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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

Dimer interface and active site ofMtbHadAB complex. (A) Position of β2 strands of HadA and HadB with respect to each other is shown. Main chain atoms of both these β-strands interact with each other. (B) Location of β2 strands of HadA and HadB and other structural elements involved in dimerization is shown. (C–E) Nature of intermolecular interactions between different regions of HadA and HadB is shown. Interacting residues are shown as sticks. Distances of all the interactions between HadA and HadB are listed in Table S2. (F) His-Asp catalytic dyad (shown as sticks) contributed by HadB is in vicinity of the fatty acid channel (grey surface representation). Aliphatic carbon chain of a fatty acid molecule modeled into the channel is shown on the right hand side in stick representation. Catalytic waters bonded to His-Asp dyad are shown as spheres (inset). (G) Surface electrostatic potential representation of AcpM (PDB code 1KLP) without showing the C-terminal tail. The surface is predominantly negatively charged. (H) Location of positively charged residues on the rear side of MtbHadAB is shown. (I) Surface electrostatic potential representation of the view shown in panel H. Location of positively charged amino acids contributed by HadA or HadB as well as entrance of fatty acid binding channel are marked. Blue and red colors represent positive and negative potential, respectively
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Fig4: Dimer interface and active site ofMtbHadAB complex. (A) Position of β2 strands of HadA and HadB with respect to each other is shown. Main chain atoms of both these β-strands interact with each other. (B) Location of β2 strands of HadA and HadB and other structural elements involved in dimerization is shown. (C–E) Nature of intermolecular interactions between different regions of HadA and HadB is shown. Interacting residues are shown as sticks. Distances of all the interactions between HadA and HadB are listed in Table S2. (F) His-Asp catalytic dyad (shown as sticks) contributed by HadB is in vicinity of the fatty acid channel (grey surface representation). Aliphatic carbon chain of a fatty acid molecule modeled into the channel is shown on the right hand side in stick representation. Catalytic waters bonded to His-Asp dyad are shown as spheres (inset). (G) Surface electrostatic potential representation of AcpM (PDB code 1KLP) without showing the C-terminal tail. The surface is predominantly negatively charged. (H) Location of positively charged residues on the rear side of MtbHadAB is shown. (I) Surface electrostatic potential representation of the view shown in panel H. Location of positively charged amino acids contributed by HadA or HadB as well as entrance of fatty acid binding channel are marked. Blue and red colors represent positive and negative potential, respectively

Mentions: A close examination of the structure of the MtbHadAB heterodimer reveals that hetero-dimerization is mediated by two types of inter-molecular interactions (Fig. 4). The first type of interactions involves the main chain backbone atoms of the β2 strands from both proteins. HadA and HadB interact with each other via their β2 strands such that the sheets from the two proteins are joined together to form one single contiguous sheet (Fig. 4A and 4B). The second type of inter-molecular interactions is mainly mediated by side chains (Fig. 4C–E; Tables S2 and S3) and is observed between: 1) amino acids of αHD of HadA and the helices α1 and α2, as well as the loop connecting these helices of HadB, 2) helix α1 of HadA and HadB, 3) β2 of HadA with αHD of HadB. PISA analysis revealed that hetero-dimerization of HadA with HadB resulted in burial of 1,453 Å2 surface area of each monomer.Figure 4


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)

Dimer interface and active site ofMtbHadAB complex. (A) Position of β2 strands of HadA and HadB with respect to each other is shown. Main chain atoms of both these β-strands interact with each other. (B) Location of β2 strands of HadA and HadB and other structural elements involved in dimerization is shown. (C–E) Nature of intermolecular interactions between different regions of HadA and HadB is shown. Interacting residues are shown as sticks. Distances of all the interactions between HadA and HadB are listed in Table S2. (F) His-Asp catalytic dyad (shown as sticks) contributed by HadB is in vicinity of the fatty acid channel (grey surface representation). Aliphatic carbon chain of a fatty acid molecule modeled into the channel is shown on the right hand side in stick representation. Catalytic waters bonded to His-Asp dyad are shown as spheres (inset). (G) Surface electrostatic potential representation of AcpM (PDB code 1KLP) without showing the C-terminal tail. The surface is predominantly negatively charged. (H) Location of positively charged residues on the rear side of MtbHadAB is shown. (I) Surface electrostatic potential representation of the view shown in panel H. Location of positively charged amino acids contributed by HadA or HadB as well as entrance of fatty acid binding channel are marked. Blue and red colors represent positive and negative potential, respectively
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Related In: Results  -  Collection

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Fig4: Dimer interface and active site ofMtbHadAB complex. (A) Position of β2 strands of HadA and HadB with respect to each other is shown. Main chain atoms of both these β-strands interact with each other. (B) Location of β2 strands of HadA and HadB and other structural elements involved in dimerization is shown. (C–E) Nature of intermolecular interactions between different regions of HadA and HadB is shown. Interacting residues are shown as sticks. Distances of all the interactions between HadA and HadB are listed in Table S2. (F) His-Asp catalytic dyad (shown as sticks) contributed by HadB is in vicinity of the fatty acid channel (grey surface representation). Aliphatic carbon chain of a fatty acid molecule modeled into the channel is shown on the right hand side in stick representation. Catalytic waters bonded to His-Asp dyad are shown as spheres (inset). (G) Surface electrostatic potential representation of AcpM (PDB code 1KLP) without showing the C-terminal tail. The surface is predominantly negatively charged. (H) Location of positively charged residues on the rear side of MtbHadAB is shown. (I) Surface electrostatic potential representation of the view shown in panel H. Location of positively charged amino acids contributed by HadA or HadB as well as entrance of fatty acid binding channel are marked. Blue and red colors represent positive and negative potential, respectively
Mentions: A close examination of the structure of the MtbHadAB heterodimer reveals that hetero-dimerization is mediated by two types of inter-molecular interactions (Fig. 4). The first type of interactions involves the main chain backbone atoms of the β2 strands from both proteins. HadA and HadB interact with each other via their β2 strands such that the sheets from the two proteins are joined together to form one single contiguous sheet (Fig. 4A and 4B). The second type of inter-molecular interactions is mainly mediated by side chains (Fig. 4C–E; Tables S2 and S3) and is observed between: 1) amino acids of αHD of HadA and the helices α1 and α2, as well as the loop connecting these helices of HadB, 2) helix α1 of HadA and HadB, 3) β2 of HadA with αHD of HadB. PISA analysis revealed that hetero-dimerization of HadA with HadB resulted in burial of 1,453 Å2 surface area of each monomer.Figure 4

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