Crystal structure of the intraflagellar transport complex 25/27

Abstract

The cilium is an important organelle that is found on many eukaryotic cells, where it serves essential functions in motility, sensory reception and signalling. Intraflagellar transport (IFT) is a vital process for the formation and maintenance of cilia. We have determined the crystal structure of Chlamydomonas reinhardtii IFT25/27, an IFT sub-complex, at 2.6 Å resolution. IFT25 and IFT27 interact via a conserved interface that we verify biochemically using structure-guided mutagenesis. IFT27 displays the fold of Rab-like small guanosine triphosphate hydrolases (GTPases), binds GTP and GDP with micromolar affinity and has very low intrinsic GTPase activity, suggesting that it likely requires a GTPase-activating protein (GAP) for robust GTP turnover. A patch of conserved surface residues contributed by both IFT25 and IFT27 is found adjacent to the GTP-binding site and could mediate the binding to other IFT proteins as well as to a potential GAP. These results provide the first step towards a high-resolution structural understanding of the IFT complex.

Figure 1

Structure of the IFT25ΔC/27 complex. Cartoon representation of the overall structure of IFT25ΔC/27 with IFT27 and IFT25 shown in green and slate, respectively. The calcium ion bound to IFT25 is shown as an orange ball. The two views of the complex are related by a 180-degree rotation around the horizontal axis. The switch I and G4 regions of IFT27 are disordered in the structure and represented with dotted lines in the figure. The N- and C-termini of both proteins are indicated.

Figure 2

Structural characterization of the IFT25ΔC/27 interface. (A) Close-up view of the protein–protein interface of the IFT25/27 complex. IFT27 secondary structure elements that do not engage in interactions with IFT25 but hinder the view of the interface have been removed for better visualization. Residues involved in calcium coordination (D30 and T35) or forming the interaction interface between the two proteins are shown in a stick representation and labelled. The salt bridge between R22_IFT25 and E191_IFT27 and the hydrogen bond between E122_IFT25 and Y95_IFT27 are indicated with dashed lines. (B) Pull-down experiments with interface mutants. GST-tagged wild-type IFT27 (GST-27^WT) or mutant IFT27 (GST-27^mut, V194R) was co-expressed with C-terminally truncated but otherwise wild-type IFT25 (25ΔC^WT) or mutant IFT25 (25ΔC^mut, V38R, T40R and T125E triple mutant) and pulled down using GSH beads. Whereas both GST-27^WT and GST-27^mut effectively pull down 25ΔC^WT, they do not pull down 25ΔC^mut, demonstrating that the interface between the two proteins has been disrupted. To ensure that 25ΔC^mut is expressed and soluble, the protein was pulled down separately using the His-affinity tag on IFT25 (right panel).

Figure 3

Conserved sequence motifs and IFT25-interacting residues of IFT27. Multiple sequence alignment of IFT27 proteins from differences species with CrIFT22 and the GTPases Rab11 and Rab8. The secondary structure derived from the IFT27 structure presented here is indicated above the sequences. Important sequence motifs of small GTPases such as switch regions, G-protein-specific sequences (G1-5) as well as Rab-specific residues (RabF1–5) are marked below the sequence. IFT27 residues that interact directly with residues from IFT25 are coloured green. The catalytic glutamine conserved in most GTPases, but not in IFT27 or IFT22, is coloured in blue and the prenylation sites at the C-termini of Rab8 and Rab11 are shown in pink. Hs: Homo sapiens, Mm: Mus musculus, Xl: Xenopus laevis, Dr: Danio rerio, Cr: Chlamydomonas reinhardtii. Tt: Tetrahymena thermophila.

Figure 4

IFT27 is lacking a catalytic glutamine residue. (A) Comparison of the GTPase sites of GDP-AlF₃-bound Ras (pdb code 1WQ1) and nucleotide-free IFT27. IFT27 is coloured green, Ras light brown and the bound nucleotide is shown as black sticks. The magnesium ion and the catalytic water are shown as a purple and red ball, respectively. The catalytic Q61 of Ras and equivalent residue of IFT27 (S79) are shown as sticks and labelled. Switch regions and the P-loop are also labelled. (B) GTPase assay. The release of inorganic phosphate upon GTP hydrolysis is followed using the EnzCheck Phosphate kit (Invitrogen) by absorbance measurements at 360 nm. Uncatalysed GTP hydrolysis in buffer as well as catalysed reactions using 500 μM of WT or mutant IFT25ΔC/27 complex over a time course of 90 min are shown. The concentration of GTP is in all cases 500 μM. As a positive control, 25 μM of the large GTPase IIGP1 is seen to hydrolyse all of the GTP in approximately 10 min. Whereas the GTP-binding mutant S30N is seen to reduce the GTPase activity, introduction of a glutamine at the position of S79 of IFT27 increases the activity significantly. Each of the curves representing WT or mutant IFT25ΔC/27 catalysed GTP turnover is the average of two independent experiments. (C) ITC measurements. ITC was used to measure the affinity of IFT25ΔC/27 for GDP and GTP nucleotides. WT IFT25ΔC/27 is seen to bind both GTP and GDP with micromolar affinities. The occupancies are close to one, demonstrating that only one nucleotide-binding site is present in the complex. The nucleotide binding can be attributed to the GTPase site of IFT27 since a point mutation (S30N) in the P-loop of IFT27 abolishes binding to GTP (right panel).

Figure 5

Surface conservation of the IFT25/27 structure. (Top) Cartoon representation of the IFT25ΔC/27 complex in a similar orientation as for Figure 1. Residues 24–38 of IFT27 that partly block the view of the GTP-binding pocket have been omitted from the figure for better visualization. (Bottom) Surface representation of the IFT25ΔC/27 complex in a similar orientation as for Figure 1. The sequence conservation between different species of IFT27 and IFT25 (same species as for the alignment in Figure 3; Supplementary Figure S2) are mapped onto the surface of the IFT25/27 heterodimeric structure. Variable residues are coloured white, whereas conserved residues are shown in orange according to the colour bar. In addition to a highly conserved GTP-binding pocket of IFT27, a neighbouring surface patch, extending across the IFT25/27 dimer, is highly conserved and likely to mediate protein–protein interactions.

Figure 6

Model of putative Rab-GAP binding to IFT25/27. Surface representation of the IFT25/27 heterodimer with the sequence conservation mapped onto the surface as for Figure 5. The model for the putative binding of Rab-GAP to the IFT25/27 complex is shown in blue as ribbon. The model has been derived by superimposing a transition-state structure of Rab33 (the Rab33-GDP-AlF₃–Rab-GAP complex) (pdb code 2G77) onto IFT27 of the IFT25/27 complex. GDP-AlF₃ from the Rab33 structure is displayed as sticks. Both the GDP-AlF₃ and the Rab-GAP binding to IFT25/27 represent models and are not experimentally determined structures.