Abstract
To investigate the roles of site I and II invariant Glu residues 41 and 77 in the functional properties and calcium-induced structural opening of skeletal muscle troponin C (TnC) regulatory domain, we have replaced them by Ala in intact F29W TnC and in wild-type and F29W N domains (TnC residues 1-90). Reconstitution of intact E41A/F29W and E77A/F29W mutants into TnC-depleted muscle skinned fibers showed that Ca(2+)-induced tension is greatly reduced compared with the F29W control. Circular dichroism measurements of wild-type N domain as a function of pCa (= -log[Ca(2+)]) demonstrated that approximately 90% of the total change in molar ellipticity at 222 nm ([theta](222 nm)) could be assigned to site II Ca(2+) binding. With E41A, E77A, and cardiac TnC N domains this [theta](222 nm) change attributable to site II was reduced to < or =40% of that seen with wild type, consistent with their structures remaining closed in +Ca(2+). Furthermore, the Ca(2+)-induced changes in fluorescence, near UV CD, and UV difference spectra observed with intact F29W are largely abolished with E41A/F29W and E77A/F29W TnCs. Taken together, the data indicate that the major structural change in N domain, including the closed to open transition, is triggered by site II Ca(2+) binding, an interpretation relevant to the energetics of the skeletal muscle TnC and cardiac TnC systems.
Highlights
The troponin complex, present in skeletal and cardiac muscle in association with tropomyosin and actin of the thin filaments, is primarily responsible for the Ca2ϩ regulation of contraction and relaxation in these tissues
These include 1) the demonstration that mutation to Ala of either of the invariant Glu residues in position 12 of the site I and II Ca2ϩ binding loops leads to virtual elimination of the Ca2ϩ sensitivity of the contractile response in skinned muscle fibers; 2) the observation that by far the major fraction (ϳ90%) of the total Ca2ϩ-induced ellipticity change of isolated N domain of wild-type sTnC is associated with binding to site
II; 3) that this latter change is substantially reduced to 30 – 40% in each of the two Glu 3 Ala mutants and in cTnC; 4) the Ca2ϩ-induced fluorescence, near UV CD, and UV difference spectral changes observed with sTnCF29W are largely eliminated in both Glu 3 Ala mutants of sTnCF29W; 5) in comparison with sTnC, sTnCF29W has minimal effect on biological function as assessed in skinned fibers, but in solution its isolated N domain shows an increase in both Ca2ϩ-induced ellipticity change and altered affinity for Ca2ϩ at sites I and II
Summary
The troponin complex, present in skeletal and cardiac muscle in association with tropomyosin and actin of the thin filaments, is primarily responsible for the Ca2ϩ regulation of contraction and relaxation in these tissues. Each of the N and C domains consists of two intimately associated EF-hand or helix-loop-helix metal binding motifs. Those of C domain (sites III and IV) have high affinity for Ca2ϩ (Kd Х 10Ϫ7 M) and bind Mg2ϩ competitively (Kd Х 10Ϫ3 M). Based on a substantial body of evidence, the C domain sites III/IV are believed to be occupied by Ca2ϩ/Mg2ϩ throughout the contraction/relaxation cycle and to serve a structural role in anchoring TnC to the other troponin components. The intact TnC (4Ca2ϩ) possesses two nonpolar surface patches, one in each domain, with surrounding constellations of negatively charged residues Evidence for these as interaction sites with regions of TnI has been reported (see Ref. 25 and references therein). In most EF-hands/loops, the highly conserved bidentate Glu residue at position 12 (ϪZ) provides two Ca2ϩ ligands through its two side chain carboxyl oxygens
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