Differences in the electrostatic surfaces of the type III secretion needle proteins PrgI, BsaL, and MxiH

Abstract

Gram-negative bacteria use a needle-like protein assembly, the type III secretion apparatus, to inject virulence factors into target cells to initiate human disease. The needle is formed by the polymerization of approximately 120 copies of a small acidic protein that is conserved among diverse pathogens. We previously reported the structure of the BsaL needle monomer from Burkholderia pseudomallei by nuclear magnetic resonance (NMR) spectroscopy and others have determined the crystal structure of the Shigella flexneri MxiH needle. Here, we report the NMR structure of the PrgI needle protein of Salmonella typhimurium, a human pathogen associated with food poisoning. PrgI, BsaL, and MxiH form similar two helix bundles, however, the electrostatic surfaces of PrgI differ radically from those of BsaL or MxiH. In BsaL and MxiH, a large negative area is on a face formed by the helix alpha1-alpha2 interface. In PrgI, the major negatively charged surface is not on the "face" but instead is on the "side" of the two-helix bundle, and only residues from helix alpha1 contribute to this negative region. Despite being highly acidic proteins, these molecules contain large basic regions, suggesting that electrostatic contacts are important in needle assembly. Our results also suggest that needle-packing interactions may be different among these bacteria and provide the structural basis for why PrgI and MxiH, despite 63% sequence identity, are not interchangeable in S. typhimurium and S. flexneri.

Figure 1

The middle regions of the needle proteins are more conserved compared to their N-termini. The structural features of PrgI, BsaL, and MxiH are indicated as well-structured helices (α₁ and α₂, solid wavy lines), regions with partial α-helical character (dashed wavy lines), and random coil regions (dashed lines). Conserved hydrophobic residues (blue) at the helix α₁-α₂ interface are boxed and prolines and glycines (red) that act as helix breakers are indicated. The PxxP motif and the last five residues of PrgI, BsaL, and BsaL that were deleted are boxed. The needle proteins are from Salmonella typhimurium (PrgI), Burkholderia pseudomallei (BsaL), Shigella flexneri (MxiH), Escherichia coli O157:H7 (EprI), Yersinia pestis (YscF), and Pseudomonas aeruginosa (PscF).

Figure 2

(A) Assigned ¹H-¹⁵N HSQC spectra of PrgI^CΔ5. The insert shows an expansion of the crowded region of the spectrum. The side chain peak of W5 (not shown) is at 10.16 ¹H ppm and 129.89 ¹⁵N ppm. (B) Stereoview of the superposition of 20 lowest energy structures of PrgI^CΔ5, showing only the backbone N, C^α, and C’ atoms. Only the region V20-V65 can be superimposed into an ensemble of NMR structures, forming the core domain of PrgI^CΔ5, however, the regions flanking this core domain, from W5-G19 and K66-I75, retained α-helical conformation in each of the 20 NMR structures.

Figure 3

PrgI^CΔ5 forms a two helix bundle stabilized by hydrophobic contacts at the helix α₁- α₂ interface. The side chains of the hydrophobic and polar residues are shown. (A) is oriented by a 180 rotation from (B). Atoms are colored as: red, oxygen; blue, nitrogen; gray, carbon.

Figure 4

The electrostatic surface of (A) PrgI, (B) BsaL, and (C) MxiH are shown together with the ribbon structures showing the relative orientation of the two helix bundle. The structures of PrgI, BsaL, and MxiH are oriented is similar manner from top to bottom to allow comparison of the surfaces. The structures on the left are rotated by 180° on the y-axis from the right.

Figure 5

Salmonella invasion assay. The number of bacterial colonies (y axis) correlates with the ability of S. typhimurium to invade a cultured human epithelial cell line (Henle 407). Wild type (WT) bacterium is invasive, whereas a S. typhimurium with a prgI null mutation (prgI-) is noninvasive. A plasmid that overexpressed a functional PrgI (prgI⁺) restored the invasiveness of the prgI null mutant strain. Point mutations were introduced in the Q26, K50, and R58 residues using the PrgI plasmid. The assays were done in triplicates.

Figure 6

(A) Model of the S. flexneri needle created using the crystal structure of MxiH^CΔ5 (PDB #2CA5) and published needle packing parameters,, and visualized with the top towards the host and below towards the bacterium, and along the 25 Å needle channel looking down towards the bacterium. (B) Two MxiH molecules within the needle with extensive intermolecular contacts are chosen for electrostatic analysis. (C) The two monomers, MxiH 1 and MxiH 2, are separated from the MxiH dimer and rotated by 90° towards the viewer to reveal the electrostatic potentials of the residues involved in the needle packing interaction. The N-terminal 19 residues of MxiH were removed because they were disordered in the MxiH crystal structure. (D) Electrostatic analysis of two PrgI molecules (PrgI 1 and PrgI 2) in the model of the S. typhimurium needle, which was created by superimposing the NMR structure of PrgI into S. flexneri needle. The N-terminal 19 residues of PrgI were removed in the calculation of electrostatic potentials because they did not converge into a single family of structures. The Coulombic electrostatic interaction energy is -120 kcal/mol for the MxiH dimer and -23 kcal/mol for the PrgI dimer. Molecular graphics were made using Pymol (A and B) and Dino (DINO: Visualizing Structural Biology (2002) http://www.dino3d.org) (C and D). The amino (N) and the carboxy (C) termini and the PxxP loop are indicated.