Wang S, George S E, Davis J P, Johnson J D
Department of Medical Biochemistry, The Ohio State University College of Medicine, Columbus 43210-1218, USA.
Biochemistry. 1998 Oct 13;37(41):14539-44. doi: 10.1021/bi9814641.
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.
心肌肌钙蛋白C（TnC）的C末端有两个Ca2+-Mg2+位点，其对Ca2+的亲和力比钙调蛋白（CaM）的两个C末端Ca2+特异性位点高约20倍。将TnC的第三个EF手结构域替换为CaM的相应EF手结构域，产生了一个突变体（CaM[3TnC]），其C末端对Ca2+和Mg2+的亲和力提高了10倍。将TnC的环3替换为CaM的环3，产生了一个突变体（CaM[loop3TnC]），其Ca2+结合速率比CaM快10倍，Ca2+解离速率快5倍。一个包含与TnC环3相同的配位氨基酸以及X和Z酸性对的突变型CaM（CaM[loop3X, Z]），其C末端对Ca2+的亲和力提高了3倍，且没有表现出CaM[loop3TnC]所具有的Ca2+交换速率增加的情况。因此，除了酸性对外的环因素必定是CaM[loop3TnC]快速Ca2+交换速率的原因。TnC第三个EF手结构域中的螺旋6和螺旋5支持TnC环3快速的Ca2+结合速率，并使其Ca2+解离速率降低约4倍，这解释了TnC第三个EF手结构域对Ca2+的高亲和力。将TnC的环3或螺旋5替换到CaM中，通过降低Mg2+解离速率增加了对Mg2+的亲和力。我们的结果与TnC第三个EF手结构域对Ca2+和Mg2+的高亲和力相一致，这是由环3中的两个（X和Z）酸性对以及与CaM第三个EF手结构域相比螺旋6和螺旋5更大的疏水性导致的。
