Abstract:
:The interaction between the calcium binding and inhibitory components of troponin is central to the regulation of muscle contraction. In this work, two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance (2D-¿1H,15N¿-HSQC NMR) spectroscopy was used to determine the stoichiometry, affinity, and mechanisms for binding of Ca2+ and two synthetic TnI peptides [TnI1-40 (or Rp40) and TnI96-115] to the isolated C-domain of skeletal troponin C (CTnC). The Ca2+ titration revealed that 2 equiv of Ca2+ binds to sites III and IV of CTnC with strong positive cooperativity and high affinity [dissociation constant (KD) = 0.1 microM]. In this process, CTnC folds from a largely unstructured state to a compact domain capable of interacting with TnI. Titration of CTnC x 2Ca2+ with Rp40 occurs with a 1:1 stoichiometry and a KD of 2 +/- 1 microM. Titration of CTnC x 2Ca2+ with a peptide corresponding to the inhibitory region of TnI (TnI96-115) also reveals a 1:1 ratio, but weaker affinity (KD = 47 +/- 7 microM). Both Rp40- and TnI96-115-induced backbone amide chemical shift changes of CTnC x 2Ca2+ are similarly distributed along the sequence, indicating that these two regions of TnI may compete for the same binding site on CTnC x 2Ca2+. The changes induced by Rp40 are much larger, however, and define the interaction sites on TnC and regions where the flexibility of hinge and terminal residues is altered. To investigate the possibility of direct competition, TnI(96-115) was titrated into the CTnC x 2Ca(2+) x Rp40 complex, whereas Rp40 was titrated into the CTnC x 2Ca2+. TnI96-115 complex. The results show that Rp40 can displace TnI96-115 completely, while TnI96-115 has no effect on CTnC x 2Ca2+ x Rp40. Recent proposals for the mechanism of muscle regulation [Tripet, B. P., Van Eyk, J. E., and Hodges, R. S. (1997) J. Mol. Biol. 271, 728-750] suggest that the N-terminal and inhibitory regions of TnI competitively bind the structural domain of TnC. The findings presented here indicate that additional factors, such as interactions between the N-domain of TnC with the C-domain of TnI or the C-domain of TnT, are required, if the inhibitory region is going to successfully compete for the structural domain of TnC.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Mercier P,Li MX,Sykes BDdoi
10.1021/bi992579nsubject
Has Abstractpub_date
2000-03-21 00:00:00pages
2902-11issue
11eissn
0006-2960issn
1520-4995pii
bi992579njournal_volume
39pub_type
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