Crystal structure of Legionella DotD: insights into the relationship between type IVB and type II/III secretion systems

Abstract

The Dot/Icm type IVB secretion system (T4BSS) is a pivotal determinant of Legionella pneumophila pathogenesis. L. pneumophila translocate more than 100 effector proteins into host cytoplasm using Dot/Icm T4BSS, modulating host cellular functions to establish a replicative niche within host cells. The T4BSS core complex spanning the inner and outer membranes is thought to be made up of at least five proteins: DotC, DotD, DotF, DotG and DotH. DotH is the outer membrane protein; its targeting depends on lipoproteins DotC and DotD. However, the core complex structure and assembly mechanism are still unknown. Here, we report the crystal structure of DotD at 2.0 Å resolution. The structure of DotD is distinct from that of VirB7, the outer membrane lipoprotein of the type IVA secretion system. In contrast, the C-terminal domain of DotD is remarkably similar to the N-terminal subdomain of secretins, the integral outer membrane proteins that form substrate conduits for the type II and the type III secretion systems (T2SS and T3SS). A short β-segment in the otherwise disordered N-terminal region, located on the hydrophobic cleft of the C-terminal domain, is essential for outer membrane targeting of DotH and Dot/Icm T4BSS core complex formation. These findings uncover an intriguing link between T4BSS and T2SS/T3SS.

Figure 1. Structure of DotDΔN.

(A) Domain structure of DotD. The green and orange boxes denotes the DotD domain and the Lid, respectively, which were visible in the electron density map. The black box denotes the signal sequence. Trypsin and V8 protease preferential cleavage sites are shown by black and grey arrows, respectively. (B) Crystal structure of DotDΔN. (C) Multiple alignment of DotD orthologs from closely related bacterial pathogens, with structural annotation gained from the DotD structure. DotD sequences were obtained from blast nonredundant protein database (nr). Lp: Legionella pneumophila (strains Philadelphia-1, Paris, Lens), Rgrylli: Rickettsiella grylli, Cb: Coxiella burnetii (strains Dugway 5J108-111, RSA 331). Conserved and similar residues were black and grey shaded, respectively. The lipidation site cysteins are red shaded. Tripsin/V8 sites are shows as in panel A.

Figure 2. Comparison of DotDΔN with secretin periplasmic subdomains.

(A) DotD (green) superimposed onto the N0 domain of ETEC secretin GspD (residue 43 to 120; PDB accession 3ezj) from the type II secretion system (blue). (B) DotD (green) superimposed onto the T3S domain of EPEC secretin EscC (residue 21 to 103; PDB accession 3gr5) from the type III secretion system (light blue). (C) Structure-based sequence alignment of the DotD domain, GspD and EscC. Conserved residues are colored. See text for explanations for yellow shaded residues and residues shown in italics.

Figure 3. Interaction between the DotD domain and the lid.

Stereo view of the interface between the DotD domain (α1 and α2 helices in green, β1 strand in yellow) and the lid (in orange).

Figure 4. The lid mutation adversely affected core assembly.

(A) The lid mutant is defective in outer membrane targeting of DotH. Total membranes were isolated from whole cell lysates of L. pneumophila strains producing wild-type, the lid mutant (DotD^AA) or no DotD. Inner and outer membranes were separated by isopycnic sucrose density gradient centrifugation as described in Materials and Methods. Whole cell lysates (Whole cell), soluble fractions (Soluble), total membranes (Membrane) and the separated membrane fractions were analyzed by Western immunoblotting using indicated antibodies. DotA and Momp were used as inner and outer membrane control, respectively. (B) Adverse effects of the lid mutation on interactions between DotC, DotD, DotF, DotG and DotH. L. pneumophila strains producing wild-type, the lid mutant (DotD^AA) or no DotD were treated with cleavable crosslinker (0.08 mM DSP) before lysate preparation. All L. pneumophila strains used in this experiment encode M45 epitope-tagged dotF on the chromosome for immunoprecipitation and detection of DotF. Cleared lysates were subjected to immunoprecipitation with antibodies against indicated proteins (IP: DotC, DotD, DotG and DotH) or anti-M45 epitope (IP: DotF) as described in Materials and Methods. Immunoprecipitants were treated with SDS-PAGE sample buffer containing reducing agent to cleave crosslinks, and were subjected to western immunoblotting analyses with antibodies against indicated proteins (IB: DotC, DotD, DotG and DotH) or anti-M45 epitope (IB: DotF). Boxed panels (IP:DotC/IB:DotC, IP:DotD/IB:DotD IP:DotF/IB:DotF, and IP:DotG/IB:DotG) show efficiency of immunoprecitation in this experimental condition. The loading amounts onto the SDS gels of these samples were reduced by 10-fold compared to other samples which show the efficiency of co-immunoprecipitation. Detection of DotH by immunoprecipitation followed by western immunoblotting was technically difficult because its mobility in the gel was similar to that of immunoglobulin heavy chain, and thus was not carried out.

Figure 5. Periplasmic ring models of DotD.

(A) Domain organizations of DotD, EscC and GspD. Green boxes: DotD/T3S/N0 domain; Blue boxes: Secretin_N domain (protein family database Pfam PF03958); Red boxes: Secretin domain (Pfam PF00263). (B) Schematic drawings of the type II/III secretin and a putative outer membrane complex containing DotD. Red, blue and green torus represents domains schematically drawn in panel A. (C) Ring models of the DotD domain having 12- and 14-fold rotation symmetry. Ring structures were modeled using SymmDock program fed with DotD atomic coordinates (excluding water molecules and the lid) and order of rotation symmetry (C12 or C14).