The crystal structures of the Salmonella type III secretion system tip protein SipD in complex with deoxycholate and chenodeoxycholate - PubMed (original) (raw)

The crystal structures of the Salmonella type III secretion system tip protein SipD in complex with deoxycholate and chenodeoxycholate

Srirupa Chatterjee et al. Protein Sci. 2011 Jan.

Abstract

The type III secretion system (T3SS) is a protein injection nanomachinery required for virulence by many human pathogenic bacteria including Salmonella and Shigella. An essential component of the T3SS is the tip protein and the Salmonella SipD and the Shigella IpaD tip proteins interact with bile salts, which serve as environmental sensors for these enteric pathogens. SipD and IpaD have long central coiled coils and their N-terminal regions form α-helical hairpins and a short helix α3 that pack against the coiled coil. Using AutoDock, others have predicted that the bile salt deoxycholate binds IpaD in a cleft formed by the α-helical hairpin and its long central coiled coil. NMR chemical shift mapping, however, indicated that the SipD residues most affected by bile salts are located in a disordered region near helix α3. Thus, how bile salts interact with SipD and IpaD is unclear. Here, we report the crystal structures of SipD in complex with the bile salts deoxycholate and chenodeoxycholate. Bile salts bind SipD in a region different from what was predicted for IpaD. In SipD, bile salts bind part of helix α3 and the C-terminus of the long central coiled coil, towards the C-terminus of the protein. We discuss the biological implication of the differences in how bile salts interact with SipD and IpaD.

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Figures

Figure 1

Crystal structure of the SipD-deoxycholate complex. Deoxycholate (shown as a stick model) binds at the interface of molecules A and B of the asymmetric unit. SipD is colored as follows: the coiled coil (helix α4 and α8), gray; the N-terminal region (helix α1-α3), blue; and the mixed α/β domain, green. The crystal structures of SipD-chenodeoxycholate complex and apo SipD (WT and C244S) are similar to the SipD-deoxycholate crystal and are shown in the Supporting Information.

Figure 2

Fo-Fc omit maps contoured at 3σ of (A) deoxycholate and (B) chenodeoxycholate in complex with SipD shown with residues within 5 Å of bile salts (color scheme: molecule A, yellow; molecule B, cyan; and another molecule A related by a crystallographic (011) translation, pink). (C) Conformational changes in SipD upon binding deoxycholate and chenodeoxycholate (color scheme: apo SipD, green; SipD-deoxycholate complex, blue; and SipD-chenodeoxycholate complex, gold). Bile salts have been removed for clarity. Upon binding bile salts, the side chain of F340 shifts by 5.8 Å and that of N104 by 2.1 Å.

Figure 3

(A) Results of Salmonella invasion assay (legends: WT, wild-type strain; _sipD_−, sipD null strain; SipD+, wild-type sipD introduced by the plasmid pRK2-sipD into the _sipD_− strain; L116A, F117A, Q124A, K188A, and C244S are SipD point mutations in pRK2-sipD; Δ116-124, Δ186-189, Δ187-188, and C_Δ_5 are SipD deletions in pRK2-sipD). (B) CD spectra of SipD and deletion mutants (insert: ratio of molar ellipticity at 222 and 208 nm). (C) CD thermal denaturation monitored by the molar ellipticity at 222 nm of SipD and various deletion mutants (insert: estimated transition temperatures, _T_m).

Figure 4

Two-dimensional 1H-15N TROSY spectra of (A) SipD (residues 39-343) and the (B) CΔ5, (C) Δ186-189 and the (D) Δ187-188 deletion mutants (boxed: tryptophan side chain peaks). Peak assignments for SipD were reported previously. The noise peak (asterisk) in the tryptophan side chain region in (C) is absent in the 2D 1H-15N HSQC spectrum.

Figure 5

Comparison of five current crystal structures of T3SS tip proteins: (A) Yersinia LcrV, (B) a SipD homolog from Chromobacterium violaceum, (C) Shigella IpaD, (D) Burkholderia BipD,, and (E) Salmonella SipD. The central coiled coil (gray) and mixed α/β domain (green) are common structural features of T3SS tip proteins. The N-terminal region in (A) forms a globular domain of α-helices and β-strands (red), whereas in (B–E), the N-terminal region forms α-helical hairpins (blue) followed by short α-helices (red) in (B, D, and E).

Figure 6

The major differences in the crystal structures of IpaD (purple) and SipD (green) are in the packing of the N-terminal α-helical hairpin with the coiled coil. (A) The SipD α-helical hairpin packs on the central coiled coil at a different angle of 18° compared to the α-helical hairpin of IpaD, and (B) the SipD helix α3 pack at a different angle of 26° compared to helix α3 of IpaD (also, helix α4 of IpaD is longer by one turn).

References

1. Cornelis GR. The type III secretion injectisome. Nat Rev Microbiol. 2006;4:811–825. - PubMed
1. Lara-Tejero M, Galan JE. Salmonella enterica serovar typhimurium pathogenicity island 1-encoded type III secretion system translocases mediate intimate attachment to nonphagocytic cells. Infect Immun. 2009;77:2635–2642. - PMC - PubMed
1. Espina M, Olive AJ, Kenjale R, Moore DS, Ausar SF, Kaminski RW, Oaks EV, Middaugh CR, Picking WD, Picking WL. IpaD localizes to the tip of the type III secretion system needle of Shigella flexneri. Infect Immun. 2006;74:4391–4400. - PMC - PubMed
1. Johnson S, Roversi P, Espina M, Olive A, Deane JE, Birket S, Field T, Picking WD, Blocker AJ, Galyov EE, Picking WL, Lea SM. Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD. J Biol Chem. 2007;282:4035–4044. - PMC - PubMed
1. Erskine PT, Knight MJ, Ruaux A, Mikolajek H, Sang NW, Withers J, Gill R, Wood SP, Wood M, Fox GC, Cooper JB. High resolution structure of BipD: an invasion protein associated with the type III secretion system of Burkholderia pseudomallei. J Mol Biol. 2006;363:125–136. - PubMed

The crystal structures of the Salmonella type III secretion system tip protein SipD in complex with deoxycholate and chenodeoxycholate - PubMed (original) (raw)