Streptococcus pneumoniae TIGR4 Flavodoxin: Structural and Biophysical Characterization of a Novel Drug Target

Abstract

Streptococcus pneumoniae (Sp) strain TIGR4 is a virulent, encapsulated serotype that causes bacteremia, otitis media, meningitis and pneumonia. Increased bacterial resistance and limited efficacy of the available vaccine to some serotypes complicate the treatment of diseases associated to this microorganism. Flavodoxins are bacterial proteins involved in several important metabolic pathways. The Sp flavodoxin (Spfld) gene was recently reported to be essential for the establishment of meningitis in a rat model, which makes SpFld a potential drug target. To facilitate future pharmacological studies, we have cloned and expressed SpFld in E. coli and we have performed an extensive structural and biochemical characterization of both the apo form and its active complex with the FMN cofactor. SpFld is a short-chain flavodoxin containing 146 residues. Unlike the well-characterized long-chain apoflavodoxins, the Sp apoprotein displays a simple two-state thermal unfolding equilibrium and binds FMN with moderate affinity. The X-ray structures of the apo and holo forms of SpFld differ at the FMN binding site, where substantial rearrangement of residues at the 91-100 loop occurs to permit cofactor binding. This work will set up the basis for future studies aiming at discovering new potential drugs to treat S. pneumoniae diseases through the inhibition of SpFld.

Fig 1. S. pneumoniae flavodoxin absorption and fluorescence spectra.

(a) UV/VIS absorbance of 39 μM holo (solid line) and 134 μM apoflavodoxin (dashed line) in 150 mM NaCl, 100 mM MES, pH 6.5. (b) Spectra related to three redox states of 43 μM SpFld in 150 mM NaCl, 100 mM MES, pH 6.5 at 10°C: Oxidized (solid line), semiquinone (dashed line) with absorption maxima at 592 nm and reduced (dotted line). (c) Fluorescence emission spectra of 20 μM SpFld holoprotein (solid line) and 20 μM apoprotein (dashed line), in 150 mM NaCl, 100 mM MES, pH 6.5, 10°C.

Fig 2. CD spectra of holo and apo SpFld in 150 mM NaCl, 100 mM MES, pH 6.5, 4°C.

(a) Far-CD spectra of 80 μM of holo and apo SpFld (continuous and dashed lines respectively) (b) Near-CD spectra of 80 μM of holo (continuous line) and apo (dashed line) SpFld. (c) Visible-CD spectrum of 80 μM of holoprotein.

Fig 3. Thermal unfolding curves monitored spectroscopically in 150 mM NaCl, 100 mM MES pH 6.5.

(a) (80 μM) holo SpFld by Fluorescence (open triangles, gray line), far-UV CD (black circles, black line) and near-UV CD (open squares, gray line). (b) (80 μM) apo SpFld by far-UV CD (black circles, black line) and near-UV CD (open squares, gray line).

Fig 4. DSC analysis in the absence of FMN (open squares) or in the presence of 40 μM of FMN (open circles) in 150 mM NaCl, 100 mM MES, pH 6.5.

The continuous line is the non-linear fitting curve obtained by using a two-state model for the unfolding of apoFld.

Fig 5. Titration of 20 μM Apo SpFld with 300 μM FMN at 25°C in 150 mM NaCl, 100 mM MES, pH 6.5.

The upper panel shows the thermogram (thermal power as function of time) and the lower panel shows the binding isotherm (normalized heat as a function of the molar ratio). The continuous line is the non-linear fitting curve obtained using a model considering a single ligand binding site. The inset in the upper panel shows a comparison of the thermodynamic binding profiles obtained by ITC for FMN interacting with apoflavodoxins from S. pneumoniae, Helicobacter pylori, and Anabaena PCC7119: ΔG (black bars) ΔH (gray bars) -TΔS (white bars). Data for the H. pylori and Anabaena complexes come from reference [28]

Fig 6

Ribbon structure representation of apo SpFl in blue (a) and holo SpFld in cyan (b). Changes in the key FMN binding loops indicated in (b) can be observed in the structural superimposition of the holo (cyan) and apo (blue) flavodoxins shown in (c). Also in (c), a red arrow points to a short 3₁₀ helix formed in the holoprotein. The FMN-SpFld hydrogen bonding interactions are highlighted in (d).

Fig 7. Stick representation of the 5-stranded β-sheet of SpFld showing a characteristic bulge in strand 5, which has been attributed [44] to originate in evolutionary splicing of two preexisting short strands, 5a and 5b that, in long-chain flavodoxins, are connected by their characteristic long loop.

Fig 8. Stick representation of the FMN binding site in the Sp apo and holoflavodoxins indicating two key glycine residues (G60 and G62) that close the gap left by FMN in the apoprotein by interacting with residues of the 90’ loop: Y95 and E97.

Fig 9. Stick representation of the aromatic/aromatic pairs established by the FMN sandwiching Y59 and Y95 residues with neighboring Y57 and F94, respectively.

Fig 10. Structural comparison of the short flavodoxins from Desulfovibrio vulgaris (pdb id: 1j8q; ribbon and residues in green) and from Streptococcus pneumoniae (this work, pdb id: 5lji; ribbon and residues in cyan).

The FMN cofactors of the two flavodoxins and the corresponding sandwiching aromatic residues (Y98 and W60 in Dv flavodoxin, and Y59 and Y95 in Sp flavodoxin) are shown in a stick representation.