Binding of IFT22 to the intraflagellar transport complex is essential for flagellum assembly
2019; Springer Nature; Volume: 38; Issue: 9 Linguagem: Inglês
10.15252/embj.2018101251
ISSN1460-2075
AutoresStefanie Wachter, Jamin Jung, Shahaan Shafiq, J. Basquin, Cécile Fort, Philippe Bastin, Esben Lorentzen,
Tópico(s)Toxoplasma gondii Research Studies
ResumoArticle2 April 2019free access Source DataTransparent process Binding of IFT22 to the intraflagellar transport complex is essential for flagellum assembly Stefanie Wachter Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Jamin Jung Trypanosome Cell Biology Unit, Institut Pasteur & INSERM U1201, Paris, France Search for more papers by this author Shahaan Shafiq Trypanosome Cell Biology Unit, Institut Pasteur & INSERM U1201, Paris, France Search for more papers by this author Jerome Basquin Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Cécile Fort Trypanosome Cell Biology Unit, Institut Pasteur & INSERM U1201, Paris, France Search for more papers by this author Philippe Bastin orcid.org/0000-0002-3042-8679 Trypanosome Cell Biology Unit, Institut Pasteur & INSERM U1201, Paris, France Search for more papers by this author Esben Lorentzen Corresponding Author [email protected] orcid.org/0000-0001-6493-7220 Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark Search for more papers by this author Stefanie Wachter Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Jamin Jung Trypanosome Cell Biology Unit, Institut Pasteur & INSERM U1201, Paris, France Search for more papers by this author Shahaan Shafiq Trypanosome Cell Biology Unit, Institut Pasteur & INSERM U1201, Paris, France Search for more papers by this author Jerome Basquin Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Cécile Fort Trypanosome Cell Biology Unit, Institut Pasteur & INSERM U1201, Paris, France Search for more papers by this author Philippe Bastin orcid.org/0000-0002-3042-8679 Trypanosome Cell Biology Unit, Institut Pasteur & INSERM U1201, Paris, France Search for more papers by this author Esben Lorentzen Corresponding Author [email protected] orcid.org/0000-0001-6493-7220 Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark Search for more papers by this author Author Information Stefanie Wachter1, Jamin Jung2, Shahaan Shafiq2, Jerome Basquin1, Cécile Fort2, Philippe Bastin2 and Esben Lorentzen *,3 1Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany 2Trypanosome Cell Biology Unit, Institut Pasteur & INSERM U1201, Paris, France 3Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark *Corresponding author. Tel: +45 87155478;; E-mail: [email protected] EMBO J (2019)38:e101251https://doi.org/10.15252/embj.2018101251 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 Intraflagellar transport (IFT) relies on motor proteins and the IFT complex to construct cilia and flagella. The IFT complex subunit IFT22/RabL5 has sequence similarity with small GTPases although the nucleotide specificity is unclear because of non-conserved G4/G5 motifs. We show that IFT22 specifically associates with G-nucleotides and present crystal structures of IFT22 in complex with GDP, GTP, and with IFT74/81. Our structural analysis unravels an unusual GTP/GDP-binding mode of IFT22 bypassing the classical G4 motif. The GTPase switch regions of IFT22 become ordered upon complex formation with IFT74/81 and mediate most of the IFT22-74/81 interactions. Structure-based mutagenesis reveals that association of IFT22 with the IFT complex is essential for flagellum construction in Trypanosoma brucei although IFT22 GTP-loading is not strictly required. Synopsis The atypical Rab-like GTPase IFT22 is a component of the intraflagellar transport (IFT) complex and regulates flagella formation. X-ray structures uncover the mechanism of IFT22 association with GTP/GDP and the mode of its incorporation into the IFT complex, which is required for proper flagellum construction in Trypanosoma brucei. Despite the lack of classical guanine nucleotide-binding G4/G5 motifs, IFT22 shows specific and conserved G-nucleotide binding properties. IFT22 X-ray structure shows an unusual GTP/GDP binding mode via the divergent G5 motif. X-ray structure reveals that IFT22 switch regions directly interact with IFT74/81 and become ordered upon IFT74/81-22 complex formation. Association of IFT22 with the IFT complex, but not with GTP, is required for correct assembly of the flagellum in T. brucei. Introduction Cilia (also known as flagella) are important organelles needed for cell motility, morphogenesis, sensory perception, and several signaling pathways, such as sonic hedgehog and PDGFRα signaling (Huangfu et al, 2003; Kohl et al, 2003; San Agustin et al, 2015; Salinas et al, 2017; Schneider et al, 2005). Cilia are tail-like appendages protruding from the cell surface of nearly every eukaryotic cell type and are found on various unicellular organisms and on almost all cells in the mammalian body. For example, the protist Trypanosoma brucei, commonly known as the parasite causing sleeping sickness, carries a motile flagellum that is required for development and disease pathogenesis (Ralston et al, 2009; Langousis & Hill, 2014; Rotureau et al, 2014). A microtubule-based axoneme extending from the centriole-like basal body at the ciliary base is the central shape-giving element of cilia. The organelle is surrounded by the ciliary membrane, which is a continuous outgrowth of the plasma membrane but hosts a unique composition of lipids and membrane proteins (Emmer et al, 2010; Serricchio et al, 2015). To date, more than 600 different proteins have been identified to reside in the ciliary compartment (Pazour et al, 2005). Cilia construction as well as maintenance of the organelle in almost all organisms relies on a conserved active transport process termed intraflagellar transport (IFT; Kozminski et al, 1993; Rosenbaum & Witman, 2002). Intraflagellar transport particles are thought to be responsible for the selective transfer of ciliary cargo proteins from the cytoplasm through the diffusion barrier at the transition zone. IFT is dependent on the motor proteins kinesin II for anterograde (base to tip; Cole et al, 1993, 1998; Prevo et al, 2015) and dynein 2 for retrograde (tip to base) movement (Pazour et al, 1999; Porter et al, 1999; Signor et al, 1999) of cargo proteins and turnover products. The IFT complex is required for construction of the flagellum and likely serves important functions in ciliary cargo selection and transport (Bhogaraju et al, 2013b) and can be divided into biochemically distinct IFT-A and IFT-B sub-complexes, consisting of 6 and at least 16 individual proteins, respectively (Piperno & Mead, 1997; Cole et al, 1998; Taschner & Lorentzen, 2016b). The IFT-B complex is organized into two stable sub-complexes, the 10-subunit IFT-B1 (IFT22, IFT25, IFT27, IFT46, IFT52, IFT56, IFT70, IFT74, IFT81, IFT88; Lucker et al, 2005; Follit et al, 2009; Ishikawa et al, 2014; Taschner et al, 2014) and the 6-subunit IFT-B2 complex (Taschner et al, 2016). While inactivation of IFT-B complex components or the kinesin motor typically leads to defects in cilium construction due to disrupted anterograde IFT (Pazour et al, 2000; Absalon et al, 2008), IFT-A protein or dynein deletions produce phenotypes associated with malfunctioning retrograde transport (Pazour et al, 1999; Blacque et al, 2006). Mutations in IFT components and other ciliary proteins are the cause for a wide range of genetic diseases and developmental abnormalities, known as ciliopathies (Reiter & Leroux, 2017). For the assembly of large complexes that bind diverse cargo proteins, most IFT proteins are composed of protein–protein interaction domains such as coiled-coils, β-propellers, and tetratricopeptide repeats (Taschner et al, 2012). However, the two IFT complex members IFT22 (Rabl5) and IFT27 (Rabl4) show significant sequence homology to small GTPases of the Rab family, which are key regulators of vesicular membrane-trafficking (Stenmark, 2009; Itzen & Goody, 2011). IFT22 and IFT27 share low sequence identity (< 15%) and may play regulatory roles in IFT (Schafer et al, 2006; Qin et al, 2007; Adhiambo et al, 2009; Bhogaraju et al, 2011). In mammalian cilia, IFT27 was shown to be required for exit of the BBSome complex and associated ciliary cargoes (Keady et al, 2012; Eguether et al, 2014). Recently, Rabl2 was identified as a potential third Rab-like member of the IFT complex as it was shown to regulate IFT initiation and to associate with the IFT74/81 sub-complex (Lo et al, 2012; Kanie et al, 2017; Nishijima et al, 2017). Interestingly, IFT22 and IFT27 also associate with the IFT74/81 sub-complex (Taschner et al, 2014) suggesting that IFT22, IFT27, and Rabl2 may be located within close proximity in the IFT B1 complex. IFT22, IFT27, and RabL2 are unusual Rab GTPases as they lack the C-terminal prenylation motif commonly found to associate Rab GTPases with membranes. Previous studies classified IFT22 as an atypical small GTPase with a high degree of sequence variance from classical Rab proteins, particularly in sequences assigned to the conserved nucleotide-binding pocket (Schafer et al, 2006; Adhiambo et al, 2009). IFT22 lacks the conventional G4 motif and contains a highly diverse G5 motif required for interaction with the guanine base of GTP/GDP (Rensland et al, 1995; Vetter & Wittinghofer, 2001; Itzen & Goody, 2011). Hence, it is unclear if IFT22 can specifically bind guanine nucleotides. Interestingly, in vivo studies in several ciliated organisms revealed functional differences of IFT22 between species. Mutation of the Caenorhabditis elegans (Ce) IFT22 homolog (called IFTA-2) does not affect cilium formation or IFT, but worms show deficiencies in the DAF-2 (insulin-IGF-1-like) signaling pathway, leading to an extended lifespan and abnormalities in dauer stage formation (Schafer et al, 2006; Blacque et al, 2018). In contrast, RNAi knockdown experiments of IFT22/Rabl5 in Trypanosoma brucei (Tb) led to a retrograde IFT inactivation phenotype that is characterized by short flagella filled with IFT material (Adhiambo et al, 2009). In Chlamydomonas reinhardtii (Cr), IFT22 was shown to control the cellular levels of both IFT-A and IFT-B proteins and to regulate availability of particles participating in IFT (Silva et al, 2012). Intriguingly, IFT22 homologs are missing in the genomes of Giardia intestinalis and Tetrahymena thermophila, although IFT is present in these ciliated organisms, whereas Drosophila melanogaster lacks both IFT22 and IFT74/81 homologs (van Dam et al, 2013). In this study, we provide insights into nucleotide specificity of IFT22, the molecular basis of incorporation into the IFT complex and a dissection of the in vivo function of IFT22 using structure-based mutations in T. brucei. We show that IFT22 specifically binds G-nucleotides and present the crystal structures of GTP- and GDP-bound IFT22, which identify a new, unusual binding mode for G-nucleotides in the absence of the classical G4 motif. The crystal structure of the trimeric IFT22/74/81 complex provides a molecular basis for IFT22 incorporation to the IFT complex via the switch regions of IFT22 and a heterodimeric coiled-coil region of IFT74/81. In vivo experiments using structure-based IFT22 mutants in T. brucei demonstrate that association of IFT22 with IFT-B1 is essential for ciliogenesis. Results IFT22 specifically binds GDP/GTP Due to the unusual G4/G5 regions, it was unclear if IFT22 is a selective guanine nucleotide-binding protein or if IFT22 may bind other purine nucleotides such as ATP (Espinosa et al, 2009; Taschner et al, 2012). To address nucleotide specificity, we overexpressed and purified TbIFT22 and removed nucleotides retained during the purification by urea treatment and refolding (Appendix Fig S1A–C). We then measured the affinities of apo TbIFT22 for GTP and GDP in titration experiments with fluorescently labeled non-hydrolyzable GTP/ATP derivatives (mant-GMPPNP/mant-AMPPNP) or GDP (mant-GDP). TbIFT22 bound the GTP analog with a Kd of 2 μM and GDP with a Kd of 20 μM (Fig 1A, left and middle panels). These weak μM affinities are in the same range as reported for GTP/GDP-binding by IFT27 (Bhogaraju et al, 2011) and suggest that nucleotide exchange does not necessarily require a guanine nucleotide exchange factor (GEF), as it is the case for some large GTPases (Uthaiah et al, 2003). No binding was observed for the ATP analog (Fig 1A, right panel). We therefore conclude that IFT22 is a specific guanine nucleotide-binding protein. Figure 1. IFT22 associates with guanine nucleotides through an unusual G5-dependent mechanism IFT22 nucleotide-binding experiments. Fluorescence measurements using increasing amounts of TbIFT22 and TbIFT22/74342–401/81397–450 core complex incubated with mant-labeled GDP (mant-GDP) or non-hydrolysable GTP/ATP analogs (mant-GMPPNP/mant-AMPPNP). The fluorescence intensity is plotted as a function of protein concentration. Data were fitted to a single-site binding equation for determination of the dissociation constant (Kd). Kd values and standard deviations are calculated from three independent experiments. Structural comparison of GTP-bound HsRab8A (light purple) and TbIFT22 (green) depicted in cartoon representation. Nucleotides are shown as sticks and Mg2+ as balls. Unstructured regions of TbIFT22 are represented with dotted lines. The zoomed-in view shows a superposition of the nucleotide-binding pocket. While classical GTPases form hydrogen bonds between a conserved aspartate of the G4 motif (NKxD) and the guanine base, IFT22 instead utilizes D175 located in the G5 loop. Topology diagrams of a classical Rab GTPase and of IFT22. Positions of the conserved nucleotide-binding G-motifs (G1–G5) as well as switch regions are indicated. Top: Cartoon representation of IFT22 (gray) with positions of two nucleotide-binding mutants highlighted. GTP is shown in stick representation and Mg2+ as a ball. D175 (blue) is the unusual residue binding the guanine base (see also Fig 1B), while S19 (pink) is a conserved residue required for coordination of the Mg2+ cation and is commonly mutated to an asparagine to prevent nucleotide binding. Bottom: Nucleotide-binding experiments of IFT22 nucleotide-binding mutants D175E, D175A, and S19N with fluorescently labeled nucleotides. Only the S19N mutation (light pink) abolished IFT22 nucleotide-binding ability completely. Superposition of GTP (green)- and GDP-bound (light green) TbIFT22 structures. Switch regions are marked in yellow and dotted lines indicate disordered loops not modeled in the structures. Download figure Download PowerPoint We also measured the affinities for mant-GMPPNP and mant-GDP of IFT22 in context of the TbIFT22/74/81 core complex (TbIFT22/74342–401/81397–450), which demonstrated a modest increase in affinities when compared to IF22 alone (Fig 1A, left and middle panels). To confirm these results, IFT22 or IFT22/74/81 core complexes from Tb, M. musculus (Mm) or C. reinhardtii (Cr) were incubated with excess of GTP and the content of bound nucleotides analyzed after size-exclusion chromatography (SEC) using an HPLC-based system (Appendix Fig S1E). IFT22/74/81 core complexes from all three species bound GTP, albeit to a different degree. The core complex from T. brucei incorporated the highest percentage of GTP, followed by C. reinhardtii and last M. musculus. Notably, TbIFT22 bound less GTP than the TbIFT22/74/81 core complex and no nucleotide could be detected for MmIFT22, which likely reflects that MmIFT22 has lower affinity for GTP than the Tb and CrIFT22 proteins. These results show that GTP-binding is a conserved property of IFT22 across species and confirm that the IFT22/74/81 core complex has higher affinity for GTP than IFT22 alone. Next, we analyzed the intrinsic GTPase activity of TbIFT22 and detected very low but measureable hydrolysis rates for both TbIFT22 and the TbIFT22/74/81 core complex (Appendix Fig S1F) comparable to reported intrinsic hydrolysis rates of other small GTPases (Simon et al, 1996; Scheffzek & Ahmadian, 2005; Bhogaraju et al, 2011). Thus, if GTP turnover is required for the cellular function of IFT22, a GTPase activating protein (GAP) is required to stimulate nucleotide hydrolysis. Structures of IFT22 with GTP or GDP reveal the molecular basis of guanine specificity To address the molecular basis of nucleotide binding by IFT22, we crystallized TbIFT22 with co-purified GTP and determined the structure at 2.3 Å resolution (Fig 1B and Table 1). To obtain a GDP-bound structure, TbIFT22 was treated with urea, dialyzed to remove bound nucleotides, and refolded in the presence of GDP. The IFT22-GDP structure was determined at 2.5 Å resolution, and the electron density clearly supports the presence of GDP (Appendix Fig S1G, compare left and middle panels). As expected, IFT22 exhibits the overall fold of a Rab GTPase, containing a mixed six-stranded β-sheet surrounded by α-helices (Fig 1B, right image). However, in contrast to classical GTPases that contain five α-helices, IFT22 lacks the α4 helix between β5 and β6 (Fig 1C). When the IFT22 structure is compared to protein structures currently available in the protein data bank using the Dali server (Holm & Sander, 1993), IFT22 is most similar to structures of other Rab family GTPases with Homo sapiens (Hs) Rab8A as the closest match (PDB ID: 4lhw), superposing with a root mean square deviation (rmsd) of 2.4 Å (see Fig 1B). Table 1. Data collection and refinement statistics TbIFT22-GTP TbIFT22-GDP TbIFT22/7479–401/811–450-GTP (SeMet) PDB code 6IA7 6IAE 6IAN Data collection Wavelength (Å) 1.00000 0.97891 0.97899 Resolution range (Å) 48.37–2.30 (2.38–2.30) 48.52–2.49 (2.57–2.49) 82.67–3.20 (3.40–3.20) Space group P 61 P 61 P 21 Unit cell (Å, °) a = 55.85 a = 56.02 a = 68.56 b = 55.85 b = 56.02 b = 228.30 c = 263.45 c = 263.09 c = 115.71 α = 90 α = 90 α = 90 β = 90 β = 90 β = 96.76 γ = 120 γ = 120 γ = 90 Total reflections 205,477 (17,635) 321,430 (27,213) 2,191,763 (226,031) Unique reflections 20,689 (1,996) 16,300 (1,558) 113,747 (18,991) Multiplicity 9.9 (8.8) 19.7 (17.5) 19.3 (11.9) Completeness (%) 99.7 (96.7) 99.3 (96.1) 100.0 (100.0) Mean I/sigma 16.7 (0.9) 18.5 (0.9) 11.5 (0.7) CC1/2 0.999 (0.497) 0.998 (0.363) 0.999 (0.395) Refinement Number of reflections 20,588 16,236 57,455 Protein residues 295 305 1,637 Number of atoms 2,311 2,354 12,090 R-work 0.212 (0.292) 0.220 (0.359) 0.241 (0.441) R-free 0.264 (0.349) 0.244 (0.385) 0.280 (0.447) Ramachandran favored (%) 93.5 94.0 96.3 Ramachandran outliers (%) 0.0 0.35 0.25 RMS bonds (Å) 0.005 0.006 0.007 RMS angles (°) 0.90 1.0 1.1 Average B-factors (Å2) 64 72 143 Statistics for the highest resolution shell are shown in parentheses. The high degree of sequence divergence of IFT22 when compared to other Rab GTPases (Appendix Fig S2) translates into a highly unconventional nucleotide-binding mode in IFT22. The classical G4 NKxD motif, which is missing in IFT22, features an aspartate residue (D124 in HsRab8A, see Fig 1B, detailed view) that interacts with the base of the nucleotide thus providing specificity for guanine over adenine (Rensland et al, 1995; Paduch et al, 2001). In the absence of a G4 aspartic acid, TbIFT22 uses D175 from the unusual G5 motif to form a bifurcated hydrogen bond with the guanine moiety (see detailed view in Fig 1B). While the classical G4 motif is positioned in a loop connecting β5 with α4, Asp175 is located between β6 and α5* (see Fig 1C). IFT22 homologs from D. rerio (Dr) and mammals (Mm, Hs) have a glutamate residue in the position of TbIFT22 D175 (Appendix Fig S2) indicating a potentially similar binding mechanism in those species. Cr and C. elegans (Ce) IFT22 contain an alanine and a glycine, respectively, at the position of TbIFT22 D175 making it unclear how or if they achieve specificity for G-nucleotides. To evaluate the importance of D175 in GTP/GDP-binding, we purified D175E and D175A mutant forms of TbIFT22 and carried out titrations with mant-GMPPNP or mant-GDP (Fig 1D). While the TbIFT22D175A mutant did not show any detectable nucleotide-binding, the TbIFT22D175E mutant bound mant-GMPPNP and mant-GDP with KD values of 18 μM and 139 μM, respectively, which is approximately one order of magnitude lower affinity than wild-type TbIFT22. Interestingly, TbIFT22D175A did bind mant-GMPPNP when in context of the IFT22D175A/74/81 core complex with a KD of 102 μM, which is approximately two orders of magnitude lower affinity than what we observed for the wild-type IFT22/74/81 core complex (Fig 1A and D). These results are in agreement with higher GTP affinity of the IFT22/74/81 core complex compared to IFT22 alone. Our data demonstrate that D175 is important for nucleotide binding in TbIFT22 and suggest that E175 can contact the guanine moiety of GTP/GDP although in a less favorable manner than D175 likely due to steric problems caused by the longer side-chain. We also introduced the classical S19N mutation in TbIFT22 that prevents Mg2+ coordination and thus abolishes nucleotide binding. As expected, our titration data show that TbIFT22S19N does not associate with mant-GMPPNP (Fig 1D). A hallmark of small GTPases are the switch regions that typically undergo major conformational changes between the active GTP-bound and the inactive GDP-bound states, which allow for binding of effectors (Vetter & Wittinghofer, 2001; Mourão et al, 2014). Surprisingly, no major conformational changes were observed when comparing the GTP- and GDP-bound states of IFT22 and the switch regions are unstructured in both GDP- and GTP-bound TbIFT22 structures (Fig 1E). While switch I and II of GDP-bound GTPases are known to be rather flexible and often unstructured, active GTP-bound forms usually exhibit ordered switch regions that provide a stable interaction surface for downstream effector binding. The switch regions of GTP-bound TbIFT22 are thus not in a pre-ordered conformation ready to associate with effectors. However, the observation that the IFT22/74/81 core binds nucleotides with higher affinity than IFT22 alone does suggest that IFT74/81 may interact with and stabilize the nucleotide-binding pocket of IFT22. Structure of the IFT22/74/81 complex To elucidate how IFT22 is incorporated into the IFT-B1 complex and determine if IFT74/81 is an effector of IFT22, we set out to obtain a structure of IFT22/74/81. IFT74 and IFT81 are both predicted to contain mostly coiled-coil structures (Fig 2A) and share 26% sequence identity in T. brucei suggesting that IFT74 and IFT81 are distant homologs. IFT74 and IFT81 interact directly with each other to form a binding platform for the IFT-B1 components IFT22, IFT25/27, and IFT46/52 (Lucker et al, 2005; Taschner et al, 2011, 2014). In addition to the coiled-coil regions, IFT74/81 contains an N-terminal tubulin-binding module contributed by both proteins (Bhogaraju et al, 2013a). Since IFT22/74/81 core complexes (TbIFT22/74342–401/81397–450, Appendix Fig S1D) did not yield crystals, we co-expressed and purified longer constructs of TbIFT74/81 with TbIFT22, spanning the N-terminal predicted coiled-coil domains. The positively charged IFT74 N-terminus is prone to degradation and was consequently removed resulting in the TbIFT7479–401 construct. Whereas complexes lacking the IFT81 CH domain did not crystallize, the GTP-bound TbIFT22/7479–401/811–450 complex containing the IFT81 CH domain crystallized and the structure was determined at 3.2 Å resolution by experimental phasing (Appendix Fig S3A–C and Table 1). Figure 2. Structure of the TbIFT22/74/81 complex Domain organization of IFT81, IFT74, and IFT22. Numbers refer to the T. brucei protein sequence and indicate different constructs used in this study. The part of the IFT81/74 sequence shown in shaded colors is not part of the construct used for structure determination. Coiled-coil boundaries are depicted based on the structure (ccI-ccVI) or prediction from the PCOILS webserver (cc). (CH = calponin homology, cc = coiled-coil). Crystal structure of TbIFT22/7479–401/811–450 in two perpendicular orientations shown in cartoon representation. GTP is shown as a stick model. IFT22 is depicted in green, IFT74 in orange, and IFT81 in gray. Coiled-coils are labeled ccI-ccVI. Zoomed-in view of the N-terminal TbIFT81 CH domain (gray) superposed onto the CrIFT81 CH domain (brick-red). Basic tubulin-binding residues are highlighted in yellow and light orange, respectively. Zoomed-in view of the IFT22-binding site on IFT74/81 ccVI with ordered switch regions of IFT22 depicted in yellow. Download figure Download PowerPoint The TbIFT22/7479–401/811–450 crystal structure reveals an elongated coiled-coil complex with the IFT81 CH domain and the IFT22 GTPase located at opposite ends (Fig 2B). Rather than forming one long coiled-coil, the IFT7479–401/811–450 structure can be subdivided into six separate heterodimeric coiled-coil regions (ccI to ccVI) separated by short loop regions (Fig 2A and B). Boundaries of these coiled-coils do not match particularly well with predicted coiled-coils from the PCOILS webserver (Alva et al, 2016). IFT74 and IFT81 interact intimately and share a large buried surface interface of 8,300 Å2, constituting both interactions within the coiled-coils and between different heterodimeric regions (Fig 2B and Appendix Fig S4). Whereas ccI and ccVI protrude from either end of the complex to interact with the IFT81 CH domain and IFT22, respectively, the central four coiled-coil regions, ccII-ccV, form a highly compact structure held together by interactions between ccII-ccIII, ccIII-ccIV, and ccII-ccIII-ccV (Appendix Fig S4). IFT74/81 ccII-ccV appears to form a rather unique compressed spring-like structure. Searches using the Dali server did not reveal any structures similar to IFT74/81 ccII-ccV in the protein data bank. The position of the N-terminal IFT81 CH domain is fixed to IFT74/81 ccI through contacts with the 15-residue linker region and the bent C-terminal helix of the IFT81 CH domain (Appendix Fig S5E). This C-terminal helix, which is bent in IFT81 CH domains (Appendix Fig S5A and B), adopts a straight conformation in the two MT-binding CH domain containing proteins NDC80 and EB1 (Slep & Vale, 2007; Ciferri et al, 2008; Appendix Fig S5C and D). This observation provides a molecular rationale for the different architecture of the IFT74/81 and the NDC80/NUF2 complexes (Alushin et al, 2010; Appendix Fig S5F). Interestingly, many of the positively charged Arg/Lys residues previously shown to mediate αβ-tubulin cargo binding in the CrIFT81 CH domain (Bhogaraju et al, 2013a) are found in structurally conserved positions in the TbIFT81 CH domain (Fig 2C). These tubulin-binding residues point toward ccII-ccIV perhaps suggesting that tubulin cargo could be sandwiched in the gap between the CH domain and ccII-IV (Fig 2B and C). Noteworthy, upon IFT74/81 association the switch regions of IFT22 become ordered and mediate binding to ccVI of IFT74/81 (Fig 2D). As observed for the IFT22/74/81 core complex, IFT22/7479–401/811–450 also co-purified with GTP (confirmed by HPLC) and was set up for crystallization with a molar excess of GTP at 4°C. Although the guanine base only displays partial electron density, the ribose and tri-phosphate moieties have clear electron density confirming that GTP is bound in the nucleotide-binding pocket of IFT22 (Appendix Fig S1G, right panel). The observation that the switch regions are ordered in the GTP-bound IFT22/74/81 complex structure but not in GTP-bound IFT22 shows that binding of IFT22 to IFT74/81 induces a fixed conformation of switch I and II (Fig 2D). There are no direct contacts between GTP and IFT74/81 suggesting that the increased nucleotide affinity of the IFT22/74/81 core complex compared to IFT22 alone (Fig 1A and E) is an indirect effect of fixing the switch regions in a conformation with higher nucleotide affinity. The switch regions of IFT22 interact with a conserved surface patch contributed by both IFT74 and IFT81 Analysis of the TbIFT22/74/81 complex structure reveals a relatively small (710 Å2 buried surface) mixed hydrophobic/hydrophilic interface of between IFT22 and IFT74/81 (Fig 3A and B). Switch I and II contribute most of the IFT22 residues to the interface with IFT74/81 with a few additional residues contributed from the β-sheet of the core GTPase fold (Fig 3B). Both IFT74 and IFT81 interact with IFT22 although IFT81 contributes about twice as many residues to the interface with IFT22 as IFT74 does (Fig 3A and B, and Appendix Fig S3D). A high degree of evolutionary conservation of residues in the interface between IFT22 and IFT74/81 (Fig 3A and Appendix Fig S3D) suggests that IFT22 associates with IFT74/81 in a similar manner in other ciliated organisms. To confirm this notion, we show that the IFT22-IFT74/81 interaction interface between Chlamydomonas and Trypanosoma is conserved to such a degree that TbIFT22 efficiently pulls down a purified CrIFT25/27/74/81 complex, thereby forming a stable pentameric IFT-B1 chimera (Fig 3C). The prevention of nucleotide-binding via the TbIFT22S19N mutant reduced the amount of CrIFT25/27/74/81 pulled down by His-tagged TbIFT22 to background levels (Fig 3C). We conclude that IFT22, usin
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