Limits...
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.

Show MeSH

Related in: MedlinePlus

PRE-NMR peak intensity ratios (Ipara/Idia) mapped onto cardiac Tn in the presence and absence of Ca2+.Four monocysteine cTnI constructs were spin-labeled with MTSL at residues (A) I57, (B) I143, (C) I151 and (D) I159. The cTnI spin labels caused distance dependant peak broadening for 15N-cTnC residues in 1H-15N-TROSY spectra of the cTnC-cTnI complexes. The magnitude of the peak broadening is determined by comparing the paramagnetic peak intensity (Ipara) to the peak intensity in the diamagnetic state (Idia) after the reduction of the MTSL spin label. Left: Peak intensity ratios (Ipara/Idia) for each assigned cTnC residue, in the absence of Ca2+ (green) and presence of Ca2+ (black). Peaks that were broadened beyond detection are assigned a peak intensity ratio of 0.05. Right: the peak intensity ratios are mapped onto the crystal structure of cardiac Tn (1J1D [3]) according to the scale bar, where red indicates a large reduction in peak intensity and blue indicates no change. cTnI is colored black, and the location of the spin labeled cTnI residue is indicated with a purple sphere and an arrow. cTnC helices N, A–H are labeled.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231091&req=5

pone-0112976-g003: PRE-NMR peak intensity ratios (Ipara/Idia) mapped onto cardiac Tn in the presence and absence of Ca2+.Four monocysteine cTnI constructs were spin-labeled with MTSL at residues (A) I57, (B) I143, (C) I151 and (D) I159. The cTnI spin labels caused distance dependant peak broadening for 15N-cTnC residues in 1H-15N-TROSY spectra of the cTnC-cTnI complexes. The magnitude of the peak broadening is determined by comparing the paramagnetic peak intensity (Ipara) to the peak intensity in the diamagnetic state (Idia) after the reduction of the MTSL spin label. Left: Peak intensity ratios (Ipara/Idia) for each assigned cTnC residue, in the absence of Ca2+ (green) and presence of Ca2+ (black). Peaks that were broadened beyond detection are assigned a peak intensity ratio of 0.05. Right: the peak intensity ratios are mapped onto the crystal structure of cardiac Tn (1J1D [3]) according to the scale bar, where red indicates a large reduction in peak intensity and blue indicates no change. cTnI is colored black, and the location of the spin labeled cTnI residue is indicated with a purple sphere and an arrow. cTnC helices N, A–H are labeled.

Mentions: For all four binary complex samples, a unique pattern of paramagnetic peak broadening effects arising from the inclusion of the spin label on cTnI were observed (Figure 2). Peak intensity ratios (Ipara/Idia) calculated for each assigned cTnC resonance in the −Ca2+ and +Ca2+ are plotted in Figure 3. The peak intensity ratio values are also mapped onto the +Ca2+ cardiac Tn crystal structure (1J1D) in a continuous color scale. Strong PRE peak broadening effects (Ipara/Idia≪1) are shaded red and are indicative of close proximity (<12 Å) of the cTnC residue to the nitroxide spin label on cTnI. cTnC resonances which were unaffected by the spin label (Ipara/Idia∼1) are shaded in blue and represent residues >25 Å away from the spin label. cTnC residues for which no peak intensity ratio could be reliably assigned, either due to uncertainty in assignment or peak overlap, are shown in white. This comparative mapping of the PRE broadening effects enables the rapid visualisation of the relative positioning of each of the functional cTnI regions under investigation with respect to cTnC. The mapping also reveals the effect of Ca2+ binding on the relative positioning of the two subunits.


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)

PRE-NMR peak intensity ratios (Ipara/Idia) mapped onto cardiac Tn in the presence and absence of Ca2+.Four monocysteine cTnI constructs were spin-labeled with MTSL at residues (A) I57, (B) I143, (C) I151 and (D) I159. The cTnI spin labels caused distance dependant peak broadening for 15N-cTnC residues in 1H-15N-TROSY spectra of the cTnC-cTnI complexes. The magnitude of the peak broadening is determined by comparing the paramagnetic peak intensity (Ipara) to the peak intensity in the diamagnetic state (Idia) after the reduction of the MTSL spin label. Left: Peak intensity ratios (Ipara/Idia) for each assigned cTnC residue, in the absence of Ca2+ (green) and presence of Ca2+ (black). Peaks that were broadened beyond detection are assigned a peak intensity ratio of 0.05. Right: the peak intensity ratios are mapped onto the crystal structure of cardiac Tn (1J1D [3]) according to the scale bar, where red indicates a large reduction in peak intensity and blue indicates no change. cTnI is colored black, and the location of the spin labeled cTnI residue is indicated with a purple sphere and an arrow. cTnC helices N, A–H are labeled.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112976-g003: PRE-NMR peak intensity ratios (Ipara/Idia) mapped onto cardiac Tn in the presence and absence of Ca2+.Four monocysteine cTnI constructs were spin-labeled with MTSL at residues (A) I57, (B) I143, (C) I151 and (D) I159. The cTnI spin labels caused distance dependant peak broadening for 15N-cTnC residues in 1H-15N-TROSY spectra of the cTnC-cTnI complexes. The magnitude of the peak broadening is determined by comparing the paramagnetic peak intensity (Ipara) to the peak intensity in the diamagnetic state (Idia) after the reduction of the MTSL spin label. Left: Peak intensity ratios (Ipara/Idia) for each assigned cTnC residue, in the absence of Ca2+ (green) and presence of Ca2+ (black). Peaks that were broadened beyond detection are assigned a peak intensity ratio of 0.05. Right: the peak intensity ratios are mapped onto the crystal structure of cardiac Tn (1J1D [3]) according to the scale bar, where red indicates a large reduction in peak intensity and blue indicates no change. cTnI is colored black, and the location of the spin labeled cTnI residue is indicated with a purple sphere and an arrow. cTnC helices N, A–H are labeled.
Mentions: For all four binary complex samples, a unique pattern of paramagnetic peak broadening effects arising from the inclusion of the spin label on cTnI were observed (Figure 2). Peak intensity ratios (Ipara/Idia) calculated for each assigned cTnC resonance in the −Ca2+ and +Ca2+ are plotted in Figure 3. The peak intensity ratio values are also mapped onto the +Ca2+ cardiac Tn crystal structure (1J1D) in a continuous color scale. Strong PRE peak broadening effects (Ipara/Idia≪1) are shaded red and are indicative of close proximity (<12 Å) of the cTnC residue to the nitroxide spin label on cTnI. cTnC resonances which were unaffected by the spin label (Ipara/Idia∼1) are shaded in blue and represent residues >25 Å away from the spin label. cTnC residues for which no peak intensity ratio could be reliably assigned, either due to uncertainty in assignment or peak overlap, are shown in white. This comparative mapping of the PRE broadening effects enables the rapid visualisation of the relative positioning of each of the functional cTnI regions under investigation with respect to cTnC. The mapping also reveals the effect of Ca2+ binding on the relative positioning of the two subunits.

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