NMR characterization of the interaction of the Salmonella type III secretion system protein SipD and bile salts

Abstract

Salmonella and Shigella bacteria require the type III secretion system (T3SS) to inject virulence proteins into their hosts and initiate infections. The tip proteins SipD and IpaD are critical components of the Salmonella and Shigella T3SS, respectively. Recently, SipD and IpaD have been shown to interact with bile salts, which are enriched in the intestines, and are hypothesized to act as environmental sensors for these enteric pathogens. Bile salts activate the Shigella T3SS but repress the Salmonella T3SS, and the mechanism of this differing response to bile salts is poorly understood. Further, how SipD binds to bile salts is currently unknown. Computer modeling predicted that IpaD binds the bile salt deoxycholate in a cleft formed by the N-terminal domain and the long central coiled coil of IpaD. Here, we used NMR methods to determine which SipD residues are affected by the interaction with the bile salts deoxycholate, chenodeoxycholate, and taurodeoxcholate. The bile salts perturbed nearly the same set of SipD residues; however, the largest chemical shift perturbations occurred away from what was predicted for the bile salt binding site in IpaD. Our NMR results indicate that that bile salt interaction of SipD will be different from what was predicted for IpaD, suggesting a possible mechanism for the differing response of Salmonella and Shigella to bile salts.

Fig. 1

(A) Overlay of four 2D ¹H-¹⁵N TROSY spectra of [²H,¹⁵N,¹³C]-labeled SipD^39–343 with increasing amounts of deoxycholate. Some of the assignments are indicated as well as the noise peaks (*). (B) Expanded regions of the 2D ¹H-¹⁵N TROSY spectra for specific residues that showed chemical shift changes with deoxycholate (arrows indicate the direction of the chemical shift change). Similar NMR titration data for SipD^39–343 with taurodeoxycholate (Fig. S7), chenodeoxycholate (Fig. S8) and cholate hydrate (Fig. S9) are shown in the Supporting Information.

Fig. 2

Secondary structures of SipD^39–343 based on the (A) C^α, (B) C^β and (C) C’ secondary NMR chemical shifts. The secondary structures of the SipD^39–343 crystal are also shown and are denoted by arrow (beta strand), wavy line (helix); solid line (loop), and broken line (disordered loop). Residues 118–133 lacked electron density in the SipD^39–343 crystal.

Fig. 3

Weighted chemical shift difference (Δδ_HN) of the ¹H and ¹⁵N resonances of SipD^39–343 when titrated with (A) deoxycholate, (B) taurodeoxycholate and (C) chenodeoxycholate (Δδ_HN = [½ (δ_H² + 1/25δ_N²)]^1/2 (25)). The ¹H and ¹⁵N chemical shifts were extracted from the 2D ¹H-¹⁵N TROSY spectra of the free protein and the protein with the highest molar ratios of titrants at (A) 3.5, (B) 3.7 and (C) 4.3, respectively. For the deoxycholate titration (A), the average Δδ_HN for all residues was 0.011 ppm with a standard deviation (σ) of 0.011 ppm. Thus, Δδ_HN values above 3σ (or 0.03 ppm, indicated by a horizontal line) were deemed significant. Horizontal lines at Δδ_HN 0.03 ppm in (B) and (C) were drawn for comparison with (A).

Fig. 4

Ribbon representation of the crystal structure of SipD^39–343 (to be reported elsewhere). The side chains of residues that showed significant chemical shift perturbation (Δδ_HN > 0.03) with deoxycholate were colored red (legend: α, α-helix; β, beta strand; dark oval represents the equivalent region in IpaD that was predicted by computer docking to be deoxycholate binding site in IpaD (7)). The various regions of SipD were colored as follows: N-terminal helix α₁ and α₂ (light blue), helix α₃ and loop 110–117 (light green), the central coiled coil formed by helix α₄ and α₈ (gray), and the rest of SipD (pale cyan).