New insights into the assembly of bacterial secretins: structural studies of the periplasmic domain of XcpQ from Pseudomonas aeruginosa

Abstract

The type II secretion system is a multiprotein assembly spanning the inner and outer membranes in Gram-negative bacteria. It is found in almost all pathogenic bacteria where it contributes to virulence, host tissue colonization, and infection. The exoproteins are secreted across the outer membrane via a large translocation channel, the secretin, which typically adopts a dodecameric structure. These secretin channels have large periplasmic N-terminal domains that reach out into the periplasm for communication with the inner membrane platform and with a pseudopilus structure that spans the periplasm. Here we report the crystal structure of the N-terminal periplasmic domain of the secretin XcpQ from Pseudomonas aeruginosa, revealing a two-lobe dimeric assembly featuring parallel subunits engaging in well defined interactions at the tips of each lobe. We have employed structure-based engineering of disulfide bridges and native mass spectrometry to show that the periplasmic domain of XcpQ dimerizes in a concentration-dependent manner. Validation of these insights in the context of cellular full-length XcpQ and further evaluation of the functionality of disulfide-linked XcpQ establishes that the basic oligomerization unit of XcpQ is a dimer. This is consistent with the notion that the dodecameric secretin assembles as a hexamer of dimers to ensure correct projection of the N-terminal domains into the periplasm. Therefore, our studies provide a key conceptual advancement in understanding the assembly principles and dynamic function of type II secretion system secretins and challenge recent studies reporting monomers as the basic subunit of the secretin oligomer.

FIGURE 1.

Structure of peri-XcpQ. A, three views of the homodimer of peri-XcpQ. Note the 2-fold symmetry axis and the extended β-sheet of the N2 interface. B, detail of the interaction interface between N0:N0′ (left panel) and extended β-sheet between N2:N2′ (middle and right panels). C, alignment of peri-XcpQ sequences against peri-GspD sequences from different species, annotated by secondary structure elements of peri-XcpQ. Residues participating at the N0:N0′ and N2:N2′ interfaces are colored in green and red, respectively. Residues that are involved via side chain interactions in the N0:N1 and N1:N2 interfaces are annotated with ^ and #, respectively (see also supplemental Table S2). The figure was made using Jalview 2 (39). D, comparison of the peri-XcpQ monomer model and peri-GspD monomer (Protein Data Bank code 3EZJ) in complex with a Nb7 nanobody. Note that the bound nanobody occupies a site that would not be compatible with dimerization as observed in peri-XcpQ.

FIGURE 2.

Cysteine disulfide-bridge engineering in peri-XcpQ. A, elution profiles of wild-type, S109C, and S210C peri-XcpQ in size exclusion chromatography and the corresponding SDS-PAGE analyses. For both wild type and S109C, the major peak (monomer, 1-m) was selected and submitted to oxidation by H₂O₂ or water as control prior the SDS-PAGE analysis. The elution profile of S210C peri-XcpQ mutant shows an extra peak that corresponds with a dimer (2-m) of peri-XcpQ as shown on SDS-PAGE. The positions of both substituted amino acids are denoted in green in the model of peri-XcpQ. B, detail of electron density map (2F_o − F_c (1.5 σ) and F_o − F_c (3.3 σ); α_c,MR) of the disulfide bridge of the S210C mutant of peri-XcpQ in comparison with the map for the wild-type protein structure. β-ME, β-mercaptoethanol.

FIGURE 3.

Peri-XcpQ forms dimers in solution in a concentration-dependent manner. Ion mobility drift time chromatograms obtained from native mass spectrometry on wild-type peri-XcpQ at three different concentrations. The clusters highlighted with ovals correspond to particular oligomeric states. Each state exhibits different m/z species. M, monomeric; D, dimeric. The numbers denote the charge of the protein.

FIGURE 4.

Dodecameric full-length XcpQ is a hexamer of dimers in vivo. A, Western blot analysis of boiled and unboiled samples of PAN1 cells under reducing and nonreducing conditions. PAN1 cells were transformed with constructs pB28, pB28_S109C, and pB28_S210C encoding for wild-type full-length XcpQ and its S109C and S210C variants, respectively. B, elastase activity assay on PAN1 transformants. Except for the parental strain, all transformants expressing XcpQ or its mutant variants show the formation of halos around the colonies as a result of proteolysis of elastin substrate in the plate. C, hexameric model of peri-XcpQ dimers obtained by manual modeling based on the molecular envelope of full-length EpsD as obtained by cryo-electron microscopy (Electron Microscopy Data Bank code 1763) (37).