STRUCTURAL DETERMINANTS OF CA2-TERMINAL OF CARDIAC TROPONIN-C( EXCHANGE AND AFFINITY IN THE C)

The C terminal of cardiac troponin C (TnC) has two Ca2+-Mg2+ sites whi ch exhibit similar to 20-fold higher Ca2+ affinity than the two C-term inal Ca2+ specific sites in calmodulin (CaM). Substitution of the thir d EF-hand of TnC for the corresponding EF-hand of CaM produced a mutan t (CaM[3TnC]) with a 10-fold higher C-terminal Ca2+ and Mg2+ affinity. Substitution of loop 3 of TnC for loop 3 of CaM produced a mutant (Ca M[loop3TnC]) with a 10-fold faster Ca2+ on rate and a 5-fold faster Ca 2+ off rate than CaM. A mutant CaM (CaM[loop3X,Z]) which contained the identical coordinating amino acids and X and Z acid pairs of TnC loop 3 had a 3-fold higher C-terminal Ca2+ affinity without the increased Ca2+ exchange rates exhibited by CaM[loop3TnC]. Thus, loop factors oth er than the acid pairs must be responsible for the rapid Ca2+ exchange rates of CaM[loop3TnC]. Helix 6 and helix 5 in the third EF-hand of T nC support the rapid Ca2+ on rate of TnC's loop 3 and produce an simil ar to 4-fold reduction in its Ca2+ off rate, explaining the high Ca2affinity of the third EF-hand of TnC. Exchanging loop 3 or helix 5 of TnC into CaM increased the Mg2+ affinity by decreasing the Mg2+ off ra te. Our results are consistent with the high Ca2+ and Mg2+ affinity of the third EF-hand of TnC resulting from the two (X and Z) acid pairs in loop 3, coupled with the greater hydrophobicity of helix 6 and heli x 5 compared to that of the third EF-hand of CaM.