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. 2013 Sep 3;21(9):1707-17.
doi: 10.1016/j.str.2013.06.027. Epub 2013 Aug 15.

Hexamers of the Type II Secretion ATPase GspE From Vibrio Cholerae With Increased ATPase Activity

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Hexamers of the Type II Secretion ATPase GspE From Vibrio Cholerae With Increased ATPase Activity

Connie Lu et al. Structure. .
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Abstract

The type II secretion system (T2SS), a multiprotein machinery spanning two membranes in Gram-negative bacteria, is responsible for the secretion of folded proteins from the periplasm across the outer membrane. The critical multidomain T2SS assembly ATPase GspE(EpsE) had not been structurally characterized as a hexamer. Here, four hexamers of Vibrio cholerae GspE(EpsE) are obtained when fused to Hcp1 as an assistant hexamer, as shown with native mass spectrometry. The enzymatic activity of the GspE(EpsE)-Hcp1 fusions is ∼20 times higher than that of a GspE(EpsE) monomer, indicating that increasing the local concentration of GspE(EpsE) by the fusion strategy was successful. Crystal structures of GspE(EpsE)-Hcp1 fusions with different linker lengths reveal regular and elongated hexamers of GspE(EpsE) with major differences in domain orientation within subunits, and in subunit assembly. SAXS studies on GspE(EpsE)-Hcp1 fusions suggest that even further variability in GspE(EpsE) hexamer architecture is likely.

Figures

Figure 1
Figure 1. Characterization of Vibrio cholerae GspEEpsE fused to Hcp1 in solution
Figure 1A. Domain bar diagram of GspEEpsE and homologous ATPases with electron microscopy reconstructions or crystal structures. VcGspE =GspE, the assembly ATPase from the T2SS in the Gram-negative Vibrio cholerae; TtPilF = PilF, an assembly ATPase from the T4PS in the Gram-negative eubacterium Thermus thermopilus; PaPilT = PilT, a retraction ATPase from the T4PS in the Gram-negative eubacterium Pseudomonas aeruginosa; AaPilT = PilT, a retraction ATPase from the T4PS in the Gramnegative eubacterium Aquifex aeolicus; AfGspE2 = one of three related ATPases in the Archaeon Archaeoglobus fulgidus; SaFlaI = FlaI, an ATPase from the archaellum assembly system in the Archaeon Sulfolobus acidocaldarius. The percentages of amino acid sequence identity given are from the comparison with GspEEpsE. Where domains are homologous in structures to GspEEpsE, the percentage sequence identity is given compared to GspEEpsE. Figure 1B. Native mass spectra of ΔN1GspEEpsE-Hcp1 fusion proteins. Left: ΔN1GspEEpsE-8aa-Hcp1 Right: ΔN1GspEEpsE-6aa-Hcp1. The data show that these proteins each assemble as hexamers in solution. The mass measured for each complex is only slightly greater than that expected assuming that each protein subunit in the hexamer contains one Zn and one nucleotide. The native mass spectra for ΔN1GspEEpsE-5aa-Hcp1 and ΔN1GspEEpsE-7aa-Hcp1, fragmentation spectra for ΔN1GspEEpsE-8aa-Hcp1, and a table of observed and expected masses are shown in Figure S1. Figure 1C. ATPase activities of V. cholerae ΔN1GspEEpsE-linker-Hcp1 variants. From left to right the activities for: ΔN1GspEEpsE (control monomer), ΔN1GspEEpsE-8aa-Hcp1, ΔN1GspEEpsE-7aa-Hcp1, ΔN1GspEEpsE-6aa(KLASGA)-Hcp1, ΔN1GspEEpsE-6aa(GSGSGS)-Hcp1 and ΔN1GspEEpsE-5aa-Hcp1. Two variant linkers were tested in the case of ΔN1GspEEpsE-6aa-Hcp1 fusions to evaluate the effect of linker sequence. As shown, both types of linkers gave the same increase in activity. The crystal structure was determined of ΔN1GspEEpsE-6aa(GSGSGS)-Hcp1 which is called ΔN1GspEEpsE-6aa-Hcp1 throughout the paper.
Figure 1
Figure 1. Characterization of Vibrio cholerae GspEEpsE fused to Hcp1 in solution
Figure 1A. Domain bar diagram of GspEEpsE and homologous ATPases with electron microscopy reconstructions or crystal structures. VcGspE =GspE, the assembly ATPase from the T2SS in the Gram-negative Vibrio cholerae; TtPilF = PilF, an assembly ATPase from the T4PS in the Gram-negative eubacterium Thermus thermopilus; PaPilT = PilT, a retraction ATPase from the T4PS in the Gram-negative eubacterium Pseudomonas aeruginosa; AaPilT = PilT, a retraction ATPase from the T4PS in the Gramnegative eubacterium Aquifex aeolicus; AfGspE2 = one of three related ATPases in the Archaeon Archaeoglobus fulgidus; SaFlaI = FlaI, an ATPase from the archaellum assembly system in the Archaeon Sulfolobus acidocaldarius. The percentages of amino acid sequence identity given are from the comparison with GspEEpsE. Where domains are homologous in structures to GspEEpsE, the percentage sequence identity is given compared to GspEEpsE. Figure 1B. Native mass spectra of ΔN1GspEEpsE-Hcp1 fusion proteins. Left: ΔN1GspEEpsE-8aa-Hcp1 Right: ΔN1GspEEpsE-6aa-Hcp1. The data show that these proteins each assemble as hexamers in solution. The mass measured for each complex is only slightly greater than that expected assuming that each protein subunit in the hexamer contains one Zn and one nucleotide. The native mass spectra for ΔN1GspEEpsE-5aa-Hcp1 and ΔN1GspEEpsE-7aa-Hcp1, fragmentation spectra for ΔN1GspEEpsE-8aa-Hcp1, and a table of observed and expected masses are shown in Figure S1. Figure 1C. ATPase activities of V. cholerae ΔN1GspEEpsE-linker-Hcp1 variants. From left to right the activities for: ΔN1GspEEpsE (control monomer), ΔN1GspEEpsE-8aa-Hcp1, ΔN1GspEEpsE-7aa-Hcp1, ΔN1GspEEpsE-6aa(KLASGA)-Hcp1, ΔN1GspEEpsE-6aa(GSGSGS)-Hcp1 and ΔN1GspEEpsE-5aa-Hcp1. Two variant linkers were tested in the case of ΔN1GspEEpsE-6aa-Hcp1 fusions to evaluate the effect of linker sequence. As shown, both types of linkers gave the same increase in activity. The crystal structure was determined of ΔN1GspEEpsE-6aa(GSGSGS)-Hcp1 which is called ΔN1GspEEpsE-6aa-Hcp1 throughout the paper.
Figure 1
Figure 1. Characterization of Vibrio cholerae GspEEpsE fused to Hcp1 in solution
Figure 1A. Domain bar diagram of GspEEpsE and homologous ATPases with electron microscopy reconstructions or crystal structures. VcGspE =GspE, the assembly ATPase from the T2SS in the Gram-negative Vibrio cholerae; TtPilF = PilF, an assembly ATPase from the T4PS in the Gram-negative eubacterium Thermus thermopilus; PaPilT = PilT, a retraction ATPase from the T4PS in the Gram-negative eubacterium Pseudomonas aeruginosa; AaPilT = PilT, a retraction ATPase from the T4PS in the Gramnegative eubacterium Aquifex aeolicus; AfGspE2 = one of three related ATPases in the Archaeon Archaeoglobus fulgidus; SaFlaI = FlaI, an ATPase from the archaellum assembly system in the Archaeon Sulfolobus acidocaldarius. The percentages of amino acid sequence identity given are from the comparison with GspEEpsE. Where domains are homologous in structures to GspEEpsE, the percentage sequence identity is given compared to GspEEpsE. Figure 1B. Native mass spectra of ΔN1GspEEpsE-Hcp1 fusion proteins. Left: ΔN1GspEEpsE-8aa-Hcp1 Right: ΔN1GspEEpsE-6aa-Hcp1. The data show that these proteins each assemble as hexamers in solution. The mass measured for each complex is only slightly greater than that expected assuming that each protein subunit in the hexamer contains one Zn and one nucleotide. The native mass spectra for ΔN1GspEEpsE-5aa-Hcp1 and ΔN1GspEEpsE-7aa-Hcp1, fragmentation spectra for ΔN1GspEEpsE-8aa-Hcp1, and a table of observed and expected masses are shown in Figure S1. Figure 1C. ATPase activities of V. cholerae ΔN1GspEEpsE-linker-Hcp1 variants. From left to right the activities for: ΔN1GspEEpsE (control monomer), ΔN1GspEEpsE-8aa-Hcp1, ΔN1GspEEpsE-7aa-Hcp1, ΔN1GspEEpsE-6aa(KLASGA)-Hcp1, ΔN1GspEEpsE-6aa(GSGSGS)-Hcp1 and ΔN1GspEEpsE-5aa-Hcp1. Two variant linkers were tested in the case of ΔN1GspEEpsE-6aa-Hcp1 fusions to evaluate the effect of linker sequence. As shown, both types of linkers gave the same increase in activity. The crystal structure was determined of ΔN1GspEEpsE-6aa(GSGSGS)-Hcp1 which is called ΔN1GspEEpsE-6aa-Hcp1 throughout the paper.
Figure 2
Figure 2. Crystal structures of hexameric ΔN1GspEEpsE from Vibrio cholerae fused to Hcp1
Insets: schematic view of the hexamer outlining all six CTD•N2D’ construction units. CTD domains in light grey, N2D’ domains in dark grey. Figure 2A. V. cholerae ΔN1GspEEpsE-6aa-Hcp1, with a quasi-C6 ΔN1GspEEpsE hexamer and V. cholerae ΔN1GspEEpsE-8aa-Hcp1 containing a C2 hexamer. Upper panel: The quasi-C6 hexamer of V. cholerae ΔN1GspEEpsE-6aa-Hcp1. This fusion forms a ΔN1GspEEpsE hexamer with quasi-C6 point group symmetry. Shown are subunits A (green), B (cyan), C (purple), D (yellow), E (blue) and F (red). The CTDs are shown in a lighter shade of the same color as the N2Ds of the same subunit. The Hcp1 assistant hexamer is shown in orange. Left: view perpendicular to the quasi sixfold depicting also the Hcp1 hexamer. Right: view along the quasi-sixfold axis of the ΔN1GspEEpsE hexamer, with the Hcp1 hexamer omitted. The nucleotides shown in spheres are AMPPNP superposed from the helical Δ90GspEEpsE structure (PDB: 1p9w (Robien et al., 2003)). The shape of the hexamer in this view is very regular. One CTD•N2D’ construction unit is outlined. Lower panel: The C2 hexamer of V. cholerae ΔN1GspEEpsE-8aa-Hcp1. This fusion forms a ΔN1GspEEpsE hexamer with C2 point group symmetry. Shown are subunits A (green), B (blue), C (red) – each occurring twice in the hexamer. The CTDs are shown in a lighter shade of the same color as the N2Ds of the same subunit. The Hcp1 assistant hexamer is shown in orange. Left: view perpendicular to the twofold depicting also the Hcp1 hexamer. Right: view along the twofold axis of the ΔN1GspEEpsE hexamer, with the Hcp1 hexamer omitted. The nucleotides shown in pink spheres are AMPPNP superposed from the helical Δ90GspEEpsE structure (PDB: 1p9w (Robien et al., 2003)). The shape of the hexamer in this view is an approximate ellipsoid of 105 Å by 150 Å. One CTD•N2D’ construction unit is outlined. See also Figure S2, S3, S4 and S7. Figure 2B. The variability of the N2D-vs-CTD orientations in V. cholerae GspEEpsE. Shown are superimposed subunits in the “canonical view” with the CTDs superimposed below and the N2Ds on top (colored as Figure 2A). For a different “orthogonal view,” see Figure 2C. The nucleotide shown for reference is AMPPNP from the helical Δ90GspEEpsE structure (PDB: 1p9w (Robien et al)). For N2D-vs-CTD orientations see also Table S1. Top left: Superposition of the six subunits of the qC6 ΔN1GspEEpsE hexamer from the ΔN1GspEEpsE-6aa-Hcp1 structure, revealing only small differences, by one to five degrees, in N2D-vs-CTD orientations. Top middle: Superposition of subunit D (yellow) from of the qC6 ΔN1GspEEpsE hexamer and the three subunits of the C2 ΔN1GspEEpsE hexamer from the ΔN1GspEEpsE-8aa-Hcp1 structure. N2D-vs-CTD orientations vary by 16 to 41 degrees. Top right: Superposition of subunit C (red) from the ΔN1C2 GspEEpsE hexamer and ΔN1GspEEpsE (grey) from the helical Δ90GspEEpsE structure (PDB: 1p9w (Robien et al)). The difference in N2Dvs-CTD orientation is only 2 degrees. Bottom left: Superposition of subunits A (green) and B (blue) of the C2 ΔN1GspEEpsE hexamer. The difference in N2D-vs-CTD orientation is 32 degrees. Bottom middle: Superposition of subunits B (blue) and C (red) of the C2 ΔN1GspEEpsE hexamer. The difference in N2D-vs-CTD orientation is 48 degrees. Bottom right: Superposition of subunits C (red) and A (green) of the C2 ΔN1GspEEpsE hexamer. The difference in N2D-vs-CTD orientation is 47 degrees. Figure 2C. “Orthogonal views” of the subunits in V. cholerae GspEEpsE. Pairwise comparison of ΔN1GspEEpsE subunits after superposition of the CTDs (grey, as background)viewed in a direction approximately perpendicular to the “canonical view” in Figure 2B. The N2D of subunit E from the qC6 ΔN1GspEEpsE hexamer (orange) is used as reference for each case.
Figure 2
Figure 2. Crystal structures of hexameric ΔN1GspEEpsE from Vibrio cholerae fused to Hcp1
Insets: schematic view of the hexamer outlining all six CTD•N2D’ construction units. CTD domains in light grey, N2D’ domains in dark grey. Figure 2A. V. cholerae ΔN1GspEEpsE-6aa-Hcp1, with a quasi-C6 ΔN1GspEEpsE hexamer and V. cholerae ΔN1GspEEpsE-8aa-Hcp1 containing a C2 hexamer. Upper panel: The quasi-C6 hexamer of V. cholerae ΔN1GspEEpsE-6aa-Hcp1. This fusion forms a ΔN1GspEEpsE hexamer with quasi-C6 point group symmetry. Shown are subunits A (green), B (cyan), C (purple), D (yellow), E (blue) and F (red). The CTDs are shown in a lighter shade of the same color as the N2Ds of the same subunit. The Hcp1 assistant hexamer is shown in orange. Left: view perpendicular to the quasi sixfold depicting also the Hcp1 hexamer. Right: view along the quasi-sixfold axis of the ΔN1GspEEpsE hexamer, with the Hcp1 hexamer omitted. The nucleotides shown in spheres are AMPPNP superposed from the helical Δ90GspEEpsE structure (PDB: 1p9w (Robien et al., 2003)). The shape of the hexamer in this view is very regular. One CTD•N2D’ construction unit is outlined. Lower panel: The C2 hexamer of V. cholerae ΔN1GspEEpsE-8aa-Hcp1. This fusion forms a ΔN1GspEEpsE hexamer with C2 point group symmetry. Shown are subunits A (green), B (blue), C (red) – each occurring twice in the hexamer. The CTDs are shown in a lighter shade of the same color as the N2Ds of the same subunit. The Hcp1 assistant hexamer is shown in orange. Left: view perpendicular to the twofold depicting also the Hcp1 hexamer. Right: view along the twofold axis of the ΔN1GspEEpsE hexamer, with the Hcp1 hexamer omitted. The nucleotides shown in pink spheres are AMPPNP superposed from the helical Δ90GspEEpsE structure (PDB: 1p9w (Robien et al., 2003)). The shape of the hexamer in this view is an approximate ellipsoid of 105 Å by 150 Å. One CTD•N2D’ construction unit is outlined. See also Figure S2, S3, S4 and S7. Figure 2B. The variability of the N2D-vs-CTD orientations in V. cholerae GspEEpsE. Shown are superimposed subunits in the “canonical view” with the CTDs superimposed below and the N2Ds on top (colored as Figure 2A). For a different “orthogonal view,” see Figure 2C. The nucleotide shown for reference is AMPPNP from the helical Δ90GspEEpsE structure (PDB: 1p9w (Robien et al)). For N2D-vs-CTD orientations see also Table S1. Top left: Superposition of the six subunits of the qC6 ΔN1GspEEpsE hexamer from the ΔN1GspEEpsE-6aa-Hcp1 structure, revealing only small differences, by one to five degrees, in N2D-vs-CTD orientations. Top middle: Superposition of subunit D (yellow) from of the qC6 ΔN1GspEEpsE hexamer and the three subunits of the C2 ΔN1GspEEpsE hexamer from the ΔN1GspEEpsE-8aa-Hcp1 structure. N2D-vs-CTD orientations vary by 16 to 41 degrees. Top right: Superposition of subunit C (red) from the ΔN1C2 GspEEpsE hexamer and ΔN1GspEEpsE (grey) from the helical Δ90GspEEpsE structure (PDB: 1p9w (Robien et al)). The difference in N2Dvs-CTD orientation is only 2 degrees. Bottom left: Superposition of subunits A (green) and B (blue) of the C2 ΔN1GspEEpsE hexamer. The difference in N2D-vs-CTD orientation is 32 degrees. Bottom middle: Superposition of subunits B (blue) and C (red) of the C2 ΔN1GspEEpsE hexamer. The difference in N2D-vs-CTD orientation is 48 degrees. Bottom right: Superposition of subunits C (red) and A (green) of the C2 ΔN1GspEEpsE hexamer. The difference in N2D-vs-CTD orientation is 47 degrees. Figure 2C. “Orthogonal views” of the subunits in V. cholerae GspEEpsE. Pairwise comparison of ΔN1GspEEpsE subunits after superposition of the CTDs (grey, as background)viewed in a direction approximately perpendicular to the “canonical view” in Figure 2B. The N2D of subunit E from the qC6 ΔN1GspEEpsE hexamer (orange) is used as reference for each case.
Figure 2
Figure 2. Crystal structures of hexameric ΔN1GspEEpsE from Vibrio cholerae fused to Hcp1
Insets: schematic view of the hexamer outlining all six CTD•N2D’ construction units. CTD domains in light grey, N2D’ domains in dark grey. Figure 2A. V. cholerae ΔN1GspEEpsE-6aa-Hcp1, with a quasi-C6 ΔN1GspEEpsE hexamer and V. cholerae ΔN1GspEEpsE-8aa-Hcp1 containing a C2 hexamer. Upper panel: The quasi-C6 hexamer of V. cholerae ΔN1GspEEpsE-6aa-Hcp1. This fusion forms a ΔN1GspEEpsE hexamer with quasi-C6 point group symmetry. Shown are subunits A (green), B (cyan), C (purple), D (yellow), E (blue) and F (red). The CTDs are shown in a lighter shade of the same color as the N2Ds of the same subunit. The Hcp1 assistant hexamer is shown in orange. Left: view perpendicular to the quasi sixfold depicting also the Hcp1 hexamer. Right: view along the quasi-sixfold axis of the ΔN1GspEEpsE hexamer, with the Hcp1 hexamer omitted. The nucleotides shown in spheres are AMPPNP superposed from the helical Δ90GspEEpsE structure (PDB: 1p9w (Robien et al., 2003)). The shape of the hexamer in this view is very regular. One CTD•N2D’ construction unit is outlined. Lower panel: The C2 hexamer of V. cholerae ΔN1GspEEpsE-8aa-Hcp1. This fusion forms a ΔN1GspEEpsE hexamer with C2 point group symmetry. Shown are subunits A (green), B (blue), C (red) – each occurring twice in the hexamer. The CTDs are shown in a lighter shade of the same color as the N2Ds of the same subunit. The Hcp1 assistant hexamer is shown in orange. Left: view perpendicular to the twofold depicting also the Hcp1 hexamer. Right: view along the twofold axis of the ΔN1GspEEpsE hexamer, with the Hcp1 hexamer omitted. The nucleotides shown in pink spheres are AMPPNP superposed from the helical Δ90GspEEpsE structure (PDB: 1p9w (Robien et al., 2003)). The shape of the hexamer in this view is an approximate ellipsoid of 105 Å by 150 Å. One CTD•N2D’ construction unit is outlined. See also Figure S2, S3, S4 and S7. Figure 2B. The variability of the N2D-vs-CTD orientations in V. cholerae GspEEpsE. Shown are superimposed subunits in the “canonical view” with the CTDs superimposed below and the N2Ds on top (colored as Figure 2A). For a different “orthogonal view,” see Figure 2C. The nucleotide shown for reference is AMPPNP from the helical Δ90GspEEpsE structure (PDB: 1p9w (Robien et al)). For N2D-vs-CTD orientations see also Table S1. Top left: Superposition of the six subunits of the qC6 ΔN1GspEEpsE hexamer from the ΔN1GspEEpsE-6aa-Hcp1 structure, revealing only small differences, by one to five degrees, in N2D-vs-CTD orientations. Top middle: Superposition of subunit D (yellow) from of the qC6 ΔN1GspEEpsE hexamer and the three subunits of the C2 ΔN1GspEEpsE hexamer from the ΔN1GspEEpsE-8aa-Hcp1 structure. N2D-vs-CTD orientations vary by 16 to 41 degrees. Top right: Superposition of subunit C (red) from the ΔN1C2 GspEEpsE hexamer and ΔN1GspEEpsE (grey) from the helical Δ90GspEEpsE structure (PDB: 1p9w (Robien et al)). The difference in N2Dvs-CTD orientation is only 2 degrees. Bottom left: Superposition of subunits A (green) and B (blue) of the C2 ΔN1GspEEpsE hexamer. The difference in N2D-vs-CTD orientation is 32 degrees. Bottom middle: Superposition of subunits B (blue) and C (red) of the C2 ΔN1GspEEpsE hexamer. The difference in N2D-vs-CTD orientation is 48 degrees. Bottom right: Superposition of subunits C (red) and A (green) of the C2 ΔN1GspEEpsE hexamer. The difference in N2D-vs-CTD orientation is 47 degrees. Figure 2C. “Orthogonal views” of the subunits in V. cholerae GspEEpsE. Pairwise comparison of ΔN1GspEEpsE subunits after superposition of the CTDs (grey, as background)viewed in a direction approximately perpendicular to the “canonical view” in Figure 2B. The N2D of subunit E from the qC6 ΔN1GspEEpsE hexamer (orange) is used as reference for each case.
Figure 3
Figure 3. SAXS studies on ΔN1GspEEpsE-8aa-Hcp1 and ΔN1GspEEpsE-6aa-Hcp1
Experimental (black and yellow circles) and calculated (red and blue) SAXS scattering curves for ΔN1GspEEpsE-8aa-Hcp1 and ΔN1GspEEpsE-6aa-Hcp1 with the same nucleotides as in the mother liquor of the crystal structures (See the right upper corner of each panel, and for specific details see methods). The calculated SAXS curve based on the ΔN1GspEEpsE-6aa-Hcp1 structure with the qC6 hexamer of ΔN1GspEEpsE shown in blue, and for the ΔN1GspEEpsE-8aa-Hcp1 structure with the C2 hexamer of ΔN1GspEEpsE shown in red. Residual plots are shown in the lower panels. See also Figure S9.
Figure 4
Figure 4. Comparison of T2SS GspEEpsE and T4PS secretion ATPases
Figure 4A. The regular and irregular GspEEpsE and PilT hexamers. NOTE: The N2D and CTD domains of the same subunit are represented with darker and lighter shades of the same color. Insets: Hexamers with the CTD•N2D’ construction units outlined Top: comparison of regular hexamers. Left: V. cholerae ΔN1GspEEpsE qC6 hexamer. Middle: A. aeolicus PilT C6 hexamer (PDB: 2EWV). Right: P. aeruginosa PilT C2 hexamer (PDB: 3JVV). Bottom: comparison of irregular hexamers. Left: V. cholerae ΔN1GspEEpsE C2 hexamer. Middle: A. aeolicus PilT qC2 hexamer (PDB: 2GSZ). See also Figure S5. Figure 4B. The variability of the N2D-vs-CTD orientations in GspEEpsE and PilT hexamers. Shown are superimposed subunits in the “canonical view” with the CTDs superimposed below and the N2Ds on top. For a different, “orthogonal view,” see Figure S6. For N2D-vs-CTD orientations see Table S1. Top: superposition of the CTD from the subunit in the AaPilT C6 hexamer (yellow; PDB: 2EWV) and the three independent subunits of the AaPilT qC2 hexamer (different shades of purple; PDB: 2GSZ) onto subunit E from the ΔN1GspEEpsE qC6 hexamer (orange). Bottom: superposition of the CTDs of PaPilT (different shades of blue; PDB: 3JVV) onto subunit E from the ΔN1GspEEpsE qC6 hexamer (orange). See also Figure S6 and S8. Figure 4C. Domain rearrangements of T2SS GspEEpsE and T4PS secretion ATPases. Rows from top to bottom: hexamers; the C1Ds only; the N2Ds in color with C1Ds in grey as background; the C2Ds only. Note how different the domain positions are in the various hexamers.
Figure 4
Figure 4. Comparison of T2SS GspEEpsE and T4PS secretion ATPases
Figure 4A. The regular and irregular GspEEpsE and PilT hexamers. NOTE: The N2D and CTD domains of the same subunit are represented with darker and lighter shades of the same color. Insets: Hexamers with the CTD•N2D’ construction units outlined Top: comparison of regular hexamers. Left: V. cholerae ΔN1GspEEpsE qC6 hexamer. Middle: A. aeolicus PilT C6 hexamer (PDB: 2EWV). Right: P. aeruginosa PilT C2 hexamer (PDB: 3JVV). Bottom: comparison of irregular hexamers. Left: V. cholerae ΔN1GspEEpsE C2 hexamer. Middle: A. aeolicus PilT qC2 hexamer (PDB: 2GSZ). See also Figure S5. Figure 4B. The variability of the N2D-vs-CTD orientations in GspEEpsE and PilT hexamers. Shown are superimposed subunits in the “canonical view” with the CTDs superimposed below and the N2Ds on top. For a different, “orthogonal view,” see Figure S6. For N2D-vs-CTD orientations see Table S1. Top: superposition of the CTD from the subunit in the AaPilT C6 hexamer (yellow; PDB: 2EWV) and the three independent subunits of the AaPilT qC2 hexamer (different shades of purple; PDB: 2GSZ) onto subunit E from the ΔN1GspEEpsE qC6 hexamer (orange). Bottom: superposition of the CTDs of PaPilT (different shades of blue; PDB: 3JVV) onto subunit E from the ΔN1GspEEpsE qC6 hexamer (orange). See also Figure S6 and S8. Figure 4C. Domain rearrangements of T2SS GspEEpsE and T4PS secretion ATPases. Rows from top to bottom: hexamers; the C1Ds only; the N2Ds in color with C1Ds in grey as background; the C2Ds only. Note how different the domain positions are in the various hexamers.
Figure 4
Figure 4. Comparison of T2SS GspEEpsE and T4PS secretion ATPases
Figure 4A. The regular and irregular GspEEpsE and PilT hexamers. NOTE: The N2D and CTD domains of the same subunit are represented with darker and lighter shades of the same color. Insets: Hexamers with the CTD•N2D’ construction units outlined Top: comparison of regular hexamers. Left: V. cholerae ΔN1GspEEpsE qC6 hexamer. Middle: A. aeolicus PilT C6 hexamer (PDB: 2EWV). Right: P. aeruginosa PilT C2 hexamer (PDB: 3JVV). Bottom: comparison of irregular hexamers. Left: V. cholerae ΔN1GspEEpsE C2 hexamer. Middle: A. aeolicus PilT qC2 hexamer (PDB: 2GSZ). See also Figure S5. Figure 4B. The variability of the N2D-vs-CTD orientations in GspEEpsE and PilT hexamers. Shown are superimposed subunits in the “canonical view” with the CTDs superimposed below and the N2Ds on top. For a different, “orthogonal view,” see Figure S6. For N2D-vs-CTD orientations see Table S1. Top: superposition of the CTD from the subunit in the AaPilT C6 hexamer (yellow; PDB: 2EWV) and the three independent subunits of the AaPilT qC2 hexamer (different shades of purple; PDB: 2GSZ) onto subunit E from the ΔN1GspEEpsE qC6 hexamer (orange). Bottom: superposition of the CTDs of PaPilT (different shades of blue; PDB: 3JVV) onto subunit E from the ΔN1GspEEpsE qC6 hexamer (orange). See also Figure S6 and S8. Figure 4C. Domain rearrangements of T2SS GspEEpsE and T4PS secretion ATPases. Rows from top to bottom: hexamers; the C1Ds only; the N2Ds in color with C1Ds in grey as background; the C2Ds only. Note how different the domain positions are in the various hexamers.

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