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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 lowest-energy conformer of the cNTnC·Ca2+·W7 complex with the first conformer of cNTnC·Ca2+·Sp (PDB entry 1MXL). The structures were aligned over the backbone heavy atoms of residues 2−87 (2.46 Å rmsd). The backbone of cNTnC·Ca2+ from 1MXL is shown as a black Cα trace. The switch peptide (cardiac TnI147−163) is shown as sticks, with carbon atoms colored light brown. W7 is shown with black sticks for carbon atoms and with the surface represented as a transparent outline. This shows that W7 serically interferes with the stabilizing cNTnC−Sp interactions originating from Ile149 and Met154. The terminal amine group of W7 may compete with Arg148 for electrostatic interactions with acidic patches on helices C and D.
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fig8: Comparison of the lowest-energy conformer of the cNTnC·Ca2+·W7 complex with the first conformer of cNTnC·Ca2+·Sp (PDB entry 1MXL). The structures were aligned over the backbone heavy atoms of residues 2−87 (2.46 Å rmsd). The backbone of cNTnC·Ca2+ from 1MXL is shown as a black Cα trace. The switch peptide (cardiac TnI147−163) is shown as sticks, with carbon atoms colored light brown. W7 is shown with black sticks for carbon atoms and with the surface represented as a transparent outline. This shows that W7 serically interferes with the stabilizing cNTnC−Sp interactions originating from Ile149 and Met154. The terminal amine group of W7 may compete with Arg148 for electrostatic interactions with acidic patches on helices C and D.

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 lowest-energy conformer of the cNTnC·Ca2+·W7 complex with the first conformer of cNTnC·Ca2+·Sp (PDB entry 1MXL). The structures were aligned over the backbone heavy atoms of residues 2−87 (2.46 Å rmsd). The backbone of cNTnC·Ca2+ from 1MXL is shown as a black Cα trace. The switch peptide (cardiac TnI147−163) is shown as sticks, with carbon atoms colored light brown. W7 is shown with black sticks for carbon atoms and with the surface represented as a transparent outline. This shows that W7 serically interferes with the stabilizing cNTnC−Sp interactions originating from Ile149 and Met154. The terminal amine group of W7 may compete with Arg148 for electrostatic interactions with acidic patches on helices C and D.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

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

fig8: Comparison of the lowest-energy conformer of the cNTnC·Ca2+·W7 complex with the first conformer of cNTnC·Ca2+·Sp (PDB entry 1MXL). The structures were aligned over the backbone heavy atoms of residues 2−87 (2.46 Å rmsd). The backbone of cNTnC·Ca2+ from 1MXL is shown as a black Cα trace. The switch peptide (cardiac TnI147−163) is shown as sticks, with carbon atoms colored light brown. W7 is shown with black sticks for carbon atoms and with the surface represented as a transparent outline. This shows that W7 serically interferes with the stabilizing cNTnC−Sp interactions originating from Ile149 and Met154. The terminal amine group of W7 may compete with Arg148 for electrostatic interactions with acidic patches on helices C and D.
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