Ca2+-induced PRE-NMR changes in the troponin complex reveal the possessive nature of the cardiac isoform for its regulatory switch.
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.
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales, Australia.
Nicole M
NM
Cordina
Department of Molecular Cardiology and Biophysics, The Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.
Chu K
CK
Liew
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales, Australia.
Phani R
PR
Potluri
School of Physics, The University of New South Wales, Sydney, New South Wales, Australia.
Paul M
PM
Curmi
Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, United States of America.
Piotr G
PG
Fajer
Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, United States of America.
Timothy M
TM
Logan
School of Molecular and Microbial Biosciences, University of Sydney, Sydney, New South Wales, Australia.
Joel P
JP
Mackay
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales, Australia.
Louise J
LJ
Brown
D000818:Animals; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D020033:Protein Isoforms; D051381:Rats; D014336:Troponin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D046912:Multiprotein Complexes; D020033:Protein Isoforms; D014336:Troponin; D002118:Calcium