Structure of a type IV secretion system core complex

Abstract

Type IV secretion systems (T4SSs) are important virulence factors used by Gram-negative bacterial pathogens to inject effectors into host cells or to spread plasmids harboring antibiotic resistance genes. We report the 15 angstrom resolution cryo-electron microscopy structure of the core complex of a T4SS. The core complex is composed of three proteins, each present in 14 copies and forming a approximately 1.1-megadalton two-chambered, double membrane-spanning channel. The structure is double-walled, with each component apparently spanning a large part of the channel. The complex is open on the cytoplasmic side and constricted on the extracellular side. Overall, the T4SS core complex structure is different in both architecture and composition from the other known double membrane-spanning secretion system that has been structurally characterized.

Fig.1

Purification of the pKM101-encoded T4SS complex core. A. SDS-PAGE analysis of the non-induced (NI) and induced (I) cells (left panel), the pure wild-type core complex (middle panel), and digested D1 and D2 complexes (right panel). In the right panel, “a” refers to the upper band that contains a C-terminal fragment of TraF/VirB10 and full-length TraO/VirB9; “b” refers to the top band that contains a C-terminal fragment of TraF/VirB10 and “c” refers to the middle band that contains a C-terminal fragment of TraO/VirB9. The content of the a, b, and c bands was assessed by mass spectrometry and N-terminal sequencing. B. Gel filtration of the wild-type, D1 and D2 complexes.

Fig.2

Cryo-EM structure of the TraN/VirB7, TraO/VirB9, and TraF/VirB10C-ST core complex. A. Side-view. B. Cut-away side view. Electron density is color-coded from red to blue to indicate regions of strong to weaker density, respectively. C. Top view (view from outside the cell). D. Bottom view (view from the cytoplasm). All features and dimensions mentioned in the text are indicated.

Fig.3

Comparison of the structures of the wild-type (negative stain (A) and cryo-EM (C)), D1 (negative stain (B)), and D2 (cryo-EM (D)) complexes. Parts removed by proteolysis are shown in cyan. The E panel shows schematic diagrams of the composition of each of the complexes. TraN, TraO, and TraF are in the same representation and color-coding as in Fig. S1A. Numbering for TraN and TraO are for the mature proteins (signal sequence excluded). A black outline encloses the parts of the proteins included in the various complexes.

Fig.4

Localization of core complex macromolecular components to the various regions of the cryo-EM structure. A. Immunolocalization of the N-terminus of TraF/VirB10 by negative stain EM. Top panel: representative class averages are shown. Lower panel: same as top panel but with additional density due to anti-His₆ antibodies colored in red. B. Localization of TraO/VirB9 (blue) and TraF/VirB10 (red) within the cryo-EM structure of the wild-type core complex.