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Conserved glutamines in SpaP and the SpaR lid together orchestrate substrate transport through the Q2-belt of the EA. a Surface representations of the EA showing the SpaR loop/lid and surrounding Q2-belt. Q2-belt residues are shown in light green, M-gate residues in yellow. Numbers correspond to residues building the SpaR lid. Phe212 and Ile114 are displayed in dark green and red, respectively. b Left: surface representations of the Q2-belt residues Gln44/45 colored by hydrophobicity. Right: close up of Q2-belt with Gln44/45 displayed in stick representation and SpaPs as ribbon diagrams. c Hydrogen-bond formation between the substrate backbone and SpaP 2 Gln45. The surfaces represent EM density (threshold: 0.016). d Representative immunoblots of three independent secretion assays of SptP and SipA effector proteins using wild-type SB905, SpaR knockout (ΔSpaR), and a strain carrying the SpaR loop mutation I114A. e Densitometry quantification of SipA and SptP secretion assay immunoblots in d. f Representative immunoblots of three independent secretion assays of SptP and SipA effector proteins using wild-type SB905, SpaP-knockout (ΔSpaP), and ΔSpaP strains reconstituted with either wild-type SpaP (WT CP), or the indicated SpaP Q2-belt Gln44,45 mutants. g Densitometry quantification of SipA and SptP secretion assay immunoblots in f. Source data for e-g are provided with this paper.
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... below, creating what has been termed a plug in the structurally-related flagellar system ( Supplementary Figs. 19 and 23) 12,13,15 . In the map of our substrate-engaged complex, the SpaR loop (or aptly named lid) adopts two distinct conformational states positioned vertically along the translocation path ( Fig. 5a and Supplementary Fig. 23 and Supplementary Video 2). In state 1 (7ah9), the predominant state in our substrate-trapped structure, the SpaR loop generates a narrow path, ~6 Å in width, to make way for the substrate on its passage to the more spacious atrium. Stabilized by the formation of an antiparallel β-sheet, the hydrophobic side ...
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... substrate-trapped structure, the SpaR loop generates a narrow path, ~6 Å in width, to make way for the substrate on its passage to the more spacious atrium. Stabilized by the formation of an antiparallel β-sheet, the hydrophobic side chain of SpaR Ile114 is exposed towards the channel lumen, where it directly faces the translocating substrate ( Fig. 5a and Supplementary Fig. 24). Mutational analysis revealed that substitution of Ile114 to alanine impairs SptP and SipA effector protein secretion, indicating that the SpaR loop actively contributes to substrate transport (Fig. 5d, ...
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... side chain of SpaR Ile114 is exposed towards the channel lumen, where it directly faces the translocating substrate ( Fig. 5a and Supplementary Fig. 24). Mutational analysis revealed that substitution of Ile114 to alanine impairs SptP and SipA effector protein secretion, indicating that the SpaR loop actively contributes to substrate transport (Fig. 5d, ...
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... state 2 (7ahi), no secondary structure elements can be observed and SpaR Ile114 is rotated away from the channel, which increases the width of the translocation path from ~6 Å to ~10 Å (Fig. 5a). Interface analyses (Proteins, Interfaces, Structures and Assemblies (PISA)) revealed that neither of the two SpaR loop conformations forms stable interactions with residues building the translocation channel (state 1: ΔG = −4.4 kcal/ mol, P = 0.86; state 2: ΔG = −4.1 kcal/mol, P = 0.85), demonstrating that the SpaR loop is a moveable ...
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... (PISA)) revealed that neither of the two SpaR loop conformations forms stable interactions with residues building the translocation channel (state 1: ΔG = −4.4 kcal/ mol, P = 0.86; state 2: ΔG = −4.1 kcal/mol, P = 0.85), demonstrating that the SpaR loop is a moveable element that enjoys conformational flexibility during substrate transport (Fig. 5a and Supplementary Table ...
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... area surrounding the substrate on the height of the upfolded SpaR loop is shaped by a loop which connects α-helices α2 and α3 in each of the five spirally organized SpaP protomers (Fig. 5b). Reminiscent of the Q1-belt function and conserved across virulent T3SSs, SpaP Gln44 and Gln45 localize within close Numbers correspond to residues building the SpaR lid. Phe212 and Ile114 are displayed in dark green and red, respectively. b Left: surface representations of the Q2-belt residues Gln44/45 colored by hydrophobicity. ...
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... quantification of SipA and SptP secretion assay immunoblots in f. Source data for e-g are provided with this paper. proximity to the substrate, together generating an extended hydrogen-bonding donor/acceptor interface to engage with the backbone and polar side chains of the SptP substrate as it emerges from the M-gate and passes SpaR Ile114 (Fig. 5c and Supplementary Figs. 12, 16, and 17). Because of the striking similarity with the Q1-belt, we decided to name this region of the translocation path Q2-belt. Mutation of the two SpaP glutamines Gln44 and Gln45 had only a moderate impact on SptP and SipA effector protein secretion, revealing that the Q2-belt plays an ancillary role in ...
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Graphical abstract
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