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Molecular basis for the inhibition of β-hydroxyacyl-ACP dehydratase HadAB complex from Mycobacterium tuberculosis by flavonoid inhibitors.

Dong Y, Qiu X, Shaw N, Xu Y, Sun Y, Li X, Li J, Rao Z - Protein Cell (2015)

Bottom Line: We show that inhibitors bind in this cavity and protrude into the substrate binding channel.Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site.The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.

ABSTRACT
Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobacterium tuberculosis (Mtb). Consequently, stalling dehydration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxyacyl-ACP dehydratase HadAB complex. How this inhibition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2',4,4'-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon mutation to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site. The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.

No MeSH data available.


Related in: MedlinePlus

Binding of flavonoids toMtbHadAB complex. (A) KD values determined for the binding of flavonoids to MtbHadAB complex by ITC. (B) Different flavonoids tested in the current study (shown as sticks) bind at the same location on MtbHadAB. Upon superimposition of the structures, the ligands overlap. They bind in the cavity (right panel, gray colored surface representation) located just beneath the fatty acid binding channel. An example of butein (BUN; cyan sticks, right panel) bound in the cavity is shown. (C) Nature of amino acid interacting with flavonoids is shown. Flavonoids and amino acids are shown as sticks, while water is depicted as spheres. 2Fo-Fc electron density contoured at 1σ for the three ligands is shown
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Fig5: Binding of flavonoids toMtbHadAB complex. (A) KD values determined for the binding of flavonoids to MtbHadAB complex by ITC. (B) Different flavonoids tested in the current study (shown as sticks) bind at the same location on MtbHadAB. Upon superimposition of the structures, the ligands overlap. They bind in the cavity (right panel, gray colored surface representation) located just beneath the fatty acid binding channel. An example of butein (BUN; cyan sticks, right panel) bound in the cavity is shown. (C) Nature of amino acid interacting with flavonoids is shown. Flavonoids and amino acids are shown as sticks, while water is depicted as spheres. 2Fo-Fc electron density contoured at 1σ for the three ligands is shown

Mentions: Flavonoids like butein (BUN), 2’,4,4’-trihydroxychalcone (HCC) and fisetin (FSE), are known to stall biosynthesis of mycolic acids by mycobacterial FAS-II system (Fig. 1). They have been shown to particularly inhibit the activity of HadB (Brown et al. 2007b). Consequently, these flavonoids inhibit the growth of Mycobacterium bovis BCG, demonstrating their utility in development of anti-TB drugs. How these flavonoids inhibit HadB is currently not understood. Results of our biophysical studies on HadA and HadB show that HadB forms a tight hetero-dimeric complex with HadA. Therefore, these flavonoids probably target the MtbHadAB complex for inhibiting mycolic acid synthesis. We tested the binding of these flavonoids to the MtbHadAB complex using an isothermal titration calorimetry (ITC) based assay. All the three flavonoids bound the MtbHadAB complex with a KD value in the 10–15 μmol/L range (Fig. 5A).Figure 5


Molecular basis for the inhibition of β-hydroxyacyl-ACP dehydratase HadAB complex from Mycobacterium tuberculosis by flavonoid inhibitors.

Dong Y, Qiu X, Shaw N, Xu Y, Sun Y, Li X, Li J, Rao Z - Protein Cell (2015)

Binding of flavonoids toMtbHadAB complex. (A) KD values determined for the binding of flavonoids to MtbHadAB complex by ITC. (B) Different flavonoids tested in the current study (shown as sticks) bind at the same location on MtbHadAB. Upon superimposition of the structures, the ligands overlap. They bind in the cavity (right panel, gray colored surface representation) located just beneath the fatty acid binding channel. An example of butein (BUN; cyan sticks, right panel) bound in the cavity is shown. (C) Nature of amino acid interacting with flavonoids is shown. Flavonoids and amino acids are shown as sticks, while water is depicted as spheres. 2Fo-Fc electron density contoured at 1σ for the three ligands is shown
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig5: Binding of flavonoids toMtbHadAB complex. (A) KD values determined for the binding of flavonoids to MtbHadAB complex by ITC. (B) Different flavonoids tested in the current study (shown as sticks) bind at the same location on MtbHadAB. Upon superimposition of the structures, the ligands overlap. They bind in the cavity (right panel, gray colored surface representation) located just beneath the fatty acid binding channel. An example of butein (BUN; cyan sticks, right panel) bound in the cavity is shown. (C) Nature of amino acid interacting with flavonoids is shown. Flavonoids and amino acids are shown as sticks, while water is depicted as spheres. 2Fo-Fc electron density contoured at 1σ for the three ligands is shown
Mentions: Flavonoids like butein (BUN), 2’,4,4’-trihydroxychalcone (HCC) and fisetin (FSE), are known to stall biosynthesis of mycolic acids by mycobacterial FAS-II system (Fig. 1). They have been shown to particularly inhibit the activity of HadB (Brown et al. 2007b). Consequently, these flavonoids inhibit the growth of Mycobacterium bovis BCG, demonstrating their utility in development of anti-TB drugs. How these flavonoids inhibit HadB is currently not understood. Results of our biophysical studies on HadA and HadB show that HadB forms a tight hetero-dimeric complex with HadA. Therefore, these flavonoids probably target the MtbHadAB complex for inhibiting mycolic acid synthesis. We tested the binding of these flavonoids to the MtbHadAB complex using an isothermal titration calorimetry (ITC) based assay. All the three flavonoids bound the MtbHadAB complex with a KD value in the 10–15 μmol/L range (Fig. 5A).Figure 5

Bottom Line: We show that inhibitors bind in this cavity and protrude into the substrate binding channel.Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site.The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.

ABSTRACT
Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobacterium tuberculosis (Mtb). Consequently, stalling dehydration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxyacyl-ACP dehydratase HadAB complex. How this inhibition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2',4,4'-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon mutation to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site. The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.

No MeSH data available.


Related in: MedlinePlus