Top view of a needle showing 11 PrgI subunits arranged in a right-handed helix with outward-facing N-termini (N-ter).
Figure courtesy of Adam Lange.
Some of the most pernicious Gram-negative bacteria use a type III secretion system (T3SS) to inject effectors into host cells. These include the bacteria that cause bubonic plague and typhoid fever, and Salmonella typhimurium, which causes food poisoning. Secretion requires the needle of the T3SS, a tubular protein filament that in S. typhimurium is composed of multiple copies of PrgI protomers.
Previous studies used X-ray crystallography and cryo-electron microscopy to study the needle assembly, but could not produce high-resolution structures. As the needle is insoluble, it is not amenable to solution NMR methods. To obtain an atomic resolution model of the T3SS needle, Lange, Becker, Baker, Kolbe and colleagues reconstituted recombinant wild-type needles and applied solid-state NMR.
The groups purified 13C -labeled S. typhimurium PrgI protomers and polymerized them in vitro. Solid-state NMR showed that the PrgI structure consists of two α-helices separated by a loop. It also revealed an extended rigid N-terminal domain and a kink in helix α1 that was omitted in previous X-ray and solution NMR studies. Sparse isotopic 13C labeling yielded a large number of cross-peaks in the solid-state NMR spectra, corresponding to 247 long-range 13C–13C and 15N–13C restraints. These restraints identified the intrasubunit hairpin, intersubunit lateral interfaces resulting from α1–α1 and α2–α2 helix–helix packing, and an axial translation of ∼24 Å between neighboring subunits.
The groups modeled the assembly by customizing a homo-oligomeric fold-and-dock protocol in the energy-guided optimization software Rosetta. A right-handed helical assembly with 11 subunits per two turns was most consistent with the restraints and microscopy data. The final model (PDB 2LPZ) is a helical assembly with a ∼80-Å outer diameter tube and a ∼25-Å lumen lined with conserved residues; each subunit i interfaces with i ± 5 and i ± 6 laterally, and with i ± 11 axially.
In contrast to previous models, the PrgI N-termini face outward in their needle structure. The researchers confirmed this orientation by immunogold staining of His-tagged PrgI, and used in vivo invasion assays with prgI mutants to validate the function of key residues in the PrgI termini predicted to participate in lateral and axial interfaces.
The combined NMR and modeling approach should help refine interfaces in the structure of other homo-oligomeric protein assemblies.