Probing the calcium-induced conformational transition of troponin C with site-directed mutants

Abstract

The contraction of skeletal muscle is regulated by calcium binding to troponin C (TnC). TnC consists of two spatially independent domains, each of which contains two metal ion binding sites. Calcium binding to the regulatory sites of the N-terminal domain triggers muscle contraction by a series of conformational changes. Site-directed mutagenesis offers a means of elucidating the links in this signal path between TnC and actin-myosin crossbridges. Such mapping is possible if the mutants shift the equilibrium between 'on' and 'off' states of the regulatory complex while maintaining the coupling between calcium binding and tension development. Candidate amino-acid residues for yielding this information would be in positions remote from the calcium-binding sites and from the site of development of tension. Analysis of the crystal structure of TnC and of the model of the calcium-activated molecule has enabled us to identify two such residues: Glu 57 and Glu 88. In separate experiments we have replaced each of these residues by lysines. The resulting reduction in calcium affinity indicates that these residues have a long-range effect on calcium binding. This result may reflect the formation of a salt bridge between positions 57 and 88 that is not present in the native molecule. Moreover, the level of tension recovery when the mutants are incorporated into muscle suggests that the interaction between TnC and other muscle components has also been altered. Thus, these residues may participate in the contraction signal transmission.

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.…”

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

“…However, the N-domain of cTnC remains essentially closed in the Ca 2ϩ bound state, because the BC unit moves away only slightly from the NAD unit (15). Therefore, analogous residues in cTnC, Phe 20 , Val 44 , Met 45 , Leu 48 , and Met 81 exhibit no increase in their solvent accessibility upon Ca 2ϩ binding (Table I) and form numerous side chain contacts with each other in both the apo and Ca 2ϩ -bound states. Thus, there was a question of whether analogous mutations in cTnC would have the same effect on Ca 2ϩ affinity and exchange rates as they did in sTnC.…”

“…This figure was drawn using Rasmol (61). The helices are labeled (N, A, B, C, and D) and shown as ribbons; hydrophobic residues that were mutated to Gln (Phe 20 , Val 44 , Met 45 , Leu 48 , and Met 81 ) are shown in a ball-and-stick format and labeled. tion rates to the N-domain of cTnC F27W (ϳ1.2-1.7 ϫ 10 8 M Ϫ1 s Ϫ1 , reported in this work) and sTnC F29W (ϳ1.1-1.6 ϫ 10 8 M Ϫ1 s Ϫ1 ; 8, 25) were nearly identical at 15°C.…”

“…We then designed five Ca 2ϩ -sensitizing cTnC F27W mutants, in which we individually substituted hydrophobic Phe 20 , Val 44 , Met 45 , Leu 48 , or Met 81 with polar Gln. None of the mutations affected the ability of cTnC F27W to competitively bind Mg 2ϩ with a physiologically relevant affinity.…”