Figure 1.
SEC/MALS/RI and electron microscopy analysis of the Hcp1 and Hcp2 proteins.
(A, B) SEC/MALS/RI chromatograms of Hcp1 (A) and of Hcp2 (B). The molar mass (left axis, solid line) and the UV280 nm absorbance (right axis, grey line) are plotted as a function of the column elution volume. (C, D) Transmission electron micrographs of negatively stained Hcp1 (C) and Hcp2 (D). Scale bar, 50 nm.
Figure 2.
Structure of the Hcp1 N93W/S158W (Hcp1WW) protein.
(A) Ribbon representation of the Hcp1WW structure. The N- and C- termini are indicated (Nt and Ct respectively). The red dots indicate positions 93 and 158 while green dots indicate the 96 and 158 positions. (B) Carbone α backbone of Hcp1WW. The side chains of the residues forming the hydrophobic core are shown in grey. (C) Section of the Hcp1WW crystal packing. 5 Hcp1WW hexameric rings are shown in different colors to aid visualization. The Trp-93 and Trp-158 side chains are represented in red and green spheres respectively.
Table 1.
Data collection and refinement statistics.
Figure 3.
Sequence alignment and structural superimposition of Hcp1WW with other crystallized Hcp proteins.
(A) Sequence alignment of the EAEC Hcp1WW protein, Hcp3 from P. aeruginosa (3HE1), Hcp from Y. pestis (3V4H), EvpC from E. tarda (3EAA) and Hcp1 from P. aeruginosa (1Y12). Residues targeted in this study are indicated by dots (red for N93, S158 and green for G96). (B) Ribbon representation of the superimposition of Hcp1WW with the indicated Hcp proteins. The color corresponding to each structure is indicated.
Table 2.
Comparison of the five Hcp of known structures.
Figure 4.
Interaction study of Hcp1/Hcp1 hexamers using surface plasmon resonance.
(A) Binding pattern of Hcp1 (3.75 to 120 µM) on Hcp1 covalently immobilized on the CM5 chip. The variation of plasmon resonance is reported on the y axis (in arbitrary unit; ΔRU) and the reaction time on the x axis. (B) Graph representing the equilibrium response level (ΔRU; y axis) plotted as a function of the Hcp1 concentration (µM, x axis), with t curve fit to 1∶1 equilibrium model for determination of the KD at 50% saturation.
Figure 5.
Hexamers of Hcp1WW and model of the hexamers stacking in the Hcp1G96C/S158C tube assembly.
(A, B) Top- (A) and bottom- (B) view surface representations of Hcp1WW hexamers. The positions of the two overhang L1,2 and L2,3 loops are indicated. The buried surface at monomer interface is indicated. (C) Surface representation of a two stacked hexamers model of Hcp1G96C/S158C. The cysteine residues implicated in disulfide bond formation are indicated, as well as the major determinants at the hexamer-hexamer interface with their surface buried areas.
Figure 6.
Hcp1G96C/S158C tube formation as shown by in vivo disulfide bond formation and Electron Microscopy.
(A) Cytoplasmic extracts from EAEC Δhcp1 cells producing the indicated cysteine Hcp1 mutant proteins after in vivo oxidative treatment with copper phenanthroline were loaded on a 12.5%-acrylamide SDS PAGE and immunodetected with the anti-FLAG monoclonal antibody. Positions of the Hcp1 monomer and multimers are indicated on the right. Molecular weight markers (in kDa) are indicated on the left. (B) Representative electron micrographs of negatively-stained Hcp1G96C/S158C particles. Hcp1G96C/S158C tubular structures are indicated by arrows. Scale bar, 100 nm.