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. 2016 Mar 14:6:22945.
doi: 10.1038/srep22945.

Structure of the fimbrial protein Mfa4 from Porphyromonas gingivalis in its precursor form: implications for a donor-strand complementation mechanism

Patrik Kloppsteck  1 , Michael Hall  1 , Yoshiaki Hasegawa  2 , Karina Persson  1
Affiliations

Structure of the fimbrial protein Mfa4 from Porphyromonas gingivalis in its precursor form: implications for a donor-strand complementation mechanism

Patrik Kloppsteck et al. Sci Rep. .

Abstract

Gingivitis and periodontitis are chronic inflammatory diseases that can lead to tooth loss. One of the causes of these diseases is the Gram-negative Porphyromonas gingivalis. This periodontal pathogen is dependent on two fimbriae, FimA and Mfa1, for binding to dental biofilm, salivary proteins, and host cells. These fimbriae are composed of five proteins each, but the fimbriae assembly mechanism and ligands are unknown. Here we reveal the crystal structure of the precursor form of Mfa4, one of the accessory proteins of the Mfa1 fimbria. Mfa4 consists of two β-sandwich domains and the first part of the structure forms two well-defined β-strands that run over both domains. This N-terminal region is cleaved by gingipains, a family of proteolytic enzymes that encompass arginine- and lysine-specific proteases. Cleavage of the N-terminal region generates the mature form of the protein. Our structural data allow us to propose that the new N-terminus of the mature protein may function as a donor strand in the polymerization of P. gingivalis fimbriae.

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Figures

Figure 1
Figure 1. Schematic diagrams of the Mfa1 gene cluster and the Mfa4 protein.
(a) The five genes that encode the Mfa1 fimbrial proteins; Mfa1 forms the shaft of the protein, Mfa2 regulates the length of the fimbriae and Mfa3-5 are tip proteins. (b) Mfa4 starts with an 18 aa long signal sequence followed by a lipidated cysteine. The mature form of the protein starts at Asn54 after gingipain cleavage at Arg53. Residues 19–53 are referred to as the N-terminal extension.
Figure 2
Figure 2. Overall structure of Mfa4.
(a) Schematic representation of Mfa4 structure in which the N-domain is depicted in light blue and the C-domain in dark blue. The N-terminal extension is coloured red. The RGP cleavage site is indicated. (b) Topology diagram of Mfa4. β-strands are represented as arrows, helices as rectangles and loops as lines. Colours are the same as in (a).
Figure 3
Figure 3. The N-terminal extension of Mfa4.
(a) The N-terminal extension shown as a stick model where the rest of Mfa4 is presented as an electrostatic surface. (b) The Mfa4 N-terminal extension is modelled in an Fo-Fc simulated-annealing omit-difference map contoured at 2.5 sigma and shown in stereo.
Figure 4
Figure 4. Structural features of Mfa4 and related proteins.
Mfa4, the putative cell adhesin protein from B. eggerthii (4gpv), and FimA from P. gingivalis W83 are compared. Their N-terminal extensions, N-, and C-domains are colored red, cyan and purple, respectively. The C-terminal strands of 4gpv and FimA are shown in green. The RGP cleavage site is marked with scissors.
Figure 5
Figure 5. Structure-based sequence alignment of the N-terminal region.
The N-terminal sequences of Mfa4 and structurally related proteins with an exposed arginine in the β1β2-loop. The conserved arginine is depicted in red. Hydrophobic amino acids in the β1/β1a strand are depicted in blue and highlighted in grey. Horizontal arrows indicate β-strands (β1b is only present in Mfa4). The location of the N-terminal extension is indicated.
Figure 6
Figure 6. Mfa4 expression and immunoblot analysis of the point mutants.
(a) Immunoblot analysis of Mfa4 protein. The whole-cell lysates were separated on SDS-PAGE and probed with an Mfa4 antibody by immunoblotting. Lanes: 1, JI-1; 2, JI-1 Mfa4WT; 3, JI-1 R53A; 4, JI-1 R53K; 5, JI-1 R50A/R53A; 6, KDP112(ΔrgpA/B); 7, FMFA4 (Δmfa4, negative control) (b) Immunoblot analysis of Mfa4 from purified Mfa1 fimbriae. Lanes: 1 JI-1; 2, JI-1 R53A; 3, JI-1R53K; 4, JI-1 R50A/R53A; 5, FMFA4. (C) SDS–PAGE of the purified Mfa1 fimbriae. Lanes: 1, JI-1; 2, JI-1 R53A; 3, JI-1 R53K; 4, JI-1 R50A/R53A; 5, FMFA4. Asterisks indicate the Mfa4 bands analysed by N-terminal sequencing.
Figure 7
Figure 7. Proposed polymerization model of Mfa1 fimbria based on a donor strand complementation mechanism where the Nmature strand is inserted into a neighboring Mfa protein.
The fimbrial proteins are transported to the inner membrane via the Sec system and to the outer membrane via the Lol pathway (Mfa1, Mfa3 and Mfa4). The accessory protein Mfa5 is transported to the outer membrane via the type IX secretion system.
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