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. 2016 Mar 23:6:23123.
doi: 10.1038/srep23123.

The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

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The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

Iñaki de Diego et al. Sci Rep. .

Abstract

In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway.

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Figures

Figure 1
Figure 1. Structure and topology diagram of the rIgSF-CTD tandem and structural superposition of the IgSF and CTD domains calculated using the Secondary Structure Matching (SSM) algorithm.
(A) Overview of the IgSF (cyan)-CTD (yellow) tandem. The structure is elongated, with both domains packing in a perpendicular fashion. The shape of the IgSF closely resembles a β-barrel, whereas the CTD is more a regular β-sandwich. (B) Topology and schematic diagrams of, respectively, IgSF (left) and CTD (right). Although both domains have an Ig-like fold, significant differences are observed between sheet composition. Whereas the N-terminal sheet of each domain is antiparallel in both cases, the C-terminal sheet differs, being mixed in IgSF. In IgSF, strands sA-sA’ and, to a lower extent, sD, are establish interactions with both sheets of the barrel, thus generating a higher curvature in the surface of the sheets. (C) IgSF (cyan, sA–sG) and CTD (yellow, s1–s7) can be superimposed with an RMSD of 2.1 Å for 63 aligned residues, thus confirming that both fragments have an Ig-like fold. However, main differences occur at the first strand (sA–sA’ for IgSF, s1 for the CTD) and in the curvature of the sheets (less prominent for the CTD). (D) Structure-based sequence alignment. Despite low sequence identity, the number, length and type of secondary structures reveals that both are Ig-like domains.
Figure 2
Figure 2. Small angle X-ray scattering (SAXS) of rIgSF-CTD.
(A) SAXS data for wild-type rIgSF-CTD (black symbols) and the r664i6H variant (red symbols) with the ensemble optimisation model fit shown as lines in the reverse colours. (B) Atom pair distribution, P(r) versus r, calculated as the Fourier transform of the I(q) versus q profiles in A, with the same colour coding. (C) Ensemble optimisation modelling (EOM) selected representative structures aligned via the IgSF domains (magenta) with the small flexible tip of N-terminal residues and the linker in yellow (surface representation only), showing the range of positions for the CTD domains within the wild-type rIgSF-CTD construct; the CTD domains coloured differently for each conformation within the set. (D) Overlay of EOM selected sets for wild-type rIgSF-CTD (cyan CTD domains) and r664i6H variant (orange CTD domains) to show the range of flexibility of the CTD with respect to the aligned IgSF domain (magenta).
Figure 3
Figure 3. The rCTD structure.
(A) Purified rCTD yielded a dimeric structure (chains in cyan and green), in which both domains interact head to back. The C-terminal strand does not fold back, running antiparalel to the penultimate strand but rather runs in extended conformation and provides the last strand of the symmetric partner. (B) Topology diagram showing the domain swap of the C-terminal strands. (C) Stick model showing the superposition of strands s6 and s7 in the CTD of the IgSF-CTD tandem structure (carbon atoms in gold) and in the dimeric standalone CTD structure (strand s6 of one monomer in green, strand s7 of the other monomer in cyan). Except for the respective linkers to the preceding strands s6, the two s7 strands nicely fit on top of each other.
Figure 4
Figure 4. Probing the structure of the wild-type rIgSF-CTD and the r665i6H variant by limited proteolysis.
Both proteins were incubated with V8 protease (V8), Lys-C endopeptidase (Lys-C) and human neutrophil elastase (HNE) at (A) different substrate:enzyme molar ratios (concentration-dependent proteolysis) for 1 h or (B) at a constant 100:1 substrate:enzyme ratio for different time intervals (time-dependent proteolysis). Boiling with SDS-PAGE reducing sample buffer terminated reaction and samples were subjected to SDS-PAGE, transferred onto PVDF membrane for N-terminal sequencing (sequences next to each panel). (C) Identified primary cleavage sites within the linker region are indicated by solid arrows: red for elastase, blue for Lys-C and green for V8 protease. Open arrows indicate secondary cleavages by the respective enzymes. Polypeptide sequence comprising the IgSF domain is in blue font; the linker region between IgSF and CTD domains is in lowercase and inserted 6×His is in red. Horizontal arrows below the sequence indicate β-strands in the IgSF-CTD tertiary structure.
Figure 5
Figure 5. Probing the structure of insertion/substitution variants of IgSF-CTD by limited proteolysis.
IgSF-CTD variants were incubated with (panels A and B) porcine pancreatic elastase (PPE) or (panel C) factor Xa (fXa) at different substrate:enzyme molar ratios (concentration-dependent proteolysis) for 1 h or at a constant 100:1 substrate:enzyme ratio for different time intervals (time-dependent proteolysis). Boiling with SDS-PAGE reducing sample buffer terminated reaction and samples were subjected to SDS-PAGE, transferred onto PVDF membrane for N-terminal sequencing (sequences next to each panel). (D) Identified primary cleavage sites within the linker region are indicated by solid arrows: red for PPE and black for fXa. Open and broken red arrows indicate secondary and minor cleavage sites for PPE. Polypeptide sequence comprising the IgSF domain is in blue font; the linker region between IgSF and CTD domains is in lowercase with inserted/substituted sequences in red font. Horizontal arrows below the sequence indicate β-strands in the IgSF-CTD tertiary structure.
Figure 6
Figure 6. Probing of full-length proRgpB variants expressed in the native organism bearing factor Xa cleavage motif (IEGRAA) in the junction between the IgSF and CTD domains.
(A) Concentration-dependent and (B) time-dependent limited proteolysis with factor Xa. (A) 1.67 μM of proRgpB with insertion (RgpB662iXa6H) or substitution (RgpB665sXa6H) of residues in the linker region with Xa cleavage motif were incubated either alone or with fXa at the molar ratio 50:1, 100:1 and 1,000:1 at 37 °C for 3 h (upper panels) or 15 h (lower panels). The proteolysis was assessed by SDS-PAGE. Ctrl, control of proRgpB variant incubated alone. (B) ProRgpB variants were incubated at 37 °C with fXa at 10:1 ratio for up to 72 h. At indicated time intervals, samples were withdrawn and analyzed by SDS-PAGE for proteolytic degradation.
Figure 7
Figure 7. Soluble rCTD is a dimer in equilibrium and the CTD cleaved off natively expressed proRgpB spontaneously dimerizes.
(A) rCTD at 1, 0.5 and 0.1 mg ml−1 was treated with glutaraldehyde and analysed by SDS-PAGE. (B) Recombinant CTD (rCTD) (red), proRgpB662iXa (black), and proRgpB662iXa preincubated with fXa (blue) were subjected to size exclusion chromatography on a Superdex 75 10/300 GL column equilibrated with 50 mM Tris, 150 mM NaCl, 2.5 mM CaCl2, 0.02% NaN3 pH 7.5 (C) Indicated fractions of resolved proteins were analysed by Western blot using anti-rCTD antibodies to reveal the CTD content in each analysed fraction.
Figure 8
Figure 8. Superimposition of the IgSF domains in the presence of other domains in RgpB, and a model of the full-length latent proRgpB.
(A) The IgSF domain of r664i6H (cyan) superimposes perfectly with the IgSF domain of mature RgpB (PDB id. 1CVR, in red) and RgpB-N-terminal prodomain complex (PDB id. 4IEF, in orange) and shows significant structural conservation with related IgSF sequences from Kgp (PDB id. 4RBM, in green) and PPAD (PDB id. 4YT9, in grey). (B) Surface representation of the chimeric, multi-domain model of the latent full-length proRgpB, transiently present in the periplasm during secretion, including the N-terminal prodomain (green), the catalytic domain (purple), the IgSF domain (cyan) and the CTD (yellow).

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