Molecular insights into the binding of coenzyme F420 to the conserved protein Rv1155 from Mycobacterium tuberculosis

Abstract

Coenzyme F420 is a deazaflavin hydride carrier with a lower reduction potential than most flavins. In Mycobacterium tuberculosis (Mtb), F420 plays an important role in activating PA-824, an antituberculosis drug currently used in clinical trials. Although F420 is important to Mtb redox metabolism, little is known about the enzymes that bind F420 and the reactions that they catalyze. We have identified a novel F420 -binding protein, Rv1155, which is annotated in the Mtb genome sequence as a putative flavin mononucleotide (FMN)-binding protein. Using biophysical techniques, we have demonstrated that instead of binding FMN or other flavins, Rv1155 binds coenzyme F420 . The crystal structure of the complex of Rv1155 and F420 reveals one F420 molecule bound to each monomer of the Rv1155 dimer. Structural, biophysical, and bioinformatic analyses of the Rv1155-F420 complex provide clues about its role in the bacterium.

Keywords: Mycobacterium tuberculosis; Rv1155; coenzyme F420; conserved hypothetical protein.

Figure 1

Coenzyme F₄₂₀ compared to the structures of three other flavins, riboflavin, FMN (flavin mononucleotide), and FAD (flavin adenine dinucleotide). Oxidized F₄₂₀ (upper) is shown. The predominant F₄₂₀ isoforms observed in mycobacteria have polyglutamate tails consisting of glutamates (n = 5 or 6) that are linked by γ-glutamyl bonds., In the lower panel are shown the oxidized structures of riboflavin, FMN, and FAD.

Figure 2

UV–visible absorbance spectra of Rv1155 and Rv2607, members of the Mtb PNPOx family, show that Rv1155 lacks the absorbance profile characteristic of an FMN-binding protein. The UV–visible spectrum of free, oxidized FMN (blue trace) has two peaks in ∼350–500 nm region. The UV–visible spectrum of recombinant Rv2607 (green trace) has two peaks in the same part of the spectrum, which is consistent with Rv2607 being a known FMN-binding protein. Rv1155 (red trace) lacks absorbance in this region of the spectrum, indicating that it does not co-purify with a flavin when expressed recombinantly in E. coli. Free oxidized FMN, Rv2607-FMN, and Rv1155 are present at 30 μM.

Figure 3

Rv1155 binds coenzyme F₄₂₀ as assessed by differential scanning fluorimetry (DSF). (A) DSF melt curves in the presence of F₄₂₀ (red trace) and in the absence of F₄₂₀ (blue trace). Melt curves for FMN, FAD, and NADPH (not shown) were almost identical to the Rv1155 curve in the absence of F₄₂₀. (B) T_m values for Rv1155 in the presence of 50 μM F₄₂₀, FMN, FAD, and NADPH. The change in T_m (ΔT_m) was calculated relative to the Rv1155 only sample. Each measurement was made in triplicate. RFU, relative fluorescence units.

Figure 4

Isothermal titration calorimetry reveals the binding of F₄₂₀ to Rv1155. The ligand, F₄₂₀ (880 μM), was titrated into a solution of the protein, Rv1155 (50 μM as monomer). The top panel shows the ITC thermogram of the raw titration data that were measured for each injection of F₄₂₀. The data points in the bottom panel form the binding isotherm, in which each integrated peak area in units of kcal/mol is plotted against the ratio of the number of moles F₄₂₀ that were injected to the number of moles of Rv115 present in the experiment. The solid line represents the best fit of the isotherm data to a one-binding-site model, which yielded a K_D value of 170 μM for the binding of F₄₂₀ to Rv1155.

Figure 5

Crystal structure of Rv1155 bound to F₄₂₀ at 2.3 Å resolution. (A) Structure of the Rv1155 homodimer with two F₄₂₀ molecules (green, purple) bound at the interface between chain A (cyan) and chain B (tan). (B) Detailed view of binding site 1. (C) Detailed view of binding site 2. Electron density for the ring system of F₄₂₀ is not visible in binding site 2 (see text). The 2F_o–F_c electron density for F₄₂₀ is contoured at 1.0σ. Hydrogen bonds are represented as yellow lines.

Figure 6

The 2F_o–F_c omit electron density of the F₄₂₀ in the crystal structure of Rv1155 contoured at 1.0σ. (A) F₄₂₀ modeled into the density found in the binding site 1 viewed from the interface of chains A and B. (B) F₄₂₀ as viewed after 90° rotation around the horizontal axis relative to panel A. The density indicates that the ring assumes a butterfly conformation, meaning the ring system is not fully planar, but that the pyrimidine and benzene rings of the deazaisoalloxazine ring system are bent below the central ring, which contains C₅ and N₁₀. The butterfly angle is 16.5°, defined as the angle between the plane of the pyrimidine ring and the plane of the benzene ring. The angle was measured using Chimera.

Figure 7

Putative active site in Rv1155–F₄₂₀. Surface representation of the Rv1155 colored by electrostatic potential (red, negative; blue, positive; white, neutral, which corresponds to areas occupied by polar or hydrophobic residues). The dashed line encircles a putative substrate-binding pocket near the F₄₂₀ deazaisoalloxazine ring system, which has a volume of 130 ų.