Central JSM Sexual Medicine Bringing Excellence in Open Access 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 Dynein Copyright © 2017 Lorentzen et al. Arms into Sperm Flagella by OPEN ACCESS Keywords the Intraflagellar Transport • Sperm • Cilium • 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 cytoplasm and subsequently trafficked into the cilium by the intraflagellar transport (IFT) system. In the case of the green alga Chlamydomonasreinhardtii, the adaptor protein 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 ciliaarms relies are onperiodically conserved molecularattached [7].structures Outer evolvedCilia toare provide conserved unicellular eukaryotic organisms organelles with that the function ability toin including a microtubule(MT)-based axoneme onto which inner move in aqueous environments. In humans, motile cilia are lightdynein chains arms that (ODAs) assemble are macro-molecularinto 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 ATPases 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 primaryand 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: Central Bringing Excellence in Open Access 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 2Bcolumn was carriedin 10mM out Hepes as previously pH 7.5, complexTg737Rpw that mutant serves asmice an adaptorcarrying for a ciliaryhypermorphic 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 proteins 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 gene, 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 ispublished highly abundant study indemonstrated the testes of micethat 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