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Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase.

Goulas T, Mizgalska D, Garcia-Ferrer I, Kantyka T, Guevara T, Szmigielski B, Sroka A, Millán C, Usón I, Veillard F, Potempa B, Mydel P, Solà M, Potempa J, Gomis-Rüth FX - Sci Rep (2015)

Bottom Line: RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis.Catalysis is based on a cysteine-histidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes.We provide a working mechanism hypothesis based on 18 structure-derived point mutants.

View Article: PubMed Central - PubMed

Affiliation: Proteolysis Lab; Department of Structural Biology ("María de Maeztu" Unit of Excellence); Molecular Biology Institute of Barcelona, CSIC; Barcelona Science Park, Helix Building; c/Baldiri Reixac, 15-21; E-08028 Barcelona Spain.

ABSTRACT
Citrullination is a post-translational modification of higher organisms that deiminates arginines in proteins and peptides. It occurs in physiological processes but also pathologies such as multiple sclerosis, fibrosis, Alzheimer's disease and rheumatoid arthritis (RA). The reaction is catalyzed by peptidylarginine deiminases (PADs), which are found in vertebrates but not in lower organisms. RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis. Uniquely among microbes, P. gingivalis secretes a PAD, termed PPAD (Porphyromonas peptidylarginine deiminase), which is genetically unrelated to eukaryotic PADs. Here, we studied function of PPAD and its substrate-free, substrate-complex, and substrate-mimic-complex structures. It comprises a flat cylindrical catalytic domain with five-fold α/β-propeller architecture and a C-terminal immunoglobulin-like domain. The PPAD active site is a funnel located on one of the cylinder bases. It accommodates arginines from peptide substrates after major rearrangement of a "Michaelis loop" that closes the cleft. The guanidinium and carboxylate groups of substrates are tightly bound, which explains activity of PPAD against arginines at C-termini but not within peptides. Catalysis is based on a cysteine-histidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes. We provide a working mechanism hypothesis based on 18 structure-derived point mutants.

No MeSH data available.


Related in: MedlinePlus

Overall structure and topology of PPAD.(A) Ribbon-type plot of PPAD in a lateral view revealing its tooth-like shape, which consists of regions assignable to cusp, crown, neck and root. The upper N-terminal cylindrical catalytic domain (CD; residues 44–359; top entry base and bottom exit base) is shown with the N-terminal segment in yellow and each of its constituting blades (I to V) in one color (blue, magenta, orange, red, and green). The C-terminal IgSF-like domain (residues 360–465) is shown in grey for its β-strands (labeled β22-β29) and white for loops and coils. A sodium ion is shown as a blue sphere and a black arrow pinpoints the Michaelis-loop. (B) Top view onto the entry base of the CD cylinder after a horizontal 90°-rotation of (A). The helices (α1-α8) and strands (β1-β20) of the CD are labeled. Catalytic-triad-residue (C351, H236 and N297) side chains are shown and labeled in red to highlight the active site in the center of the α/β-propeller. A black arrow pinpoints the Michaelis-loop. (C) Topology scheme of the five-bladed PPAD CD with strands as arrows and helices as cylinders with their respective limiting residues; coloring as in panels (A) and (B). The three catalytic residues of (B) are shown as pink asterisks, and the Michaelis-loop is denoted by a black arrow.
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f1: Overall structure and topology of PPAD.(A) Ribbon-type plot of PPAD in a lateral view revealing its tooth-like shape, which consists of regions assignable to cusp, crown, neck and root. The upper N-terminal cylindrical catalytic domain (CD; residues 44–359; top entry base and bottom exit base) is shown with the N-terminal segment in yellow and each of its constituting blades (I to V) in one color (blue, magenta, orange, red, and green). The C-terminal IgSF-like domain (residues 360–465) is shown in grey for its β-strands (labeled β22-β29) and white for loops and coils. A sodium ion is shown as a blue sphere and a black arrow pinpoints the Michaelis-loop. (B) Top view onto the entry base of the CD cylinder after a horizontal 90°-rotation of (A). The helices (α1-α8) and strands (β1-β20) of the CD are labeled. Catalytic-triad-residue (C351, H236 and N297) side chains are shown and labeled in red to highlight the active site in the center of the α/β-propeller. A black arrow pinpoints the Michaelis-loop. (C) Topology scheme of the five-bladed PPAD CD with strands as arrows and helices as cylinders with their respective limiting residues; coloring as in panels (A) and (B). The three catalytic residues of (B) are shown as pink asterisks, and the Michaelis-loop is denoted by a black arrow.

Mentions: The PPAD two-domain moiety (CD plus IgSF; Fig. 1) shows approximate maximal dimensions of 55 Å(height)×57 Å(width)×50 Å(depth) according to the orientation of Fig. 1a and lacks any bound calcium ion, thus explaining why it is not needed for activity. Overall, it resembles a tooth—with the 316-residue CD featuring the crown and IgSF the root—, which is reminiscent of the gross overall shape of Kgp and RgpB despite completely different functions and CD architectures (see Fig. 2b in22 and Fig. 2a in23). The neck is the interface between the two domains, and the active site is at the cusp, on the grinding surface (see below). The CD (A44-K359; see Fig. 1a–c) comprises eight helices and 20 β-strands and is a flat cylinder made up by a distorted five-fold α/β-propeller arranged around a central shaft. The PPAD CD cylinder has an upper entry base, which coincides with the tooth cusp, and an opposite lower exit base at the neck (Fig. 1a). Around the shaft, five propeller blades (I to V) spanning between 47 (blade III) and 76 (blade I) residues are sequentially arranged counterclockwise according to Fig. 1b,c. Each blade starts on the entry base with a loop connected to the previous blade and consists at least of a three-stranded twisted β-sheet with an inner, a middle and an outer strand, plus one helix. The inner strand runs across the cylinder to the exit base paralleling the central shaft. A short loop links the inner strand with the antiparallel middle strand, which runs in the opposite direction towards the entry base. This strand is connected through another loop with the helix, which lines the cylinder side wall. Finally, the helix is linked to the outer strand, which parallels the middle strand and likewise lines the cylinder side wall. Into this minimal architecture—found only in blade V (Fig. 1c)—, additional structural elements are inserted in each blade, thus accounting for overall blade asymmetry and chain lengths. In particular, a sodium ion is pinched by the inner strand and the consensus helix of blade II and is bound in an octahedral manner by six oxygens at distances of 2.30–2.63 Å: D148O, D158O, and two solvent molecules coplanar with the cation; and apically by D147Oδ1 and D158Oδ1.


Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase.

Goulas T, Mizgalska D, Garcia-Ferrer I, Kantyka T, Guevara T, Szmigielski B, Sroka A, Millán C, Usón I, Veillard F, Potempa B, Mydel P, Solà M, Potempa J, Gomis-Rüth FX - Sci Rep (2015)

Overall structure and topology of PPAD.(A) Ribbon-type plot of PPAD in a lateral view revealing its tooth-like shape, which consists of regions assignable to cusp, crown, neck and root. The upper N-terminal cylindrical catalytic domain (CD; residues 44–359; top entry base and bottom exit base) is shown with the N-terminal segment in yellow and each of its constituting blades (I to V) in one color (blue, magenta, orange, red, and green). The C-terminal IgSF-like domain (residues 360–465) is shown in grey for its β-strands (labeled β22-β29) and white for loops and coils. A sodium ion is shown as a blue sphere and a black arrow pinpoints the Michaelis-loop. (B) Top view onto the entry base of the CD cylinder after a horizontal 90°-rotation of (A). The helices (α1-α8) and strands (β1-β20) of the CD are labeled. Catalytic-triad-residue (C351, H236 and N297) side chains are shown and labeled in red to highlight the active site in the center of the α/β-propeller. A black arrow pinpoints the Michaelis-loop. (C) Topology scheme of the five-bladed PPAD CD with strands as arrows and helices as cylinders with their respective limiting residues; coloring as in panels (A) and (B). The three catalytic residues of (B) are shown as pink asterisks, and the Michaelis-loop is denoted by a black arrow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487231&req=5

f1: Overall structure and topology of PPAD.(A) Ribbon-type plot of PPAD in a lateral view revealing its tooth-like shape, which consists of regions assignable to cusp, crown, neck and root. The upper N-terminal cylindrical catalytic domain (CD; residues 44–359; top entry base and bottom exit base) is shown with the N-terminal segment in yellow and each of its constituting blades (I to V) in one color (blue, magenta, orange, red, and green). The C-terminal IgSF-like domain (residues 360–465) is shown in grey for its β-strands (labeled β22-β29) and white for loops and coils. A sodium ion is shown as a blue sphere and a black arrow pinpoints the Michaelis-loop. (B) Top view onto the entry base of the CD cylinder after a horizontal 90°-rotation of (A). The helices (α1-α8) and strands (β1-β20) of the CD are labeled. Catalytic-triad-residue (C351, H236 and N297) side chains are shown and labeled in red to highlight the active site in the center of the α/β-propeller. A black arrow pinpoints the Michaelis-loop. (C) Topology scheme of the five-bladed PPAD CD with strands as arrows and helices as cylinders with their respective limiting residues; coloring as in panels (A) and (B). The three catalytic residues of (B) are shown as pink asterisks, and the Michaelis-loop is denoted by a black arrow.
Mentions: The PPAD two-domain moiety (CD plus IgSF; Fig. 1) shows approximate maximal dimensions of 55 Å(height)×57 Å(width)×50 Å(depth) according to the orientation of Fig. 1a and lacks any bound calcium ion, thus explaining why it is not needed for activity. Overall, it resembles a tooth—with the 316-residue CD featuring the crown and IgSF the root—, which is reminiscent of the gross overall shape of Kgp and RgpB despite completely different functions and CD architectures (see Fig. 2b in22 and Fig. 2a in23). The neck is the interface between the two domains, and the active site is at the cusp, on the grinding surface (see below). The CD (A44-K359; see Fig. 1a–c) comprises eight helices and 20 β-strands and is a flat cylinder made up by a distorted five-fold α/β-propeller arranged around a central shaft. The PPAD CD cylinder has an upper entry base, which coincides with the tooth cusp, and an opposite lower exit base at the neck (Fig. 1a). Around the shaft, five propeller blades (I to V) spanning between 47 (blade III) and 76 (blade I) residues are sequentially arranged counterclockwise according to Fig. 1b,c. Each blade starts on the entry base with a loop connected to the previous blade and consists at least of a three-stranded twisted β-sheet with an inner, a middle and an outer strand, plus one helix. The inner strand runs across the cylinder to the exit base paralleling the central shaft. A short loop links the inner strand with the antiparallel middle strand, which runs in the opposite direction towards the entry base. This strand is connected through another loop with the helix, which lines the cylinder side wall. Finally, the helix is linked to the outer strand, which parallels the middle strand and likewise lines the cylinder side wall. Into this minimal architecture—found only in blade V (Fig. 1c)—, additional structural elements are inserted in each blade, thus accounting for overall blade asymmetry and chain lengths. In particular, a sodium ion is pinched by the inner strand and the consensus helix of blade II and is bound in an octahedral manner by six oxygens at distances of 2.30–2.63 Å: D148O, D158O, and two solvent molecules coplanar with the cation; and apically by D147Oδ1 and D158Oδ1.

Bottom Line: RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis.Catalysis is based on a cysteine-histidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes.We provide a working mechanism hypothesis based on 18 structure-derived point mutants.

View Article: PubMed Central - PubMed

Affiliation: Proteolysis Lab; Department of Structural Biology ("María de Maeztu" Unit of Excellence); Molecular Biology Institute of Barcelona, CSIC; Barcelona Science Park, Helix Building; c/Baldiri Reixac, 15-21; E-08028 Barcelona Spain.

ABSTRACT
Citrullination is a post-translational modification of higher organisms that deiminates arginines in proteins and peptides. It occurs in physiological processes but also pathologies such as multiple sclerosis, fibrosis, Alzheimer's disease and rheumatoid arthritis (RA). The reaction is catalyzed by peptidylarginine deiminases (PADs), which are found in vertebrates but not in lower organisms. RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis. Uniquely among microbes, P. gingivalis secretes a PAD, termed PPAD (Porphyromonas peptidylarginine deiminase), which is genetically unrelated to eukaryotic PADs. Here, we studied function of PPAD and its substrate-free, substrate-complex, and substrate-mimic-complex structures. It comprises a flat cylindrical catalytic domain with five-fold α/β-propeller architecture and a C-terminal immunoglobulin-like domain. The PPAD active site is a funnel located on one of the cylinder bases. It accommodates arginines from peptide substrates after major rearrangement of a "Michaelis loop" that closes the cleft. The guanidinium and carboxylate groups of substrates are tightly bound, which explains activity of PPAD against arginines at C-termini but not within peptides. Catalysis is based on a cysteine-histidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes. We provide a working mechanism hypothesis based on 18 structure-derived point mutants.

No MeSH data available.


Related in: MedlinePlus