Limits...
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

Overall structure ofMtbHadAB complex. (A) Cartoon representation of the structure of MtbHadAB hetero-dimer is shown. HadA (red color) sits antiparallel to HadB (blue color). (B) Topology diagram of the structure of HadA and HadB. A short pair of strands in HadA was undefined in the native structure because of poor density. (C) Differences in positioning of αHD of HadA and HadB are shown. Direction of the axis of the β-sheet w.r.t. αHD is shown with arrows. Difference in conformation of loop connecting β2 with αHD is pointed using a magenta colored arrow. (D) Location of pockets in the active site of HadB is shown. The fatty acid binding channel and a vertical cavity traversing the fatty acid binding channel is shown in grey colored transparent surface representation. Location of catalytic dyad, His41-Asp36, is shown. Cavities were identified using CAVER software
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4491049&req=5

Fig3: Overall structure ofMtbHadAB complex. (A) Cartoon representation of the structure of MtbHadAB hetero-dimer is shown. HadA (red color) sits antiparallel to HadB (blue color). (B) Topology diagram of the structure of HadA and HadB. A short pair of strands in HadA was undefined in the native structure because of poor density. (C) Differences in positioning of αHD of HadA and HadB are shown. Direction of the axis of the β-sheet w.r.t. αHD is shown with arrows. Difference in conformation of loop connecting β2 with αHD is pointed using a magenta colored arrow. (D) Location of pockets in the active site of HadB is shown. The fatty acid binding channel and a vertical cavity traversing the fatty acid binding channel is shown in grey colored transparent surface representation. Location of catalytic dyad, His41-Asp36, is shown. Cavities were identified using CAVER software

Mentions: The crystal structure of MtbHadAB complex was solved by single wavelength anomalous dispersion (SAD) method using X-ray diffraction data collected from crystals of seleno-methionine labeled protein. The crystal belonged to space group P41212, with unit-cell parameters a = b = 82.0 Å, c = 139.8 Å, α = β = γ = 90.0°. A Matthews coefficient of 3.56 Å3 Da−1 (Matthews, 1968; Potterton et al., 2003), corresponding to a solvent content of 65.49%, coupled with the previous biophysical identification indicated the presence of both one molecule of HadA and HadB per asymmetric unit. The final model encompassing residues 3–146 of HadA and residues 1–142 of HadB was refined to 1.75 Å resolution with an Rwork (Rfree) value of 15.7% (18.5%) (Fig. 3A, Table S1).Figure 2


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)

Overall structure ofMtbHadAB complex. (A) Cartoon representation of the structure of MtbHadAB hetero-dimer is shown. HadA (red color) sits antiparallel to HadB (blue color). (B) Topology diagram of the structure of HadA and HadB. A short pair of strands in HadA was undefined in the native structure because of poor density. (C) Differences in positioning of αHD of HadA and HadB are shown. Direction of the axis of the β-sheet w.r.t. αHD is shown with arrows. Difference in conformation of loop connecting β2 with αHD is pointed using a magenta colored arrow. (D) Location of pockets in the active site of HadB is shown. The fatty acid binding channel and a vertical cavity traversing the fatty acid binding channel is shown in grey colored transparent surface representation. Location of catalytic dyad, His41-Asp36, is shown. Cavities were identified using CAVER software
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4491049&req=5

Fig3: Overall structure ofMtbHadAB complex. (A) Cartoon representation of the structure of MtbHadAB hetero-dimer is shown. HadA (red color) sits antiparallel to HadB (blue color). (B) Topology diagram of the structure of HadA and HadB. A short pair of strands in HadA was undefined in the native structure because of poor density. (C) Differences in positioning of αHD of HadA and HadB are shown. Direction of the axis of the β-sheet w.r.t. αHD is shown with arrows. Difference in conformation of loop connecting β2 with αHD is pointed using a magenta colored arrow. (D) Location of pockets in the active site of HadB is shown. The fatty acid binding channel and a vertical cavity traversing the fatty acid binding channel is shown in grey colored transparent surface representation. Location of catalytic dyad, His41-Asp36, is shown. Cavities were identified using CAVER software
Mentions: The crystal structure of MtbHadAB complex was solved by single wavelength anomalous dispersion (SAD) method using X-ray diffraction data collected from crystals of seleno-methionine labeled protein. The crystal belonged to space group P41212, with unit-cell parameters a = b = 82.0 Å, c = 139.8 Å, α = β = γ = 90.0°. A Matthews coefficient of 3.56 Å3 Da−1 (Matthews, 1968; Potterton et al., 2003), corresponding to a solvent content of 65.49%, coupled with the previous biophysical identification indicated the presence of both one molecule of HadA and HadB per asymmetric unit. The final model encompassing residues 3–146 of HadA and residues 1–142 of HadB was refined to 1.75 Å resolution with an Rwork (Rfree) value of 15.7% (18.5%) (Fig. 3A, Table S1).Figure 2

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