Purification and three-dimensional electron microscopy structure of the Neisseria meningitidis type IV pilus biogenesis protein PilG

Abstract

Type IV pili are surface-exposed retractable fibers which play a key role in the pathogenesis of Neisseria meningitidis and other gram-negative pathogens. PilG is an integral inner membrane protein and a component of the type IV pilus biogenesis system. It is related by sequence to the extensive GspF family of secretory proteins, which are involved in type II secretion processes. PilG was overexpressed and purified from Escherichia coli membranes by detergent extraction and metal ion affinity chromatography. Analysis of the purified protein by perfluoro-octanoic acid polyacrylamide gel electrophoresis showed that PilG formed dimers and tetramers. A three-dimensional (3-D) electron microscopy structure of the PilG multimer was determined using single-particle averaging applied to samples visualized by negative staining. Symmetry analysis of the unsymmetrized 3-D volume provided further evidence that the PilG multimer is a tetramer. The reconstruction also revealed an asymmetric bilobed structure approximately 125 A in length and 80 A in width. The larger lobe within the structure was identified as the N terminus by location of Ni-nitrilotriacetic acid nanogold particles to the N-terminal polyhistidine tag. We propose that the smaller lobe corresponds to the periplasmic domain of the protein, with the narrower "waist" region being the transmembrane section. This constitutes the first report of a 3-D structure of a member of the GspF family and suggests a physical basis for the role of the protein in linking cytoplasmic and periplasmic protein components of the type II secretion and type IV pilus biogenesis systems.

FIG. 1.

SDS-PAGE and PFO-PAGE of purified PilG. (A) SDS-PAGE of purified PilG on a 4 to 20% continuous-gradient gel. Molecular mass markers are indicated on the right of the gel. (B) PFO-PAGE of PilG on a 4 to 12% gradient gel. Lanes: 1, ferritin (440 kDa); 2, immunoglobulin G (150 kDa); 3, bovine serum albumin (66 kDa); 4, PilG. Both gels were stained with Coomassie blue.

FIG. 2.

CD spectrum of purified PilG. degM, •••.

FIG. 3.

Analysis of PilG by TEM. (A) Sample TEM data of PilG negatively stained with 2% (wt/vol) uranyl acetate. PilG particles are highlighted in square boxes. The image has been CTF corrected and contrast enhanced for presentation. Scale bar = 500 Å. (B) Selected class averages generated from SPA as applied to the PilG data set. The 14 average pairs shown are representative samples from the total classes and show a back-projection from the C4-symmetrized 3-D volume paired with the corresponding unsymmetrized class average used in the calculation. Excellent visual correlation between the pairs is apparent. Box size is 234 Å by 234 Å.

FIG. 4.

Rotational symmetry analysis of SPA PilG volume. Real-space self-correlation analysis of the unsymmetrized PilG volume is shown as a function of the angle of rotation about the main axis of the complex. Correlation coefficients were calculated from a 3-D volume produced with no symmetry averaging applied (C1). The volume was calculated using procedures described previously (12). The inset shows a top-view back-projection of the unsymmetrized volume.

FIG. 5.

3-D reconstruction of PilG. (A) Surface-rendered symmetrized volumes of the PilG complex: the structure in the left column has C2 symmetry imposed, and the structure on the right has C4 symmetry. Both volumes are displayed at a threshold of 2.6 σ above the mean density. The volume was low-pass filtered to a 22-Å resolution. The main features and dimensions of the complex are indicated. (B) Slabs taken through the C4-symmetrized 3-D volume from panel A, highlighting structural features at 3 σ (red), 3.5 σ (orange), 4 σ (yellow), 4.5 σ (pale blue), and 5 σ (dark blue) above the mean density.

FIG. 6.

Nanogold labeling of PilG and proposed orientation in the inner membrane. (A) Montage of raw multiple gold-labeled PilG complexes. Data were CTF corrected for presentation. Box size is 234 Å by 234 Å. (B) 3-D reconstruction of the PilG-nanogold complex, filtered to a 30-Å resolution. The PilG volume is shown by green netting contoured at 3 σ above the mean density. Peaks associated with nanogold particles are shown in yellow at −4.1 σ below the mean density. C4 symmetry has been imposed. The volume was calculated using the PilG reconstruction determined previously (Fig. 5) as a start model for the nanogold-labeled data set (n = 1,100) and processed as described previously (12). The volume is displayed following low-pass filtering to 25 Å. Scale bar = 50 Å. (C) The left panel shows a composite figure, with the volume from the reconstruction of PilG alone displayed at 2.6 σ (light green) and 5 σ (dark green) above the mean density in green surface rendering with 50% opacity. Superimposed on this density are the peaks associated with the bound nanogold particles, shown at −5 σ below the mean density in yellow surface rendering. Both volumes had C4 symmetry imposed. The cartoon on the right shows the PilG topology, with transmembrane helices predicted using PHDhtm (32), matched approximately to the distribution of mass in the PilG 3-D structure on the left.