Structural determinants of Ca2+ exchange and affinity in the C terminal of cardiac troponin C.
The C terminal of cardiac troponin C (TnC) has two Ca2+-Mg2+ sites which exhibit approximately 20-fold higher Ca2+ affinity than the two C-terminal Ca2+ specific sites in calmodulin (CaM). Substitution of the third EF-hand of TnC for the corresponding EF-hand of CaM produced a mutant (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 (CaM[loop3TnC]) with a 10-fold faster Ca2+ on rate and a 5-fold faster Ca2+ 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 other 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 TnC support the rapid Ca2+ on rate of TnC's loop 3 and produce an approximately 4-fold reduction in its Ca2+ off rate, explaining the high Ca2+ affinity 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 rate. 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 helix 5 compared to that of the third EF-hand of CaM.
Wang
S
S
Department of Medical Biochemistry, The Ohio State University College of Medicine, Columbus 43210-1218, USA.
George
S E
SE
Davis
J P
JP
Johnson
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002645:Chickens; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D002147:Calmodulin; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019209:Troponin C; D008274:Magnesium; D002118:Calcium