No abstract

Structures of the type IV pili secretin complexes from Neisseria gonorrhoeae and Neisseria meningitidis, embedded in outer membranes were investigated by transmission electron microscopy. Single particle averaging revealed additional domains not observed previously. Secretin complexes of N. gonorrhoeae showed a double ring structure with a 14–15-fold symmetry in the central ring, and a 14-fold symmetry of the peripheral ring with 7 spikes protruding. In secretin complexes of N. meningitidis, the spikes were absent and the peripheral ring was partly or completely lacking. When present, it had a 19-fold symmetry. The structures of the complexes in several pil mutants were determined. Structures obtained from the pilC1/C2 adhesin and the pilW minor pilin deletion strains were similar to wild-type, whereas deletion of the homologue of N. meningitidis PilW resulted in the absence of secretin structures. Remarkably, the pilE pilin subunit and pilP lipoprotein deletion mutants showed a change in the symmetry of the peripheral ring from 14 to 19 and loss of spikes. The pilF ATPase mutant also lost the spikes, but maintained 14-fold symmetry. These results show that secretin complexes contain previously unidentified large and flexible extra domains with a probable role in stabilization or assembly of type IV pili.

The structure of three secondary transporter proteins, GltT of Bacillus stearothermophilus, CitS of Klebsiella pneumoniae, and GltS of Escherichia coli, was studied. The proteins were purified to homogeneity in detergent solution by Ni 2þ -NTA affinity chromatography, and the complexes were determined by BN-PAGE to be trimeric, dimeric, and dimeric for GltT, CitS, and GltS, respectively. The subunit stoichiometry correlated with the binding affinity of the Ni 2þ -NTA resin for the protein complexes. Projection maps of negatively stained transporter particles were obtained by single-particle electron microscopy. Processing of the GltT particles revealed a projection map possessing 3-fold rotational symmetry, in good agreement with the trimer observed in the crystal structure of a homologous protein, Glt Ph of Pyrococcus horikoshii. The CitS protein showed up in two main views: as a kidney-shaped particle and a biscuit-shaped particle, both with a long axis of 160 A ˚. The latter has a width of 84 A ˚, the former of 92 A ˚. Symmetry considerations identify the biscuit shape as a top view and the kidney shape as a side view from within the membrane. Combining the two images shows that the CitS dimer is a protein with a strong curvature at one side of the membrane and, at the opposite side, an indentation in the middle at the subunit interface. The GltS protein was shaped like CitS with dimensions of 145 A ˚Â 84 A ˚. The shapes and dimensions of the CitS and GltS particles are consistent with a similar structure of these two unrelated proteins.

We have studied Hansenula polymorpha Pex5p and Pex20p, peroxins involved in peroxisomal matrix protein import.