The heme-sensitive regulator SbnI has a bifunctional role in staphyloferrin B production by Staphylococcus aureus

Abstract

Staphylococcus aureus infection relies on iron acquisition from its host. S. aureus takes up iron through heme uptake by the iron-responsive surface determinant (Isd) system and by the production of iron-scavenging siderophores. Staphyloferrin B (SB) is a siderophore produced by the 9-gene sbn gene cluster for SB biosynthesis and efflux. Recently, the ninth gene product, SbnI, was determined to be a free l-serine kinase that produces O-phospho-l-serine (OPS), a substrate for SB biosynthesis. Previous studies have also characterized SbnI as a DNA-binding regulatory protein that senses heme to control sbn gene expression for SB synthesis. Here, we present crystal structures at 1.9-2.1 Å resolution of a SbnI homolog from Staphylococcus pseudintermedius (SpSbnI) in both apo form and in complex with ADP, a product of the kinase reaction; the latter confirmed the active-site location. The structures revealed that SpSbnI forms a dimer through C-terminal domain swapping and a dimer of dimers through intermolecular disulfide formation. Heme binding had only a modest effect on SbnI enzymatic activity, suggesting that its two functions are independent and structurally distinct. We identified a heme-binding site and observed catalytic heme transfer between a heme-degrading protein of the Isd system, IsdI, and SbnI. These findings support the notion that SbnI has a bifunctional role contributing precursor OPS to SB synthesis and directly sensing heme to control expression of the sbn locus. We propose that heme transfer from IsdI to SbnI enables S. aureus to control iron source preference according to the sources available in the environment.

Keywords: crystal structure; heme; iron metabolism; kinetics; siderophore.

Figure 1.

Overall structure of SpSbnI. a, SpSbnI monomer colored by domain: N-terminal domain I (yellow), domain II (green), and C-terminal dimerization domain III (purple). b, SpSbnI crystallographic dimer formed by C-terminal domain swapping. c, SpSbnI dimer of dimers linked by two pairs of intermolecular disulfides.

Figure 2.

SpSbnI disulfide bonds and conservation with S. aureus SbnI. a, intermolecular and b, intramolecular disulfide bonds of SpSbnI. F_o–F_c electron density omit maps are contoured at 3σ. SpSbnI residues are drawn as sticks with separate protomers colored teal or pink. Oxygen, nitrogen, and sulfur atoms colored red, blue, and yellow, respectively. c, Superimposition of SbnI^1–240 (blue, residue labels in bold font) and SpSbnI (teal and pink) homologous strands at the dimer of dimers interface seen in the SpSbnI crystal structure. d, a superimposition of SbnI^1–240 (blue, residue labels in bold font) and SpSbnI (teal), where SpSbnI forms an intramolecular bond between Cys⁹⁹ and Cys¹⁵⁶. Cysteine residues are represented as sticks with carbon colored teal, blue, or light pink and sulfur atoms colored yellow.

Figure 3.

Structure of SpSbnI bound to ADP. a, SpSbnI crystallographic dimer formed by intermolecular disulfide bonds. Protomer A is colored light blue and protomer B is yellow. b, SpSbnI dimer of dimers formed by C-terminal domain swapping. c and d, F_o–F_c electron density omit maps (contoured to 3σ) of active sites for protomer A and B. SpSbnI residues are drawn as sticks with separate protomers colored light blue or yellow. ADP carbon is colored gray, water molecules are red, and magnesium ions are green. Oxygen, nitrogen, and phosphorus atoms colored red, blue, and orange, respectively.

Figure 4.

Amino acid conservation and surface electrostatics of SpSbnI. a, conservation pattern of the SpSbnI protomer and dimer generated using ConSurf. The color-coding bar shows the coloring scheme; conserved amino acids are colored bordeaux, residues of average conservation are white, and variable amino acids are turquoise. b, electrostatic potential mapped on the SpSbnI protomer and dimer molecular surface; a blue color indicates regions of positive potential (> +5 kT/e), whereas red represents negative potential (< −5 kT/e) values.

Figure 5.

UV-visible absorption and MCD spectra of heme-bound SbnI and heme-bound SpSbnI in the oxidized, reduced, and reduced plus CO-bound forms. UV-visible absorption spectrum of 5 μm (a) S. aureus SbnI or (g) SpSbnI mixed with equimolar heme. d, MCD spectrum of 10 μm SbnI mixed with 0.8 eq heme. UV-visible absorption spectrum of 5 μm (b) SbnI or (h) SpSbnI incubated with equimolar heme immediately after reduction with dithionite. e, MCD spectrum of 10 μm SbnI incubated with 0.8 eq heme immediately after reduction with DTT. UV-visible absorption spectrum of 5 μm (c) SbnI or (i) SpSbnI with equimolar heme after exposure to CO and subsequent reduction with dithionite. f, MCD spectrum of 10 μm SbnI with 0.8 eq heme after reduction with DTT and exposure to CO. All reactions were carried out in 50 mm HEPES (pH 7.4), 100 mm NaCl, and 5% (v/v) glycerol.

Figure 6.

Fraction of heme transferred from holo-IsdI to SbnI and SbnI variants. Quantification of IsdI heme transfer to SbnI variants was determined using a pulldown assay where strep-tagged holo-IsdI was bound to streptactin resin. SbnI variant or buffer (as no transfer control) was added, incubated for 1 min, separated by centrifugation, and the supernatant was removed. The fraction of IsdI heme transferred was calculated as the amount of holo-IsdI eluted when incubated with buffer compared with incubation with the SbnI variant. The amount of holo-IsdI was calculated based on the A₄₁₂ to A₂₈₀ ratio of the IsdI eluent. No heme transfer is defined as the A₄₁₂/A₂₈₀ of holo-IsdI incubated with buffer. Statistics are calculated based on multiple comparisons with WT SbnI. **, p value < 0.0021; ***, p value < 0.0002.