Troponin‐C mutants with increased calcium affinity

Abstract

Binding of two Ca2+ to the regulatory sites I and II of troponin C (TnC) induces a conformational transition believed to be responsible for the activation of muscle contraction. Based on the known crystal structure (2Ca2+ state), a model for the transition to the 4Ca2+ state has been proposed [Herzberg, O., Moult, J. & James, M. N. G. (1986) J. Biol. Chem. 261, 2638–2644]. The proposed conformational transition predicts that during Ca2+ binding a number of nonpolar residues become exposed to the solvent, creating a hydrophobic patch. Such a model implies that mutation of the hydrophobic to polar residues should increase the Ca2+ affinity at the regulatory sites and reduce the Ca2+ concentration necessary for muscle activation. To test this prediction, we have constructed and functionally characterized two troponin‐C mutants (V45T and M48A mutations). Direct calcium‐binding measurements in the mutants demonstrate an increase in the Ca2+ affinity for two low‐affinity sites. Replacement of endogenous troponin C in skinned muscle fibers by TnC with mutations V45T or M48A increased the Ca2+ sensitivity of their tension development. These results show that the model can be used to construct mutants that regulate muscle contraction at lower Ca2+ concentrations. They provide further experimental support for the proposed calcium‐induced conformational change of troponin C and suggest that the predicted transition plays a central role in the activation of the thin filament.

Cloning and expression of chicken skeletal muscle troponin I in Escherichia coli: The role of rare codons on the expression level

“…2B), but the dissociation constants obtained for the two complexes are not statistically different (Table l). The dissociation constants obtained for recombinant chicken TnC are in agreement with the ones we determined by equilibrium dialysis (Fujimori et al, 1990) and flow dialysis (Silva et al, 1993). The constants reported here, in Fujimori et al (1990), and in Silva et al (1993) are lower than the ones reported for rabbit TnC and rabbit TnC/TnI complex (Potter & Gergely, 1975) probably due to differences in protein sequence between rabbit and chicken TnC and TnI.…”

“…The fact that the three troponin subunits can refold and reassociate in vitro (Greaser & Gergely, 1971) has allowed the use of troponin subunits produced in Escherichia coli to study the molecular mechanism of this regulatory complex. Expression of TnC in bacteria (Chen et al, 1988;Reinach & Karlsson, 1988;Xu & HitchcockDeGregori, 1988) and the analysis of site-directed mutants (Fujimori et al, 1990;Grabarek et al, 1990;Putkey et al, 1991;Sheng et a]., 1991;Negele et al, 1992;Pearlstone et al, 1992;Silva et al, 1993) in conjunction with the determination of the crystal structure of TnC (Herzberg & James, 1985;Sundarlingam et al, 1985) has provided a better understanding of the calcium-induced conformational change in TnC (Silva & Reinach, 1991;Grabarek et al, 1992). This conformational change is responsible for the modulation of the inhibitory action of Tnl.…”

Mutations of Hydrophobic Residues in the N-terminal Domain of Troponin C Affect Calcium Binding and Exchange with the Troponin C-Troponin I96–148 Complex and Muscle Force Production

“…9). Previous experiments with TnC mutants in which the Ca 2ϩ sensitivity of the regulatory domain in solution was increased or decreased demonstrated similar qualitative shifts in the force-pCa relationship upon reconstitution in skeletal muscle fibers (8,12,34,35,39,40 Furthermore, the Ca 2ϩ sensitivity of force development generated with F78QTnC F29W was dramatically lower than that generated with M81QTnC F29W or TnC F29W , even though both Phe 78 3 Gln and Met 81 3 Gln mutations in isolated TnC F29W decreased the Ca 2ϩ sensitivity of the regulatory domain to a similar extent (6). These results are consistent with the fact that the Phe 78 3 Gln but not Met 81 3 Gln mutation leads to a dramatic decrease in Ca 2ϩ affinity of the TnC F29W -TnI 96 -148 complex.…”

Designing Calcium-sensitizing Mutations in the Regulatory Domain of Cardiac Troponin C

“…Indeed, the mutants exhibited ϳ2.1-15.2-fold increases in their Ca 2ϩ affinity, ranging from 3.4 M for F20QcTnC F27W to 0.46 M for V44QcTnC F27W . In this respect, the regulatory domain of cTnC behaved similarly to the regulatory domains of both sTnC and CaM, in which decreasing the hydrophobic interactions between the BC and (N)AD units in the apo state, through similar mutations, led to increased Ca 2ϩ affinity (22,23,25,43). Complementary to these findings, stabilizing the interactions between the BC and (N)AD units of sTnC or CaM, either through disulfide bond, salt bridge formation, or substitution of buried polar residues with hydrophobic Leu or Ile, led to decreased Ca 2ϩ affinity (44 -48).…”

“…Previous studies demonstrated that mutation of a single hydrophobic residue, which does not directly ligate Ca 2ϩ , can either dramatically increase or decrease Ca 2ϩ affinity, depending on the location of the residue within the tertiary structure of the EF-hand protein (22)(23)(24)(25). Ca 2ϩ binding to the second regulatory EF-hand of cTnC, its mutants, and its isoforms has been studied using the Phe 27 3 Trp substitution immediately preceding the first defunct Ca 2ϩ -binding loop (26 -29).…”