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 a-helical hairpins and a short helix a3 that pack against the coiled coil. Using AutoDock, others have predicted that the bile salt deoxycholate binds IpaD in a cleft formed by the a-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 a3. 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 a3 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.
Hantaviruses are distributed worldwide and can cause a hemorrhagic fever or a cardiopulmonary syndrome in humans. Mature virions consist of RNA genome, nucleocapsid protein, RNA polymerase, and two transmembrane glycoproteins, G1 and G2. The ectodomain of G1 is surface-exposed; however, it has a 142-residue C-terminal cytoplasmic tail that plays important roles in viral assembly and host-pathogen interaction. Here we show by NMR, circular dichroism spectroscopy, and mutagenesis that a highly conserved cysteine/histidine-rich region in the G1 tail of hantaviruses forms two CCHC-type classical zinc fingers. Unlike classical zinc fingers, however, the two G1 zinc fingers are intimately joined together, forming a compact domain with a unique fold. We discuss the implication of the hantaviral G1 zinc fingers in viral assembly and host-pathogen interaction.
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.
The bacterial mechanosensitive channel of large conductance (MscL) directly senses and responds to membrane tension. It serves as an “emergency release valve” upon acute decreases in the osmotic environment, thus preventing cell lysis. It is one of the best studied mechanosensitive channels and serves as a paradigm of how a channel senses and responds to its membrane environment. The MscL protein is highly conserved, found throughout the bacterial kingdom, and has been shown to encode a functional mechanosensitive channel in all species where it has been studied. However, channels from different species have shown some functional variance; an extreme example is the Mycobacterium tuberculosis MscL, which when heterologously expressed in Escherichia coli requires significantly more membrane tension for gating than the endogenous E. coli MscL. We previously speculated that the membrane environment or factors not found in E. coli promoted the proper gating of the M. tuberculosis MscL channel in its native environment. Here, by reconstituting the M. tuberculosis and E. coli MscL channels in various lipids, we demonstrate that inclusion of phosphatidylinositol, a lipid found in M. tuberculosis but not E. coli, is sufficient for gating of the M. tuberculosis MscL channel within a physiological range of membrane tension.
Background:The structure of the protein family of the bacterial inner rod proteins is unknown. Results: CD and NMR were used to determine the structural properties of the Salmonella PrgJ inner rod protein.
Conclusion:PrgJ is a partially folded protein and contains only a short helix. Significance: Protein flexibility is a hallmark of the inner rod proteins.
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