The C terminus of cardiac troponin I stabilizes the Ca2+-activated state of tropomyosin on actin filaments.
BACKGROUND
Ca(2+) control of troponin-tropomyosin position on actin regulates cardiac muscle contraction. The inhibitory subunit of troponin, cardiac troponin (cTn)I is primarily responsible for maintaining a tropomyosin conformation that prevents crossbridge cycling. Despite extensive characterization of cTnI, the precise role of its C-terminal domain (residues 193 to 210) is unclear. Mutations within this region are associated with restrictive cardiomyopathy, and C-terminal deletion of cTnI, in some species, has been associated with myocardial stunning.


OBJECTIVE
We sought to investigate the effect of a cTnI deletion-removal of 17 amino acids from the C terminus- on the structure of troponin-regulated tropomyosin bound to actin.


RESULTS
A truncated form of human cTnI (cTnI(1-192)) was expressed and reconstituted with troponin C and troponin T to form a mutant troponin. Using electron microscopy and 3D image reconstruction, we show that the mutant troponin perturbs the positional equilibrium dynamics of tropomyosin in the presence of Ca(2+). Specifically, it biases tropomyosin position toward an "enhanced C-state" that exposes more of the myosin-binding site on actin than found with wild-type troponin.


CONCLUSIONS
In addition to its well-established role of promoting the so-called "blocked-state" or "B-state," cTnI participates in proper stabilization of tropomyosin in the "Ca(2+)-activated state" or "C-state." The last 17 amino acids perform this stabilizing role. The data are consistent with a "fly-casting" model in which the mobile C terminus of cTnI ensures proper conformational switching of troponin-tropomyosin. Loss of actin-sensing function within this domain, by pathological proteolysis or cardiomyopathic mutation, may be sufficient to perturb tropomyosin conformation.
Galińska
Agnieszka
A
Department of Physiology & Biophysics, Boston University School of Medicine, 72 E Concord St., Boston, MA 02118, USA.
Hatch
Victoria
V
Craig
Roger
R
Murphy
Anne M
AM
Van Eyk
Jennifer E
JE
Wang
C-L Albert
CL
Lehman
William
W
Foster
D Brian
DB
D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D006801:Humans; D021621:Imaging, Three-Dimensional; D008854:Microscopy, Electron; D008958:Models, Molecular; D046912:Multiprotein Complexes; D009154:Mutation; D009200:Myocardial Contraction; D009206:Myocardium / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D014335:Tropomyosin / Q000378:metabolism* / Q000648:ultrastructure; D019209:Troponin C / Q000378:metabolism; D019210:Troponin I / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D020107:Troponin T / Q000378:metabolism
D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D014335:Tropomyosin; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D002118:Calcium