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Crystal Structure of Fad35R from Mycobacterium tuberculosis H37Rv in the Apo-State.

Singh AK, Manjasetty B, Balasubramani GL, Koul S, Kaushik A, Ekka MK, Singh V, Kumaran S - PLoS ONE (2015)

Bottom Line: Two DBDs are packed asymmetrically, creating an alternative dimer interface which coincides with the possible tetramer interface that connects the two canonical dimers.Quaternary state of alternative dimer mimics a closed-state structure in which two recognition helices are distanced at ~ 35 Å and ligand binding pockets are inaccessible.Results of biophysical studies indicate that Fad35R has the propensity to oligomerize in solution in the presence of tetracycline.

View Article: PubMed Central - PubMed

Affiliation: Council of Scientific and Industrial Research (CSIR), Institute of Microbial Technology, G.N.Ramachandran Protein Centre, Chandigarh, 160036, India.

ABSTRACT
Fad35R from Mycobacterium tuberculosis binds to the promoter site of Fad35 operon and its DNA binding activities are reduced in the presence of tetracycline and palmitoyl-CoA. We resolved the crystal structure of Fad35R using single-wavelength anomalous diffraction method (SAD). Fad35R comprises canonical DNA binding domain (DBD) and ligand binding domain (LBD), but displays several distinct structural features. Two recognition helices of two monomers in the homodimer are separated by ~ 48 Å and two core triangle-shaped ligand binding cavities are well exposed to solvent. Structural comparison with DesT and QacR structures suggests that ligand binding-induced movement of α7, which adopts a straight conformation in the Fad35R, may be crucial to switch the conformational states between repressive and derepressive forms. Two DBDs are packed asymmetrically, creating an alternative dimer interface which coincides with the possible tetramer interface that connects the two canonical dimers. Quaternary state of alternative dimer mimics a closed-state structure in which two recognition helices are distanced at ~ 35 Å and ligand binding pockets are inaccessible. Results of biophysical studies indicate that Fad35R has the propensity to oligomerize in solution in the presence of tetracycline. We present the first structure of a FadR homologue from mycobacterium and the structure reveals DNA and ligand binding features of Fad35R and also provides a view on alternative quaternary states that mimic open and closed forms of the regulator.

No MeSH data available.


Related in: MedlinePlus

Structural view of ligand binding pocket.A & B) The open and opposite orientations of two triangular ligand binding cavities of two monomers (blue and yellow), superimposed by 180° rotation around vertical axis followed by 65° rotation of x-axis. C) The structural view of ligand binding pocket of one monomer. The glycerol is shown as space filling and predicted ligand binding residues are shown in sticks. D) View of Fad35R (yellow) ligand binding pocket superimposed to that of DesT (cyan) bound to palmitoyl Co-A (PCA) (shown as sticks, orange) indicate that fatty acid tail can fit well into the cavity and L140, I132, and S92 may form interactions with PCA.
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pone.0124333.g002: Structural view of ligand binding pocket.A & B) The open and opposite orientations of two triangular ligand binding cavities of two monomers (blue and yellow), superimposed by 180° rotation around vertical axis followed by 65° rotation of x-axis. C) The structural view of ligand binding pocket of one monomer. The glycerol is shown as space filling and predicted ligand binding residues are shown in sticks. D) View of Fad35R (yellow) ligand binding pocket superimposed to that of DesT (cyan) bound to palmitoyl Co-A (PCA) (shown as sticks, orange) indicate that fatty acid tail can fit well into the cavity and L140, I132, and S92 may form interactions with PCA.

Mentions: The C-terminal domain is formed of residues, 71–215 and it is composed of six major helices, α4 to α9, which encompass the ligand binding site and also provide the dimerization interface. The fourth helix, α4 which extends from the base of DBD, runs parallel to α7. The seventh helix is usually kinked in most TTRs, but it adopts a straight conformation in Fad35R. The straight conformation enables α7 to mediate multiple contacts with α4 and also, the absence of curvature creates a large ligand binding cavity within the LBD. The triangular ligand binding cavity is formed by helices α5, α6, and α7 in each monomer and ttwo cavities are solvent accessible but face opposite sides of the dimer. A rotation of 180° along the y-axis, parallel to the two-fold NCS axis, followed by rotation around x-axis by 65°, superposes the ligand binding cavity of molA onto molB (Fig 2A and 2B). Structural dispositions of the two ligand binding cavities at opposite sides of the dimer would facilitate ligand access.


Crystal Structure of Fad35R from Mycobacterium tuberculosis H37Rv in the Apo-State.

Singh AK, Manjasetty B, Balasubramani GL, Koul S, Kaushik A, Ekka MK, Singh V, Kumaran S - PLoS ONE (2015)

Structural view of ligand binding pocket.A & B) The open and opposite orientations of two triangular ligand binding cavities of two monomers (blue and yellow), superimposed by 180° rotation around vertical axis followed by 65° rotation of x-axis. C) The structural view of ligand binding pocket of one monomer. The glycerol is shown as space filling and predicted ligand binding residues are shown in sticks. D) View of Fad35R (yellow) ligand binding pocket superimposed to that of DesT (cyan) bound to palmitoyl Co-A (PCA) (shown as sticks, orange) indicate that fatty acid tail can fit well into the cavity and L140, I132, and S92 may form interactions with PCA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418694&req=5

pone.0124333.g002: Structural view of ligand binding pocket.A & B) The open and opposite orientations of two triangular ligand binding cavities of two monomers (blue and yellow), superimposed by 180° rotation around vertical axis followed by 65° rotation of x-axis. C) The structural view of ligand binding pocket of one monomer. The glycerol is shown as space filling and predicted ligand binding residues are shown in sticks. D) View of Fad35R (yellow) ligand binding pocket superimposed to that of DesT (cyan) bound to palmitoyl Co-A (PCA) (shown as sticks, orange) indicate that fatty acid tail can fit well into the cavity and L140, I132, and S92 may form interactions with PCA.
Mentions: The C-terminal domain is formed of residues, 71–215 and it is composed of six major helices, α4 to α9, which encompass the ligand binding site and also provide the dimerization interface. The fourth helix, α4 which extends from the base of DBD, runs parallel to α7. The seventh helix is usually kinked in most TTRs, but it adopts a straight conformation in Fad35R. The straight conformation enables α7 to mediate multiple contacts with α4 and also, the absence of curvature creates a large ligand binding cavity within the LBD. The triangular ligand binding cavity is formed by helices α5, α6, and α7 in each monomer and ttwo cavities are solvent accessible but face opposite sides of the dimer. A rotation of 180° along the y-axis, parallel to the two-fold NCS axis, followed by rotation around x-axis by 65°, superposes the ligand binding cavity of molA onto molB (Fig 2A and 2B). Structural dispositions of the two ligand binding cavities at opposite sides of the dimer would facilitate ligand access.

Bottom Line: Two DBDs are packed asymmetrically, creating an alternative dimer interface which coincides with the possible tetramer interface that connects the two canonical dimers.Quaternary state of alternative dimer mimics a closed-state structure in which two recognition helices are distanced at ~ 35 Å and ligand binding pockets are inaccessible.Results of biophysical studies indicate that Fad35R has the propensity to oligomerize in solution in the presence of tetracycline.

View Article: PubMed Central - PubMed

Affiliation: Council of Scientific and Industrial Research (CSIR), Institute of Microbial Technology, G.N.Ramachandran Protein Centre, Chandigarh, 160036, India.

ABSTRACT
Fad35R from Mycobacterium tuberculosis binds to the promoter site of Fad35 operon and its DNA binding activities are reduced in the presence of tetracycline and palmitoyl-CoA. We resolved the crystal structure of Fad35R using single-wavelength anomalous diffraction method (SAD). Fad35R comprises canonical DNA binding domain (DBD) and ligand binding domain (LBD), but displays several distinct structural features. Two recognition helices of two monomers in the homodimer are separated by ~ 48 Å and two core triangle-shaped ligand binding cavities are well exposed to solvent. Structural comparison with DesT and QacR structures suggests that ligand binding-induced movement of α7, which adopts a straight conformation in the Fad35R, may be crucial to switch the conformational states between repressive and derepressive forms. Two DBDs are packed asymmetrically, creating an alternative dimer interface which coincides with the possible tetramer interface that connects the two canonical dimers. Quaternary state of alternative dimer mimics a closed-state structure in which two recognition helices are distanced at ~ 35 Å and ligand binding pockets are inaccessible. Results of biophysical studies indicate that Fad35R has the propensity to oligomerize in solution in the presence of tetracycline. We present the first structure of a FadR homologue from mycobacterium and the structure reveals DNA and ligand binding features of Fad35R and also provides a view on alternative quaternary states that mimic open and closed forms of the regulator.

No MeSH data available.


Related in: MedlinePlus