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Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis .

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ABSTRACT

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.

No MeSH data available.


Related in: MedlinePlus

Overall crystal structure of PorZ.(a) Ribbon-type plot in cross-eye stereo of the crystal structure to 2.9 Å resolution of PorZ depicting domains βD1, βD2 and CTD, and the three domain-connecting linkers (white ribbons; labeled LβD2-βD1, LβD1-βD2, and LβD2-CTD). Each of the seven blades of propellers βD1 and βD2 (labeled counter-clockwise I to VII) is colored in yellow, orange, red, magenta, blue, turquoise and green, respectively; the CTD is in pink. A structural calcium-binding site (green sphere) is found within βD1-blade IV, and a tetraethylene glycol (TG) and a diethylene glycol (DG) were tentaively assigned on the protein surface (brown stick-models). Other (functionally probably irrelevant) ions and ligands were omitted for clarity. The central shafts of βD1 and βD2 are pinpointed on the entry and exit sides of the propellers by red and purple arrows, respectively. For labels and extension of regular secondary structure elements, see (b). (b) Topology scheme of PorZ, with β-strands as arrows and helices as cylinders, colored as in (a). The polypeptide chain spans residues G29—R776 and the three constituting domains plus the linkers (in grey) are indicated with the residues delimiting each structural element (strands, bulges, helices, β-ribbons, blades and domains). The nomenclature adopted in the text for structure elements is “domain-blade-structural element”, e.g. βD1-VI-β3 or βD2-IV-β-ribbon. (c) Structural calcium-binding site framed by segment D520—D530 within loop Lβ3β4 of βD1-blade IV. The ion is octahedrally coordinated by D520O, T523O, T523Oγ, T526O, D529Oδ1 and D530Oδ1, which are at binding distances of ~2.4 Å.
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f5: Overall crystal structure of PorZ.(a) Ribbon-type plot in cross-eye stereo of the crystal structure to 2.9 Å resolution of PorZ depicting domains βD1, βD2 and CTD, and the three domain-connecting linkers (white ribbons; labeled LβD2-βD1, LβD1-βD2, and LβD2-CTD). Each of the seven blades of propellers βD1 and βD2 (labeled counter-clockwise I to VII) is colored in yellow, orange, red, magenta, blue, turquoise and green, respectively; the CTD is in pink. A structural calcium-binding site (green sphere) is found within βD1-blade IV, and a tetraethylene glycol (TG) and a diethylene glycol (DG) were tentaively assigned on the protein surface (brown stick-models). Other (functionally probably irrelevant) ions and ligands were omitted for clarity. The central shafts of βD1 and βD2 are pinpointed on the entry and exit sides of the propellers by red and purple arrows, respectively. For labels and extension of regular secondary structure elements, see (b). (b) Topology scheme of PorZ, with β-strands as arrows and helices as cylinders, colored as in (a). The polypeptide chain spans residues G29—R776 and the three constituting domains plus the linkers (in grey) are indicated with the residues delimiting each structural element (strands, bulges, helices, β-ribbons, blades and domains). The nomenclature adopted in the text for structure elements is “domain-blade-structural element”, e.g. βD1-VI-β3 or βD2-IV-β-ribbon. (c) Structural calcium-binding site framed by segment D520—D530 within loop Lβ3β4 of βD1-blade IV. The ion is octahedrally coordinated by D520O, T523O, T523Oγ, T526O, D529Oδ1 and D530Oδ1, which are at binding distances of ~2.4 Å.

Mentions: We produced PorZ without its predicted signal peptide (residues Q26-R776) by recombinant overexpression in Escherichia coli, and succeeded in crystallizing and solving its structure by single-wavelength anomalous diffraction with a selenomethionine derivative. The structure was refined with data to 2.9 Å resolution and consists of three domains. The first two are consecutive N-terminal seven-stranded β-propeller or circular-leaflet moieties (βD1: residues K39—M322; and βD2: G29—L34+Y335—T679 373839; PorZ residue numbering in superscript notation according to UP Q9S3Q8), each featuring a shallow cylinder or thick disk with an “entry side” and an “exit side”38. These domains are succeeded by a C-terminal domain (CTD, V692—R776). The domains are connected by linkers (L): LβD2-βD1 (L35—H38), LβD1-βD2 (P323—F334), and LβD2-CTD (G680—G691). The two propellers are offset from one another by a ~90° rotation about the intersection axis of the propellers’ planes. This causes the overall molecular structure to be reminiscent of an easy chair of approx. maximal dimensions 95 × 80 × 55 Å, with βD1 as the seat, βD2 the backrest, and CTD the backrest support (Fig. 5a). The two entry-side surfaces of the PorZ propellers mimic, respectively, the seating and reclining surfaces of the chair.


Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis .
Overall crystal structure of PorZ.(a) Ribbon-type plot in cross-eye stereo of the crystal structure to 2.9 Å resolution of PorZ depicting domains βD1, βD2 and CTD, and the three domain-connecting linkers (white ribbons; labeled LβD2-βD1, LβD1-βD2, and LβD2-CTD). Each of the seven blades of propellers βD1 and βD2 (labeled counter-clockwise I to VII) is colored in yellow, orange, red, magenta, blue, turquoise and green, respectively; the CTD is in pink. A structural calcium-binding site (green sphere) is found within βD1-blade IV, and a tetraethylene glycol (TG) and a diethylene glycol (DG) were tentaively assigned on the protein surface (brown stick-models). Other (functionally probably irrelevant) ions and ligands were omitted for clarity. The central shafts of βD1 and βD2 are pinpointed on the entry and exit sides of the propellers by red and purple arrows, respectively. For labels and extension of regular secondary structure elements, see (b). (b) Topology scheme of PorZ, with β-strands as arrows and helices as cylinders, colored as in (a). The polypeptide chain spans residues G29—R776 and the three constituting domains plus the linkers (in grey) are indicated with the residues delimiting each structural element (strands, bulges, helices, β-ribbons, blades and domains). The nomenclature adopted in the text for structure elements is “domain-blade-structural element”, e.g. βD1-VI-β3 or βD2-IV-β-ribbon. (c) Structural calcium-binding site framed by segment D520—D530 within loop Lβ3β4 of βD1-blade IV. The ion is octahedrally coordinated by D520O, T523O, T523Oγ, T526O, D529Oδ1 and D530Oδ1, which are at binding distances of ~2.4 Å.
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f5: Overall crystal structure of PorZ.(a) Ribbon-type plot in cross-eye stereo of the crystal structure to 2.9 Å resolution of PorZ depicting domains βD1, βD2 and CTD, and the three domain-connecting linkers (white ribbons; labeled LβD2-βD1, LβD1-βD2, and LβD2-CTD). Each of the seven blades of propellers βD1 and βD2 (labeled counter-clockwise I to VII) is colored in yellow, orange, red, magenta, blue, turquoise and green, respectively; the CTD is in pink. A structural calcium-binding site (green sphere) is found within βD1-blade IV, and a tetraethylene glycol (TG) and a diethylene glycol (DG) were tentaively assigned on the protein surface (brown stick-models). Other (functionally probably irrelevant) ions and ligands were omitted for clarity. The central shafts of βD1 and βD2 are pinpointed on the entry and exit sides of the propellers by red and purple arrows, respectively. For labels and extension of regular secondary structure elements, see (b). (b) Topology scheme of PorZ, with β-strands as arrows and helices as cylinders, colored as in (a). The polypeptide chain spans residues G29—R776 and the three constituting domains plus the linkers (in grey) are indicated with the residues delimiting each structural element (strands, bulges, helices, β-ribbons, blades and domains). The nomenclature adopted in the text for structure elements is “domain-blade-structural element”, e.g. βD1-VI-β3 or βD2-IV-β-ribbon. (c) Structural calcium-binding site framed by segment D520—D530 within loop Lβ3β4 of βD1-blade IV. The ion is octahedrally coordinated by D520O, T523O, T523Oγ, T526O, D529Oδ1 and D530Oδ1, which are at binding distances of ~2.4 Å.
Mentions: We produced PorZ without its predicted signal peptide (residues Q26-R776) by recombinant overexpression in Escherichia coli, and succeeded in crystallizing and solving its structure by single-wavelength anomalous diffraction with a selenomethionine derivative. The structure was refined with data to 2.9 Å resolution and consists of three domains. The first two are consecutive N-terminal seven-stranded β-propeller or circular-leaflet moieties (βD1: residues K39—M322; and βD2: G29—L34+Y335—T679 373839; PorZ residue numbering in superscript notation according to UP Q9S3Q8), each featuring a shallow cylinder or thick disk with an “entry side” and an “exit side”38. These domains are succeeded by a C-terminal domain (CTD, V692—R776). The domains are connected by linkers (L): LβD2-βD1 (L35—H38), LβD1-βD2 (P323—F334), and LβD2-CTD (G680—G691). The two propellers are offset from one another by a ~90° rotation about the intersection axis of the propellers’ planes. This causes the overall molecular structure to be reminiscent of an easy chair of approx. maximal dimensions 95 × 80 × 55 Å, with βD1 as the seat, βD2 the backrest, and CTD the backrest support (Fig. 5a). The two entry-side surfaces of the PorZ propellers mimic, respectively, the seating and reclining surfaces of the chair.

View Article: PubMed Central - PubMed

ABSTRACT

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.

No MeSH data available.


Related in: MedlinePlus