Structures of the Porphyromonas gingivalis OxyR regulatory domain explain differences in expression of the OxyR regulon in Escherichia coli and P. gingivalis

Abstract

OxyR transcriptionally regulates Escherichia coli oxidative stress response genes through a reversibly reducible cysteine disulfide biosensor of cellular redox status. Structural changes induced by redox changes in these cysteines are conformationally transmitted to the dimer subunit interfaces, which alters dimer and tetramer interactions with DNA. In contrast to E. coli OxyR regulatory-domain structures, crystal structures of Porphyromonas gingivalis OxyR regulatory domains show minimal differences in dimer configuration on changes in cysteine disulfide redox status. This locked configuration of the P. gingivalis OxyR regulatory-domain dimer closely resembles the oxidized (activating) form of the E. coli OxyR regulatory-domain dimer. It correlates with the observed constitutive activation of some oxidative stress genes in P. gingivalis and is attributable to a single amino-acid insertion in P. gingivalis OxyR relative to E. coli OxyR. Modelling of full-length P. gingivalis, E. coli and Neisseria meningitidis OxyR-DNA complexes predicts different modes of DNA binding for the reduced and oxidized forms of each.

Keywords: OxyR; Porphyromonas gingivalis; regulatory domain.

Figure 1

Stereoview of representative 2F _o − F _c electron density for the disulfide loop and downstream region of (a) oxidized and (b) C199S mutant P. gingivalis OxyR contoured at 1σ. (c) Stereoview of the backbone trace of the disulfide loop of the C199S (cyan) and oxidized wild-­type (red) OxyR RD monomer backbone folds. The two structures are aligned by superposition of the C^α atoms of those residues that fall below a cutoff of 2.0 Å resolution in the unfiltered fit. The r.m.s.d. values are 1.71 Å without rejection and 0.749 Å for the alignment of 186 residues, which excluded residues 186–226. The redox-active cysteines are shown in ball-and-stick representation.

Figure 2

Pairwise structure and sequence alignment of OxyR RD homologues. C^α ribbon-diagram alignment of (a) C199S (red) and oxidized (orange) P. gingivalis OxyR RD monomer sub­units (orange spheres are the disulfide-linked Cys199 and Cys208 in oxidized OxyR RD, and the α-helix of the Cys199–Cys208 disulfide loop in C199S mutant OxyR RD is shown in pink); (b) E. coli C199S OxyR RD (yellow with the Cys199–Cys208 disulfide loop highlighted in green) and oxidized form (magenta); (c) C199S (red) P. gingivalis and reduced (gray) N. meningitidis OxyR RD (the α-helix of the C199S mutant P. gingivalis OxyR RD disulfide loop is shown in pink and the α-helix of the reduced N. meningitidis OxyR RD disulfide loop is shown in blue); (d) oxidized P. gingivalis (orange) and E. coli (magenta) OxyR RD; (e) C199S P. gingivalis (red) and C199S E. coli (yellow) OxyR RD; and (f) C199S E. coli OxyR RD (yellow) and reduced N. meningitidis (gray) OxyR RD. (g) Sequence alignment of E. coli, P. gingivalis and N. meningitidis OxyR (see also Supplementary Fig. S2).

Figure 3

Reversible reduced–oxidized structural transitions around the disulfide loops of the three structurally characterized OxyR RDs. Reduced forms are in the left column and oxidized forms are in the right column. (a) P. gingivalis OxyR RD, (b) E. coli OxyR RD, (c) N. meningitidis OxyR RD (oxidized form modeled). The redox-active cysteines Cys(Ser)199 and Cys208 and some neighboring residues are shown in ball-and-stick representation. Yellow dashed lines show inferred hydrogen bonds and distances between the main-chain C^α atoms of Cys199/Ser199 and Cys208.

Figure 4

Dimer configurations of P. gingivalis and E. coli reduced and oxidized forms. (a, c) Reduced and oxidized dimers of P. gingivalis OxyR show no major changes in the relative orientation of monomers on oxidation/reduction; (b, d) reduced and oxidized dimers of E. coli OxyR showing relative rotational dislocation (30°) of monomers on oxidation/reduction.

Figure 5

Dimer interface of P. gingivalis and E. coli OxyR in reduced and oxidized forms. (a, b) Reduced forms of P. gingivalis and E. coli OxyR showing opposed chains (ball-and-stick representation) at the interface of monomers; (c, d) oxidized forms of P. gingivalis and E. coli OxyR. Color coding and overall orientation is the same as in Fig. 3 ▶. Dashed green lines indicate a hydrogen-bonding network which is absent in the E. coli reduced dimer.

Figure 6

Summary of predicted full-length OxyR dimer (2) and tetramer (4) forms and their DNA complexes from structure-based model building. The exclusion of structures based on unfavorable OxyR–OxyR and OxyR–DNA contacts leaves a unique set of OxyR forms (dimer, tetramer; oxidized, reduced; open, closed) for each of the three species. Ec, E. coli; Pg, P. gingivalis; Nm, N. meningitidis.

Figure 7

Full-length OxyR–DNA complex models. Modeled structures of reduced (left) P. gingivalis OxyR closed tetramer and oxidized (right) dimer bound to DNA. (a) Tetramer (monomer chains color-coded blue, cyan, orange and red) of reduced dimers (cyan with blue and orange with red). These are stabilized by interactions between extended helices of DNA-binding domains (lower left arrow) from a monomer of each dimer (blue with red and orange with cyan) and by interaction of Cys199–Cys208 α-helices between regulatory domains (cyan with red and blue with orange; upper left arrow); (b) oxidized dimer, formed through hydrophobic interactions between extended helices (lower right arrow) in DNA-binding domains, bound to two turns of DNA. Middle arrows indicate the reversible transition from reduced tetramer (left) to oxidized dimer (right).