Research article
The activation mechanism of Irga6, an interferon-inducible GTPase contributing to mouse resistance against Toxoplasma gondii
- † Equal contributors
1 Institute for Genetics, Department of Cell Genetics, University of Cologne, Zülpicher Strasse 47a, 50674 Cologne, Germany
2 Max-Planck-Institute for Molecular Physiology, Department of Structural Biology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany
3 Current address: Institute of Biochemistry II, Medical Faculty of the Goethe University, University Hospital Building 75, Theodor-Stern_Kai 7, 60528 Frankfurt am Main, Germany
4 Current address: Crucell Holland BV, Archimedesweg 6, 2333 CN Leiden, Netherlands
5 Current address: National University of Singapore, Division of Bioengineering, Block E3A, #07-02 7, Engineering Drive 1, 117576 Singapore
6 Current address: The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA
7 Current address: Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9/3, 1030 Vienna, Austria
8 Current address: Max-Planck-Institute of Biochemistry, Department of Structural Cell Biology, Am Klopferspitz 18, 82152 Martinsried, Germany
BMC Biology 2011, 9:7 doi:10.1186/1741-7007-9-7
Published: 28 January 2011Additional files
Additional file 1:
The three-dimensional structure of Irga6. The Crystal structure of Irga6-M173A GppNHp (PDB 1TQ6) [14] is shown. Protein domains are shown as indicated in the Figure 1. (a to f) The same orientations of the molecule are shown as in Figure 1.
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Additional file 2:
Amino acid sequence alignment of mouse IRGs. Amino acid sequence alignment of Irga1, Irga2, Irga3, Irga4, Irga6, Irga7, Irga8, Irgb1, Irgb2, Irgb3, Irgb4, Irgb5, Irgb6, Irgb8, Irgb9, Irgb10, Irgd, Irgm1, Irgm2 and Irgm3 from the C57BL/6 mouse. Irgc is not induced by IFNγ, Irga5 and Irgb7 are pseudogenes [4] and were thus excluded. Residues relevant for the crystal dimer interface (CDI) (Additional file 8 and 8) are highlighted (yellow 1 - red 6; indication how often they form part of the crystal dimer interface in the three available dimeric structures of Irga6 [14]). Residues that are part of the catalytic interface (CI) (Additional file 8) are marked (black X). Residues mutagenised (MUT) in this study and by Steinfeldt et al. [22] (Figure 1) are indicated: no inhibition of oligomerisation (green 0), inhibition of oligomerisation and part of the secondary patch (yellow 1), inhibition of oligomerisation and part of the secondary patch or the catalytic interface (orange 2), inhibition of oligomerisation and part of the catalytic interface (red 3). The calculated conservation score (CON) (Additional file 20) is displayed: variable (cyan 1) - conserved (magenta 9). The G1, G3, G4 and G5-motifs are highlighted by a red box. The GKS and GMS subfamilies are separated by a green line.
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Additional file 3:
Mutants of the secondary patch reduce oligomerisation. Mutagenesis of surface residues. (a) Oligomerisation of partially purified (see Methods) 80 μM WT or mutant Irga6 proteins was monitored by light scattering in the presence of 10 mM GTP at 37°C. Left panel: positive (WT) and negative (K196D) control (Figure 2a). Right panel: investigated mutants. Five mutants R31E-K32E, K169E, K176E, R210E and K246E inhibited the oligomerisation of Irga6, whereas many others had no significant effect. The mutants were fully purified. (b) Oligomerisation of 80 μM WT or mutant Irga6 proteins was monitored by light scattering in the presence of 10 mM GTP at 37°C. (c) Hydrolysis of 10 mM GTP (with traces α32P-GTP) was measured in the presence of 80 μM WT or mutant Irga6 proteins at 37°C. Samples were assayed by TLC and autoradiography.
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Additional file 4:
Oligomerisation of the catalytic interface mutants. Oligomerisation of 80 μM Irga6 mutant proteins was monitored in the presence of 10 mM GDP or GTP by DLS at 37°C.
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Additional file 5:
Oligomerisation of the secondary patch mutants. Oligomerisation of 80 μM Irga6 mutant proteins was monitored in the presence of 10 mM GDP or GTP by DLS at 37°C.
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Additional file 6:
Nucleotide-binding affinities of oligomerisation impaired Irga6 mutants. Dissociation constant (Kd) measured by equilibrium titration. The mean values and the standard deviation of at least two independent experiments are shown.
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Additional file 7:
Position of mutated residues in the crystal dimer. The Irga6 crystal dimer (PDB 1TPZ) [14] is shown. Protein domains and mutated residues are shown as indicated in the Figure 1. Lys9, Ser10, Lys196 of both subunits and Lys202 of the second subunit are not resolved in the crystal structure. (a) Top view. (b) Front view of the two G-domains; Additional file 7 rotated by 90° around the x-axis. (c) Left view; Additional file 7 rotated by 90° around the y-axis.
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Additional file 8:
Relative position of catalytic and crystal dimer interface. The structure of Irga6-M173A [14] is shown. Protein domains are shown as indicated in the Figure 1. (a and b) Residues buried in the interface of the Irga6 dimer model were calculated with CNSsolve [59] module buried surface [60] with a probe radius of 1.4 Å. The surface formed by Glu77, Thr78, Gly79, Asn94, Glu95, Lys101, Thr102, Gly103, Glu106, Val107, Gly131, Ser132, Thr133, Pro136, Pro137, Ala157, Thr158, Arg159, Phe160, Lys161, Lys162, Asn163, Asp166, Lys184, Asp186, Ser187, Asp188, Thr190, Asn191, Asp194, Gly195 and Lys233 is shown in magenta. (c and d) Residues buried in the crystal dimer interface were calculated by the same method. The two surfaces formed by Asn14, Ser18, Gln36, Glu37, Asn40, Leu41, Glu43, Leu44, Arg47, Lys48, Pro137, Asn138, Thr139, Leu141, Glu142, Tyr147, Asp166, Ala168, Lys169, Ala170, Ser172, Ala173 (instead of Met173), Met174, Lys175, Lys176, Glu177, Phe178, Arg218, Gly221, Ile222, Ala223 and Glu224 are shown. Three dimeric crystal structures of Irga6 are available (PDB 1TPZ, 1TQ2 and 1TQD) [14] therefore each residue can be maximum six time involved in this interface. Residues highly relevant for the crystal dimer interface are shown in red, less relevant in yellow. (a and c) Front view of the G-domain (Figure 1a). (b and d) Left view (Figure 1f).
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Additional file 9:
Mutations of the crystal dimer interface do not prevent oligomerisation. (a) Oligomerisation of 80 μM WT or mutant Irga6 proteins was monitored by light scattering in the presence of 10 mM GTP at 37°C. (b) Hydrolysis of 10 mM GTP (with traces α32P-GTP) was measured in the presence of 80 μM WT or mutant Irga6 proteins at 37°C. Samples were assayed by TLC and autoradiography.
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Additional file 10:
Irga6 dimer model. Atomic coordinates (structural PDB file) of the constructed (Figure 3) Irga6 dimer model (Figure 4).
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Additional file 11:
Asp164 and Arg159 participate in oligomerisation. For the construction of the Irga6 dimer model a rigid crystal structure was used. In the model the side chains of the Arg159 residues of the two subunits collide. Arg159 is located close to Asp164 on the other subunit. Asp164 forms the bottom of a pocket, derived from two loops. One loop is located between Glu77 and Ser80 and contains a part of the G1-motif. The other loop is located between Ile155 and Asn163. The conformation of Arg159 is relatively unconstrained [14]. A conformational change may occur during complex formation, reorienting Arg159 and inserting the side chain into the pocket on the opposed molecule to form a salt bridge with Asp164 in trans. Arg159 is part of the catalytic interface (Figure 1a). Consistent with this, mutations of Arg159 had deleterious effects on oligomerisation (Additional file 12). Asp164 is not solvent exposed, but withdrawn from the surface of the protein at the bottom of a pocket. It is therefore striking that even a mild mutation like D164N prevented oligomerisation (Additional file 13). (a and b) View of the nucleotide-binding region. (a) The Irga6 dimer model (Figure 4) is shown. Arg159, Asp164 (cyan subunit) and Arg159 (magenta subunit) are shown. (b) A molecule of Irga6-M173A [14] is shown. Asp164 and the molecular surface formed by the residues Glu77, Thr78, Gly79, Ser80, Ile155, Ser156, Ala157, Thr158, Arg159, Phe160, Lys161, Lys162 and Asn163 are shown. (c) Oligomerisation of 80 μM WT or mutant Irga6 proteins was monitored by light scattering in the presence of 10 mM GTP at 37°C. (d) Hydrolysis of 10 mM GTP (with traces α32P-GTP) was measured in the presence of 80 μM WT or mutant Irga6 proteins at 37°C. Samples were assayed by TLC and autoradiography.
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Additional file 12:
Oligomerisation of Arg159 mutants. Oligomerisation of 80 μM Irga6 mutant proteins was monitored in the presence of 10 mM GDP or GTP by DLS at 37°C.
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Additional file 13:
Oligomerisation of Asp164 mutants. Oligomerisation of 80 μM Irga6 mutant proteins was monitored in the presence of 10 mM GDP or GTP by DLS at 20°C or 37°C.
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Additional file 14:
Binding of guanine and xanthine nucleotides to WT and Irga6-D186N. Kd value (μM) measured by equilibrium titration. The mean values and the standard deviation of at least two independent experiments are shown.
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Additional file 15:
The Irga6 G4-motif mutant hydrolyses GTP faster than XTP. (a) Hydrolysis of 10 mM GTP (with traces α32P-GTP) was measured in the presence of 80 μM WT or mutant Irga6 at 37°C. Samples were assayed by TLC and autoradiography. (b) Hydrolysis of 10 mM XTP was measured in the presence of 80 μM WT or mutant Irga6 at 37°C. Samples were assayed by HPLC.
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Additional file 16:
The 3'OH of the GTP ribose is essential for oligomerisation; the 2'OH is not required for cooperative hydrolysis. (a) Oligomerisation of 80 μM WT Irga6 protein was monitored in the presence of 10 mM GTP, 2'dGTP, 3'dGTP or 2'3'ddGTP by DLS at 37°C. (b) Hydrolysis of 10 mM GTP or 2'dGTP was measured after 30 min. in the presence of various concentrations of WT Irga6 protein at 37°C. Samples were assayed by HPLC.
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Additional file 17:
Oligomerisation of Glu106 mutants. Oligomerisation of 80 μM Irga6 mutant proteins was monitored in the presence of 10 mM GDP or GTP by DLS at 37°C.
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Additional file 18:
Recruitment of Irga6 mutants to the T. gondii PVM. Irga6-deficient MEFs were stimulated with IFNγ and transiently transfected with Irga6-cTag1 WT and mutant constructs. The cells were infected with avirulent T. gondii strain ME49. Intracellular parasites were detected with anti-GRA7 monoclonal antibody (red) and ectopically expressed Irga6-cTag1 with anti-cTag1 antiserum (green). Nuclei were stained with DAPI (blue). Irga6-cTag1 coated (arrowhead) and non-coated (arrow) parasites are indicated. Weakly coated parasites, counted as Irga6-cTag1 positive (Figure 10b), are marked with open arrowheads. Scale bar, 10 μm.
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Additional file 19:
Amino acid sequence alignment of selected G-domains. Amino acid sequence alignment of the G-domains of Irga6, Irgb6 and Irgm3 form Mus musculus (MM), Ffh and FtsY from Thermus aquaticus (TA). The positions of G1, G3, G4 and G5 were fixed manually. Irga6 residues of the catalytic interface (Additional file 8 and 8) are highlighted in red. Residues buried in the interface of the Ffh-FtsY dimer (PDB 1RJ9) [20] were calculated with CNSsolve [59] module buried surface [60] with a probe radius of 1.4 Å and are highlighted in red. The tendency of the interface residues to align reflects the almost equal relative spatial orientation of the G-domains in the complexes and conserved structural features of G-domains. The G1, G3, G4 and G5-motifs are highlighted by a green box.
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Additional file 20:
Conservation of the Irga6 surface. The molecular surface of Irga6-M173A [14] is shown. ConSurf [62,63] was used with an alignment of IRGs (Additional file 2) to calculate the conservation score of Irga6 residues. Conserved residues are coloured in magenta, variable in cyan. (a to f) The same orientations of the molecule are shown as in Figure 1.
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Additional file 21:
Sequences of primers used for site directed mutagenesis. List of primers (sequences 5' - 3') used for generation of the Irga6 mutants.
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