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Ca2+-induced PRE-NMR changes in the troponin complex reveal the possessive nature of the cardiac isoform for its regulatory switch.

Cordina NM, Liew CK, Potluri PR, Curmi PM, Fajer PG, Logan TM, Mackay JP, Brown LJ - PLoS ONE (2014)

Bottom Line: Comparison of 1H-15N-TROSY spectra of Ca2+-bound and free states for the spin labeled cTnC-cTnI binary constructs demonstrated the release and modest movement of the cTnI switch region (∼10 Å) away from the hydrophobic N-lobe of troponin C (cTnC) upon the removal of Ca2+.Our data supports a model where the non-bound regulatory switch region of cTnI is highly flexible in the absence of Ca2+ but remains in close vicinity to cTnC.We speculate that the close proximity of TnI to TnC in the cardiac complex is favourable for increasing the frequency of collisions between the N-lobe of cTnC and the regulatory switch region, counterbalancing the reduction in collision probability that results from the incomplete opening of the N-lobe of TnC that is unique to the cardiac isoform.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales, Australia.

ABSTRACT
The interaction between myosin and actin in cardiac muscle, modulated by the calcium (Ca2+) sensor Troponin complex (Tn), is a complex process which is yet to be fully resolved at the molecular level. Our understanding of how the binding of Ca2+ triggers conformational changes within Tn that are subsequently propagated through the contractile apparatus to initiate muscle activation is hampered by a lack of an atomic structure for the Ca2+-free state of the cardiac isoform. We have used paramagnetic relaxation enhancement (PRE)-NMR to obtain a description of the Ca2+-free state of cardiac Tn by describing the movement of key regions of the troponin I (cTnI) subunit upon the release of Ca2+ from Troponin C (cTnC). Site-directed spin-labeling was used to position paramagnetic spin labels in cTnI and the changes in the interaction between cTnI and cTnC subunits were then mapped by PRE-NMR. The functionally important regions of cTnI targeted in this study included the cTnC-binding N-region (cTnI57), the inhibitory region (cTnI143), and two sites on the regulatory switch region (cTnI151 and cTnI159). Comparison of 1H-15N-TROSY spectra of Ca2+-bound and free states for the spin labeled cTnC-cTnI binary constructs demonstrated the release and modest movement of the cTnI switch region (∼10 Å) away from the hydrophobic N-lobe of troponin C (cTnC) upon the removal of Ca2+. Our data supports a model where the non-bound regulatory switch region of cTnI is highly flexible in the absence of Ca2+ but remains in close vicinity to cTnC. We speculate that the close proximity of TnI to TnC in the cardiac complex is favourable for increasing the frequency of collisions between the N-lobe of cTnC and the regulatory switch region, counterbalancing the reduction in collision probability that results from the incomplete opening of the N-lobe of TnC that is unique to the cardiac isoform.

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Spin-labeled constructs designed to examine Ca2+ induced structural changes in troponin.(A) Structural model of the cTn switch mechanism for thin filament Ca2+-mediated activation based on the crystal structure of the core cardiac Tn complex (PDB 1J1D [3]). Ca2+ binding to the regulatory domain of the cTnC subunit (grey) triggers a large conformational change, moving the cTnI inhibitory region away from the thin filament and relieving the acto-myosin inhibition (+Ca2+ state). (B) Functional regions of cTnI (N-region: blue, IT-arm: green, inhibitory region: yellow, and switch region: red) with cTnC (colored grey, surface representation) in the binary cTnC-cTnI complex (PDB 1J1D [3]). The N-terminal region (blue), inhibitory region (yellow) and the switch region (red) are labeled. Note that the inhibitory region (residues 137–148 of cTnI) is not present in the crystal structures of cardiac Tn [3]; a possible conformation of the missing region is shown here for clarity. (C) Location of residues mutated to cysteine for attachment of spin labels on cardiac TnI: I57 (N-region), I143 (inhibitory region), I151 or I159 (switch region).
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pone-0112976-g001: Spin-labeled constructs designed to examine Ca2+ induced structural changes in troponin.(A) Structural model of the cTn switch mechanism for thin filament Ca2+-mediated activation based on the crystal structure of the core cardiac Tn complex (PDB 1J1D [3]). Ca2+ binding to the regulatory domain of the cTnC subunit (grey) triggers a large conformational change, moving the cTnI inhibitory region away from the thin filament and relieving the acto-myosin inhibition (+Ca2+ state). (B) Functional regions of cTnI (N-region: blue, IT-arm: green, inhibitory region: yellow, and switch region: red) with cTnC (colored grey, surface representation) in the binary cTnC-cTnI complex (PDB 1J1D [3]). The N-terminal region (blue), inhibitory region (yellow) and the switch region (red) are labeled. Note that the inhibitory region (residues 137–148 of cTnI) is not present in the crystal structures of cardiac Tn [3]; a possible conformation of the missing region is shown here for clarity. (C) Location of residues mutated to cysteine for attachment of spin labels on cardiac TnI: I57 (N-region), I143 (inhibitory region), I151 or I159 (switch region).

Mentions: The troponin complex (Figure 1A) is composed of three different subunits; the 18 kDa Ca2+ binding subunit (TnC), the 24 kDa thin filament binding inhibitory subunit (TnI), and the 35 kDa tropomyosin anchoring subunit (TnT). TnC consists of two globular metal binding domains, the C-lobe and N-lobe, which have structural and regulatory roles, respectively. It is the binding of Ca2+ to the regulatory N-lobe that is responsible for initiating contraction. Following Ca2+ binding, the signal is propagated from the N-lobe to the TnI subunit and subsequently to the other members of the thin filament (tropomyosin and actin). This cascade of conformational events modifies the interaction between the actin thin filament and the myosin thick filament, leading to muscle contraction.


Ca2+-induced PRE-NMR changes in the troponin complex reveal the possessive nature of the cardiac isoform for its regulatory switch.

Cordina NM, Liew CK, Potluri PR, Curmi PM, Fajer PG, Logan TM, Mackay JP, Brown LJ - PLoS ONE (2014)

Spin-labeled constructs designed to examine Ca2+ induced structural changes in troponin.(A) Structural model of the cTn switch mechanism for thin filament Ca2+-mediated activation based on the crystal structure of the core cardiac Tn complex (PDB 1J1D [3]). Ca2+ binding to the regulatory domain of the cTnC subunit (grey) triggers a large conformational change, moving the cTnI inhibitory region away from the thin filament and relieving the acto-myosin inhibition (+Ca2+ state). (B) Functional regions of cTnI (N-region: blue, IT-arm: green, inhibitory region: yellow, and switch region: red) with cTnC (colored grey, surface representation) in the binary cTnC-cTnI complex (PDB 1J1D [3]). The N-terminal region (blue), inhibitory region (yellow) and the switch region (red) are labeled. Note that the inhibitory region (residues 137–148 of cTnI) is not present in the crystal structures of cardiac Tn [3]; a possible conformation of the missing region is shown here for clarity. (C) Location of residues mutated to cysteine for attachment of spin labels on cardiac TnI: I57 (N-region), I143 (inhibitory region), I151 or I159 (switch region).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231091&req=5

pone-0112976-g001: Spin-labeled constructs designed to examine Ca2+ induced structural changes in troponin.(A) Structural model of the cTn switch mechanism for thin filament Ca2+-mediated activation based on the crystal structure of the core cardiac Tn complex (PDB 1J1D [3]). Ca2+ binding to the regulatory domain of the cTnC subunit (grey) triggers a large conformational change, moving the cTnI inhibitory region away from the thin filament and relieving the acto-myosin inhibition (+Ca2+ state). (B) Functional regions of cTnI (N-region: blue, IT-arm: green, inhibitory region: yellow, and switch region: red) with cTnC (colored grey, surface representation) in the binary cTnC-cTnI complex (PDB 1J1D [3]). The N-terminal region (blue), inhibitory region (yellow) and the switch region (red) are labeled. Note that the inhibitory region (residues 137–148 of cTnI) is not present in the crystal structures of cardiac Tn [3]; a possible conformation of the missing region is shown here for clarity. (C) Location of residues mutated to cysteine for attachment of spin labels on cardiac TnI: I57 (N-region), I143 (inhibitory region), I151 or I159 (switch region).
Mentions: The troponin complex (Figure 1A) is composed of three different subunits; the 18 kDa Ca2+ binding subunit (TnC), the 24 kDa thin filament binding inhibitory subunit (TnI), and the 35 kDa tropomyosin anchoring subunit (TnT). TnC consists of two globular metal binding domains, the C-lobe and N-lobe, which have structural and regulatory roles, respectively. It is the binding of Ca2+ to the regulatory N-lobe that is responsible for initiating contraction. Following Ca2+ binding, the signal is propagated from the N-lobe to the TnI subunit and subsequently to the other members of the thin filament (tropomyosin and actin). This cascade of conformational events modifies the interaction between the actin thin filament and the myosin thick filament, leading to muscle contraction.

Bottom Line: Comparison of 1H-15N-TROSY spectra of Ca2+-bound and free states for the spin labeled cTnC-cTnI binary constructs demonstrated the release and modest movement of the cTnI switch region (∼10 Å) away from the hydrophobic N-lobe of troponin C (cTnC) upon the removal of Ca2+.Our data supports a model where the non-bound regulatory switch region of cTnI is highly flexible in the absence of Ca2+ but remains in close vicinity to cTnC.We speculate that the close proximity of TnI to TnC in the cardiac complex is favourable for increasing the frequency of collisions between the N-lobe of cTnC and the regulatory switch region, counterbalancing the reduction in collision probability that results from the incomplete opening of the N-lobe of TnC that is unique to the cardiac isoform.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales, Australia.

ABSTRACT
The interaction between myosin and actin in cardiac muscle, modulated by the calcium (Ca2+) sensor Troponin complex (Tn), is a complex process which is yet to be fully resolved at the molecular level. Our understanding of how the binding of Ca2+ triggers conformational changes within Tn that are subsequently propagated through the contractile apparatus to initiate muscle activation is hampered by a lack of an atomic structure for the Ca2+-free state of the cardiac isoform. We have used paramagnetic relaxation enhancement (PRE)-NMR to obtain a description of the Ca2+-free state of cardiac Tn by describing the movement of key regions of the troponin I (cTnI) subunit upon the release of Ca2+ from Troponin C (cTnC). Site-directed spin-labeling was used to position paramagnetic spin labels in cTnI and the changes in the interaction between cTnI and cTnC subunits were then mapped by PRE-NMR. The functionally important regions of cTnI targeted in this study included the cTnC-binding N-region (cTnI57), the inhibitory region (cTnI143), and two sites on the regulatory switch region (cTnI151 and cTnI159). Comparison of 1H-15N-TROSY spectra of Ca2+-bound and free states for the spin labeled cTnC-cTnI binary constructs demonstrated the release and modest movement of the cTnI switch region (∼10 Å) away from the hydrophobic N-lobe of troponin C (cTnC) upon the removal of Ca2+. Our data supports a model where the non-bound regulatory switch region of cTnI is highly flexible in the absence of Ca2+ but remains in close vicinity to cTnC. We speculate that the close proximity of TnI to TnC in the cardiac complex is favourable for increasing the frequency of collisions between the N-lobe of cTnC and the regulatory switch region, counterbalancing the reduction in collision probability that results from the incomplete opening of the N-lobe of TnC that is unique to the cardiac isoform.

Show MeSH
Related in: MedlinePlus