A). The fact that no complex formation structure (Table 1). The structure shown in Figure 1 reveals an elutedwas observed as a stoichiometric for HsODA16 complex and HsIFT46during SEC in [23]. SEC isInteraction in sharp 8-bladed β−propeller corresponding to residues 81-415. The contrast to what was observed for CrIFT46 and CrODA16, which overall structure of the β-propeller is highly similar to that of studies using various salt concentrations demonstrated that the the previously determined CrODA16 protein as the two models superimpose with a root-mean-square-deviation of 0.8Å. Despite CrODA16-IFT46 complex was stable at 200-300mM NaCl, partly the fact that the full-length HsODA16 was crystallized, no electron dissociated at 500-750mM NaCl and almost completely broken density is observed for the N-terminal 80 residues, which in apart at 1000mM NaCl [23]. The SEC experiment for HsODA16 case of CrODA16 folded into a small 3-stranded β-sheet domain and HsIFT46 shown in Figure 2A was carried out with 250mM positioned on top of the β-propeller. The N-terminal domain NaCl in the buffer, a condition where the Cr proteins formed a of HsODA16 shares 46% sequence identity with the CrODA16 stoichiometric complex [23]. The binding region of CrIFT46 counterpart, suggesting that it adopts the same fold. However, the for CrODA16 was mapped to the N-terminal 147 residues fact that residues 1-82 of HsODA16 were disordered in the crystal of CrIFT46 and the dissociation constant for the CrODA16- indicates that they are not stably attached to the β-propeller IFT46 complexes was measured to be ~200nM [23]. Generally, in contrast to what was observed in the case of CrODA16. This proteins that form stable complexes in SEC interact with at least structural difference is significant as the cleft formed between single digit uM affinity. To investigate if HsODA16 and HsIFT46 the N- and C-terminal domains of CrODA16 was mapped as the form a weak, lower affinity complex, we carried out pull-down binding site for CrIFT46 (Figure 1) [23], which questions the experiments with purified components. Additionally,E.coli we produced decided notion of a direct physical interaction between human ODA16 to purify GFP tagged HsIFT46 from human HEK293 cells in case and IFT46. posttranslational modifications missing in the E.coli We thus set out to test if HsODA16 interacts directly with protein might be important for the interaction with HsODA16. subjectingHsIFT46. HsIFT46 the protein was recombinantly mix to size exclusionexpressed chromatographyin , purified The results of this experiment demonstrated no detectable and incubated with HsODA16 purified from insect cells before direct interaction between GFP tagged HsIFT46 and HsODA16, even when 10X HsODA16 was incubated with HsIFT46 in buffer (SEC) (Figure 2A). The result of this experiment revealed that conditions containing 150mM NaCl (Figure 2B). As this type of HsIFT46 and HsODA16 eluted from SEC as two separate peaks pull-down experiment routinely detects affinities in the double suggesting that no complex was formed under the experimental digituM range [30],Cr counterpart. we conclude that a putative HsODA16- conditions (Figure 2A). As the elution peaks for HsODA16 and HsIFT46 complex, if it exists at all, is at least 2 orders of magnitude HsIFT46 partly overlap, the SEC profile shown in Figure 2A does weaker than the

Figure 1 Chlamydomonas

(left) Crystal structure of human ODA16 (HsODA16) shown as a cartoon and colored cyan. Comparison with the ODA16 (CrODA16) structure (shown in red and orange) reveals that both proteins consist of an 8-bladed β-propeller but HsODA16 does not have the small N-terminal domain positioned on top of the β-propeller. (right) After rotating 90 degrees around the horizontal axis, the C-terminal face of the Cr- and Hs-ODA16 structure is shown with the conserved surface residues displayed as sticks. The nine residues shown are completely conserved between CrODA16 and HsODA16 and likely constitute a common binding site for ODAs (numbering according to the HsODA16 sequence). JSM Sexual Med 2(2): 1008 (2017) 3/5 Lorentzen et al. (2017) Email:

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with IFT46 or stabilize the N-terminal domain of ODA16 in a confirmationCONCLUSION that allow for IFT46 binding.

Chlamydomonas and human, only the ChlamydomonasHere we show but that not despite the human the evolutionary proteins interact conservation directly toof ODA16 and IFT46 between

form a stable complex. This finding suggests that the coupling of ODA16 to the IFT system, and thus the trafficking of ODAs, is different in the two species. It is possible that other IFT proteins, yet uncharacterized factors or post-translational modifications in sperm cells are required to anchor the ODA-ODA16 complex to the ciliary trafficking machinery. Alternatively, the association Futurebetween experiments HsODA16 and will HsIFT46 be directed could at be investigation isoform specific these as several IFT46 isoforms are reported in the uniprot database.

betweenpossibilities. the human However, and Chlamydomonas the fact that we structures do observe suggests that that the C-terminal face of the ODA16 β-propeller is highly conserved conserved mechanism. the recognition of ODAs by ODA16 likely follows an evolutionarily ACKNOWLEDGEMENTS

We thank the Novo Nordisk Foundation (grant no. NNF15OC0014164) for funding this project. Structural coordinatesREFERENCES have been deposited at PDB, accession code 5NNZ. 1.

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Cite this article Taschner M, Basquin J, Bhogaraju S, Vetter M, Andersen MB, et al. (2017) Purification and Structure of Human ODA16: Implications for the Import of Outer Dynein Arms into Sperm Flagella by the Intraflagellar Transport Machinery. JSM Sexual Med 2(2): 1008.

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