Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD

Abstract

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence: IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intramolecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure.

Figure 1

Crystal structures of IpaD and BipD.(a) Ribbon diagram of the structure of IpaD (residues 39-322) colored by domain; N-domain (blue), central coiled-coil (green), C-domain (red). (b) Topology diagram of IpaD, colored as in (a). (c) Structure of BipD (residues 35-111 and 127-301) colored as in a. All figures produced using PyMOL(26). (d) Structure based sequence alignment of IpaD, SipD and BipD using the ESPript server(30), colored according to residue conservation. Secondary structure elements of IpaD and BipD are shown above and below the alignment respectively. The Salmonella enterica typhimurium homologue SipD is more closely related to IpaD.

Figure 2

Stereo views of IpaD and BipD electron density (a and c) and main chain traces (b and d) are shown. The electron density is calculated with coefficients 2F_O-wF_C and phases derived from the final models. The IpaD density in panel (a) is contoured at 1.0 σ around residues 284-304 in the C-terminal coiled-coil helix and the BipD density in (c) around residues 154-172 in the N-terminal coiled-coil helix. (b) and (d) show complete main chain traces oriented so that the N- and C-termini are at the bottom of the picture.

Figure 3

The N-terminal domain is a chaperone. (a) Ribbon diagram of IpaD (green) superposed on Aquifex aeolicus FliS (purple, 1ORJ-A, (rmsd = 2.0 Å over 103 Cα atoms)(39). (b) Ribbon diagram of IpaD (green) superposed on Yersinia pestis YscE (light blue, 1ZW0-A, rmsd = 1.8 Å over 57 Cα atoms)(35). (c) Ribbon diagram of IpaD (green) superposed on Bacillus subtilis FliS (purple, 1VH6) demonstrating the opened four-helix bundle.

Figure 4

Consequences of removal of the N-terminal domain. (a) Proteolytic sensitivity of the N-terminal domain of IpaD. IpaD was incubated with trypsin at various ratios (w:w) and the resulting digests were resolved on SDS-PAGE. Band 1 (24 kDa) begins at residue 120 and Band 2 (20 kDa) at residue 138. (b) Surface representation of IpaD_39-130 (left) and IpaD_131-322 (right) with hydrophobic residues colored green and brown respectively. IpaD_39-130 is rotated through 180° along the long axis relative to IpaD_131-322 in order to demonstrate the complementary hydrophobic surfaces. The surface is presented as transparent to allow visualization of the secondary structure. (c) Analytical gel filtration chromatography (Superdex 200, HR 10/30) of IpaD before and after Subtilisin treatment. Elution volume of each species is noted along with the M_r calculated from SDS-PAGE. (d) Overlay of 5 structures of the IpaD coiled-coil demonstrating the flexibility of the helices in the absence of the N-terminal domain. The conformation of the coil in the presence of the N-terminal domain is shown in green. The C-terminal domain has been removed to aid clarity.

Figure 5

Oligomerization of IpaD. (a) Ribbon diagram of the non-crystallographic dimer found in crystal form 2 (18). Molecule A is shown in green, molecule B in blue. (b) Detailed view of the dimer interaction site with sidechains displayed, colored as in a. Only structural elements that contribute to the binding site are displayed for clarity. c) Pentamer produced using the non-crystallographic symmetry from crystal form 2. At the top are ribbon representations and at the bottom surface views. The panels on the left are related to the panels on the right by a rotation of 90°.