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Structure of the inhibitor W7 bound to the regulatory domain of cardiac troponin C.

Hoffman RM, Sykes BD - Biochemistry (2009)

Bottom Line: The protein-W7 interface is defined through a three-dimensional {(1)H,(13)C}-edited-{(1)H,(12)C}-detected NOESY NMR experiment, and other aspects of the structure are modeled as perturbations to previously known coordinates and restraints.The structure supports the previously proposed troponin I blocking mechanism for the activity of W7 in striated muscle and suggests a role for the flexible tail of W7 in stabilization of the bound state.This clarifies the structure-activity relationships of W7 and implicates an electrostatically mediated component of activity in common analogues of W7, including the antipsychotic trifluoroperazine and the cardiotonic levosimendan.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

ABSTRACT
The calmodulin antagonist W7 binds to troponin C in the presence of Ca(2+) and inhibits striated muscle contraction. This study integrates multiple data into the structure of the regulatory domain of human cardiac troponin C (cNTnC) bound to Ca(2+) and W7. The protein-W7 interface is defined through a three-dimensional {(1)H,(13)C}-edited-{(1)H,(12)C}-detected NOESY NMR experiment, and other aspects of the structure are modeled as perturbations to previously known coordinates and restraints. The structure determination protocol optimizes the protein-W7 contacts prior to the introduction of protein-W7 steric interactions or conformational changes in the protein. The structure determination protocol gives families of conformers that all have an optimal docking as assessed by satisfaction of the target function. The structure supports the previously proposed troponin I blocking mechanism for the activity of W7 in striated muscle and suggests a role for the flexible tail of W7 in stabilization of the bound state. This clarifies the structure-activity relationships of W7 and implicates an electrostatically mediated component of activity in common analogues of W7, including the antipsychotic trifluoroperazine and the cardiotonic levosimendan.

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Comparison of the cNTnC·Ca2+·W7 structure with the cNTnC·Ca2+·Sp·bepridil structure (PDB entry 1LXF). The lowest-energy conformer from the plainQ calculation (black and gray carbon atoms) is aligned to the first conformer of 1LXF (light brown carbon atoms, backbone rmsd of 1.50 Å). The Sp component of 1LXF is not shown. W7’s binding site only partially overlaps with bepridil’s. The primary amine of W7 colocalizes to one of bepridil’s tertiary amines. W7 penetrates more deeply into the hydrophobic pocket of cNTnC·Ca2+ than bepridil when bound to cNTnC·Ca2+·Sp.
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fig9: Comparison of the cNTnC·Ca2+·W7 structure with the cNTnC·Ca2+·Sp·bepridil structure (PDB entry 1LXF). The lowest-energy conformer from the plainQ calculation (black and gray carbon atoms) is aligned to the first conformer of 1LXF (light brown carbon atoms, backbone rmsd of 1.50 Å). The Sp component of 1LXF is not shown. W7’s binding site only partially overlaps with bepridil’s. The primary amine of W7 colocalizes to one of bepridil’s tertiary amines. W7 penetrates more deeply into the hydrophobic pocket of cNTnC·Ca2+ than bepridil when bound to cNTnC·Ca2+·Sp.

Mentions: Previous work has motivated and supported a “switch-peptide-occlusion” mechanism for W7’s inhibition of striated muscle contraction (7,9,10). This means that W7 binds to cNTnC·Ca2+ and impedes the subsequent binding of Sp. These results substantiate such a mechanism because the overall binding site of W7 is unambiguously localized to the previously proposed Sp binding site (see Figure 8). As shown in Figure 9, the W7 binding site is similar but not identical to the location of bepridil when bound to cNTnC·Ca2+·Sp. Both bepridil and W7 bind to cNTnC·Ca2+·Sp with weaker affinity than to cNTnC·Ca2+4,10; however, Sp binding does not preclude W7 binding (10), so one could assume that Sp, when bound, shifts the binding site for W7 to one more analogous to bepridil in the cNTnC·Ca2+·Sp·bepridil complex (4).


Structure of the inhibitor W7 bound to the regulatory domain of cardiac troponin C.

Hoffman RM, Sykes BD - Biochemistry (2009)

Comparison of the cNTnC·Ca2+·W7 structure with the cNTnC·Ca2+·Sp·bepridil structure (PDB entry 1LXF). The lowest-energy conformer from the plainQ calculation (black and gray carbon atoms) is aligned to the first conformer of 1LXF (light brown carbon atoms, backbone rmsd of 1.50 Å). The Sp component of 1LXF is not shown. W7’s binding site only partially overlaps with bepridil’s. The primary amine of W7 colocalizes to one of bepridil’s tertiary amines. W7 penetrates more deeply into the hydrophobic pocket of cNTnC·Ca2+ than bepridil when bound to cNTnC·Ca2+·Sp.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2697600&req=5

fig9: Comparison of the cNTnC·Ca2+·W7 structure with the cNTnC·Ca2+·Sp·bepridil structure (PDB entry 1LXF). The lowest-energy conformer from the plainQ calculation (black and gray carbon atoms) is aligned to the first conformer of 1LXF (light brown carbon atoms, backbone rmsd of 1.50 Å). The Sp component of 1LXF is not shown. W7’s binding site only partially overlaps with bepridil’s. The primary amine of W7 colocalizes to one of bepridil’s tertiary amines. W7 penetrates more deeply into the hydrophobic pocket of cNTnC·Ca2+ than bepridil when bound to cNTnC·Ca2+·Sp.
Mentions: Previous work has motivated and supported a “switch-peptide-occlusion” mechanism for W7’s inhibition of striated muscle contraction (7,9,10). This means that W7 binds to cNTnC·Ca2+ and impedes the subsequent binding of Sp. These results substantiate such a mechanism because the overall binding site of W7 is unambiguously localized to the previously proposed Sp binding site (see Figure 8). As shown in Figure 9, the W7 binding site is similar but not identical to the location of bepridil when bound to cNTnC·Ca2+·Sp. Both bepridil and W7 bind to cNTnC·Ca2+·Sp with weaker affinity than to cNTnC·Ca2+4,10; however, Sp binding does not preclude W7 binding (10), so one could assume that Sp, when bound, shifts the binding site for W7 to one more analogous to bepridil in the cNTnC·Ca2+·Sp·bepridil complex (4).

Bottom Line: The protein-W7 interface is defined through a three-dimensional {(1)H,(13)C}-edited-{(1)H,(12)C}-detected NOESY NMR experiment, and other aspects of the structure are modeled as perturbations to previously known coordinates and restraints.The structure supports the previously proposed troponin I blocking mechanism for the activity of W7 in striated muscle and suggests a role for the flexible tail of W7 in stabilization of the bound state.This clarifies the structure-activity relationships of W7 and implicates an electrostatically mediated component of activity in common analogues of W7, including the antipsychotic trifluoroperazine and the cardiotonic levosimendan.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

ABSTRACT
The calmodulin antagonist W7 binds to troponin C in the presence of Ca(2+) and inhibits striated muscle contraction. This study integrates multiple data into the structure of the regulatory domain of human cardiac troponin C (cNTnC) bound to Ca(2+) and W7. The protein-W7 interface is defined through a three-dimensional {(1)H,(13)C}-edited-{(1)H,(12)C}-detected NOESY NMR experiment, and other aspects of the structure are modeled as perturbations to previously known coordinates and restraints. The structure determination protocol optimizes the protein-W7 contacts prior to the introduction of protein-W7 steric interactions or conformational changes in the protein. The structure determination protocol gives families of conformers that all have an optimal docking as assessed by satisfaction of the target function. The structure supports the previously proposed troponin I blocking mechanism for the activity of W7 in striated muscle and suggests a role for the flexible tail of W7 in stabilization of the bound state. This clarifies the structure-activity relationships of W7 and implicates an electrostatically mediated component of activity in common analogues of W7, including the antipsychotic trifluoroperazine and the cardiotonic levosimendan.

Show MeSH
Related in: MedlinePlus