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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

Proposed peptide citrullinating mechanism of PPAD.(A) Composite picture in stereo of the active site of PPAD (see also Fig. 2) based on the substrate-mimic complex ribbon plot colored as in Fig. 2b. Only elements engaged in substrate binding and catalysis are depicted. Residue side chains taken from the substrate-mimic complex are shown with carbons in light blue (C351), those from the substrate complex in white (Y233, H236, D238, N297, R152, R154, and W217), and those from the unbound structure in pink (Y233 and H236). The Michaelis loop is shown in the open conformation of the unbound structure in pink and in the occluded conformation of the substrate(-mimic) complexes in red, a purple straight arrow highlights the rearrangement upon substrate binding. The substrate arginine depicted belongs to the substrate complex (carbons in turquoise). Solvent molecules from the substrate-mimic complex in light blue highlight the NH3-exit/H2O-entry channel on the left and those in purple the hydroxide-entry channel on the right. The rotation of the H236 side chain from the substrate-unbound to the bound conformation is pinpointed by a curved purple arrow. (B) Proposed biochemical mechanism of action of an enzymatic activity cycle in seven steps (I to VII). The substrate arginine and product citrulline are shown with bonds in bold, hydrogen bonds are shown as dashed lines.
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f4: Proposed peptide citrullinating mechanism of PPAD.(A) Composite picture in stereo of the active site of PPAD (see also Fig. 2) based on the substrate-mimic complex ribbon plot colored as in Fig. 2b. Only elements engaged in substrate binding and catalysis are depicted. Residue side chains taken from the substrate-mimic complex are shown with carbons in light blue (C351), those from the substrate complex in white (Y233, H236, D238, N297, R152, R154, and W217), and those from the unbound structure in pink (Y233 and H236). The Michaelis loop is shown in the open conformation of the unbound structure in pink and in the occluded conformation of the substrate(-mimic) complexes in red, a purple straight arrow highlights the rearrangement upon substrate binding. The substrate arginine depicted belongs to the substrate complex (carbons in turquoise). Solvent molecules from the substrate-mimic complex in light blue highlight the NH3-exit/H2O-entry channel on the left and those in purple the hydroxide-entry channel on the right. The rotation of the H236 side chain from the substrate-unbound to the bound conformation is pinpointed by a curved purple arrow. (B) Proposed biochemical mechanism of action of an enzymatic activity cycle in seven steps (I to VII). The substrate arginine and product citrulline are shown with bonds in bold, hydrogen bonds are shown as dashed lines.

Mentions: We propose the following chemical mechanism of function of PPAD, which includes a catalytic triad (C351-H236-N297) and seven steps proceeding over two tetrahedral and one planar-thiouronium covalent reaction intermediates (Fig. 4a,b).


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)

Proposed peptide citrullinating mechanism of PPAD.(A) Composite picture in stereo of the active site of PPAD (see also Fig. 2) based on the substrate-mimic complex ribbon plot colored as in Fig. 2b. Only elements engaged in substrate binding and catalysis are depicted. Residue side chains taken from the substrate-mimic complex are shown with carbons in light blue (C351), those from the substrate complex in white (Y233, H236, D238, N297, R152, R154, and W217), and those from the unbound structure in pink (Y233 and H236). The Michaelis loop is shown in the open conformation of the unbound structure in pink and in the occluded conformation of the substrate(-mimic) complexes in red, a purple straight arrow highlights the rearrangement upon substrate binding. The substrate arginine depicted belongs to the substrate complex (carbons in turquoise). Solvent molecules from the substrate-mimic complex in light blue highlight the NH3-exit/H2O-entry channel on the left and those in purple the hydroxide-entry channel on the right. The rotation of the H236 side chain from the substrate-unbound to the bound conformation is pinpointed by a curved purple arrow. (B) Proposed biochemical mechanism of action of an enzymatic activity cycle in seven steps (I to VII). The substrate arginine and product citrulline are shown with bonds in bold, hydrogen bonds are shown as dashed lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Proposed peptide citrullinating mechanism of PPAD.(A) Composite picture in stereo of the active site of PPAD (see also Fig. 2) based on the substrate-mimic complex ribbon plot colored as in Fig. 2b. Only elements engaged in substrate binding and catalysis are depicted. Residue side chains taken from the substrate-mimic complex are shown with carbons in light blue (C351), those from the substrate complex in white (Y233, H236, D238, N297, R152, R154, and W217), and those from the unbound structure in pink (Y233 and H236). The Michaelis loop is shown in the open conformation of the unbound structure in pink and in the occluded conformation of the substrate(-mimic) complexes in red, a purple straight arrow highlights the rearrangement upon substrate binding. The substrate arginine depicted belongs to the substrate complex (carbons in turquoise). Solvent molecules from the substrate-mimic complex in light blue highlight the NH3-exit/H2O-entry channel on the left and those in purple the hydroxide-entry channel on the right. The rotation of the H236 side chain from the substrate-unbound to the bound conformation is pinpointed by a curved purple arrow. (B) Proposed biochemical mechanism of action of an enzymatic activity cycle in seven steps (I to VII). The substrate arginine and product citrulline are shown with bonds in bold, hydrogen bonds are shown as dashed lines.
Mentions: We propose the following chemical mechanism of function of PPAD, which includes a catalytic triad (C351-H236-N297) and seven steps proceeding over two tetrahedral and one planar-thiouronium covalent reaction intermediates (Fig. 4a,b).

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