Volume 3, Issue 3, Pages 709-715 (March 2013) Substrate-Activated Conformational Switch on Chaperones Encodes a Targeting Signal in Type III Secretion  Li Chen, Xuanjun Ai, Athina G. Portaliou, Conceicao A.S.A. Minetti, David P. Remeta, Anastassios Economou, Charalampos G. Kalodimos  Cell Reports  Volume 3, Issue 3, Pages 709-715 (March 2013) DOI: 10.1016/j.celrep.2013.02.025 Copyright © 2013 The Authors Terms and Conditions

Cell Reports 2013 3, 709-715DOI: (10.1016/j.celrep.2013.02.025) Copyright © 2013 The Authors Terms and Conditions

Figure 1 Structures of CesAB, CesAB-EspA, and CesABs (A) The solution structure of the homodimeric CesAB, which adopts a molten-globule-like structure in solution (Chen et al., 2011). (B) The 1H-15N HSQC spectrum of CesAB. (C) The crystal structure of the heterodimeric CesAB-EspA (Yip et al., 2005). Regions of the proteins that were not crystallographically resolved are represented as dotted lines. (D) The 1H-15N HSQC spectrum of CesAB-EspA. The CesAB subunit is 15N labeled, whereas the EspA subunit is unlabeled. (E) The solution structure of the CesABs variant (D14L-R18D-E20L), as determined in this work. (F) The 1H-15N HSQC spectrum of CesABs. (G) Superposition of the CesAB subunit of CesAB, CesAB-EspA, and CesABs. Cell Reports 2013 3, 709-715DOI: (10.1016/j.celrep.2013.02.025) Copyright © 2013 The Authors Terms and Conditions

Figure 2 Interaction of CesAB-EspA with EscN (A) Overlaid 1H-15N HSQC spectra of the titration of U-2H-15N-labeled CesAB-EspA with unlabeled hexameric EscN. Stepwise addition of EscN results in gradual resonance broadening of the interacting residues in CesAB-EspA. Spectra recorded at ten different titration points are overlaid. The CesAB residues most affected by EscN binding are shown. The resonances not affected by EscN binding even at saturating concentrations of EscN are located in flexible regions of EspA that were crystallographically unresolved. (B) CesAB-EspA residues (shown in red sticks) identified by NMR to be most affected upon binding to EscN. All residues are located in helices α2 and α3 in CesAB. (C) Superposition of the CesAB subunit of the homodimeric CesAB (blue) and the heterodimeric CesAB-EspA complex (green). The residues identified to mediate the binding of CesAB-EspA to EscN are shown. Cell Reports 2013 3, 709-715DOI: (10.1016/j.celrep.2013.02.025) Copyright © 2013 The Authors Terms and Conditions

Figure 3 Disruption of CesAB-EspA Binding to EscN Gives Rise to Secretion and Functional Defects (A) The effect of the Y64A-R68A substitution on the interaction of CesAB-EspA with EscN. Overlaid 1H-15N HSQC spectra of CesABY64A-R68A-EspA in the absence (blue) and presence (magenta) of EscN. In comparison to wild-type CesAB-EspA binding to EscN, the NMR data indicate a significant decrease in the affinity of the ternary complex (Kd is larger than 80 μM). The boxed areas show the corresponding regions of the spectra of CesAB-EspA (green) superimposed on the spectra of its complex with EscN (red). Whereas the majority of the peaks are broadened beyond detection in the CesAB-EspA-EscN complex, they are still present at a substantial intensity in the CesABY64A-R68A-EspA-EscN complex. (B) In vivo secretion of EspA from EPECΔcesAB strains complemented with pASK-IBA7 plasmids expressing wild-type or mutated CesAB. The graph reports the total amount of EspA secreted in 90 min after CesAB expression. Errors were calculated from a triplicate experiment. (C) In vivo infection of HeLa cells from EPECΔcesAB or EPECΔescN strains complemented with pASK-IBA7 plasmids expressing wild-type or mutated CesAB. The graph reports the percentage of HeLa cells infected after being inoculated with bacteria for 90 min. Errors were calculated from a triplicate experiment. (D) The infection of HeLa cells by bacterial EPECΔcesAB strains complemented with plasmids expressing wild-type or mutant CesAB. The results show very little actin pedestal formation, indicating uninfected HeLa cells, after being inoculated with bacteria for 90 min. Cell Reports 2013 3, 709-715DOI: (10.1016/j.celrep.2013.02.025) Copyright © 2013 The Authors Terms and Conditions

Figure 4 Targeting of CesAB-EspA to the ATPase Although CesAB carries the targeting signal (shown in red), this is presented to EscN only when EspA is bound to CesAB by means of an induced conformational switch on CesAB. As a result, EscN recognizes the restructured CesAB region and engages the CesAB-EspA complex. Cell Reports 2013 3, 709-715DOI: (10.1016/j.celrep.2013.02.025) Copyright © 2013 The Authors Terms and Conditions

Figure S1 Structural Characterization of CesABs, Related to Figure 1 (A and B) Overlaid 1H-15N HSQC and 1H-13C HMQC spectra of CesAB and CesABs. The dramatic improvement in resonance dispersion indicates that, in contrast to CesAB, CesABs is a well folded protein. (C and D) Far-UV and near-UV CD data of CesAB and CesABs demonstrate that the helicity is substantially enhanced and aromatic packing is greatly improved in CesABs. (E) Solution structure of CesABs. The ensemble of the 20-lowest energy conformers of CesABs is shown. Two orthogonally-related views of CesABs of the lowest-energy conformer with residues mediating the dimer displayed as yellow sticks are also shown. (F) Superposition of the structures of CesABs and wild-type CesAB. (G) Superposition of the structures of CesABs and CesAB−EspA. Cell Reports 2013 3, 709-715DOI: (10.1016/j.celrep.2013.02.025) Copyright © 2013 The Authors Terms and Conditions

Figure S2 Interaction of Hexameric EscN with CesAB, CesAB-EspA, and CesABs, Related to Figure 2 (A) Size exclusion chromatography of EscN. At concentrations higher than ∼20 μM, EscN exists as a hexamer in solution. (B) Size exclusion chromatography of EscNΔN, a variant that lacks the N-terminal region (residues 1-97) and exists exclusively as a monomer in solution. (C) ATPase activity of EscN, EscN-R366D, and EscNΔN. Arg366 plays the role of the so-called “arginine finger” residue, which is required for stimulation of the ATPase activity. Arg366 from a different subunit is thought to poke into the ATP-binding pocket when EscN forms functional oligomers (Zarivach et al., 2007). No detectable ATPase activity was measured for EscN-R366D and EscNΔN. The profile for EscN-R366D is slightly offset for clarity. (D) Overlaid 1H-13C HMQC spectra of CesAB in the absence (blue) and presence (red) of EscN. No binding was detected. Due to the poor sensitivity of the 1H-15N HSQC spectrum of CesAB (Figure 1E) the side-chain methyl spectrum, wherein all expected peaks are present, was used to probe for the interaction. (E) Overlaid 1H-15N HSQC spectra of CesAB−EspA in the absence (green) and presence (red) of EscN. The data indicate formation of the ternary CesAB−EspA−EscN complex. Several residues of the CesAB−EspA complex are unaffected by EscN binding. All these residues are located in flexible regions of the heterodimer that are not immobilized upon formation of the ternary complex. (F) Overlaid 1H-15N HSQC spectra of CesAB−EspA in the absence (green) and presence (orange) of EscNΔN. The data show that there is no interaction between the two proteins. (G) Overlaid 1H-15N HSQC spectra of CesABs in the absence (purple) and presence (red) of EscN indicating binding. The residues that are not affected by EscN binding are located in the flexible C-terminal tail of CesAB. (H) Residues identified by NMR to mediate the interaction of CesABs with EscN. (I) Residues identified by NMR to mediate the interaction of CesAB−EspA with EscN. The EscN-binding surfaces in CesAB−EspA and CesABs are very similar and consist in both cases of helices 2 and 3 of CesAB. (J) Overlaid 1H-15N HSQC spectra of EspA1–34 in the absence (blue) and presence (orange) of EscN. No binding was detected. (K) Overlaid 1H-15N HSQC spectra of CesAB−EspA (green) and CesAB−EspAΔ29 (orange). Truncation of the N-terminal 29 residues of EspA does not affect the fold of the heterocomplex and has a minimal effect on its structure. (L) Overlaid 1H-15N HSQC spectra of CesAB−EspAΔ29 in the absence (green) and presence of EscN (red). Truncation of the N-terminal 29 residues of EspA does not abrogate binding of CesAB−EspAΔ29 to EscN. Cell Reports 2013 3, 709-715DOI: (10.1016/j.celrep.2013.02.025) Copyright © 2013 The Authors Terms and Conditions

Figure S3 EspA Secretion Is Dependent on the Presence of the EscN ATPase, Related to Figure 3 In vivo infection of HeLa cells in wild-type and ΔescN strains. EscN deletion results in complete abrogation of EspA secretion and therefore no infection is observed after 120 min inoculation with bacteria. Cell Reports 2013 3, 709-715DOI: (10.1016/j.celrep.2013.02.025) Copyright © 2013 The Authors Terms and Conditions