Persechini A, Gansz K J, Paresi R J
Department of Physiology, University of Rochester Medical Center, New York 14642, USA.
Biochemistry. 1996 Jan 9;35(1):224-8. doi: 10.1021/bi952383x.
We have constructed three engineered calmodulins (CaMs) in which the two EF hand pairs have been substituted for one another or exchanged: CaMNN, the C-terminal EF hand pair (residues 82-148) has been replaced by a duplication of the N-terminal pair (residues 9-75); CaMCC, the N-terminal pair has been replaced by a duplication of the C-terminal pair; CaMCN, the two EF had pairs have been exchanged. Skeletal muscle myosin light chain kinase (skMLCK) activity is activated to 75% of the maximum level by CaMCC and to 45% of the maximum level by CaMCN and is not significantly activated by CaMNN; Kact or Ki values for the engineered CaMs are 2-3.5 nM. Smooth muscle myosin light chain kinase activity (gMLCK) is fully activated by CaMCN and is not significantly activated by either CaMNN or CaMCC; the Kact value for CaMCN is 2 nM and the Ki values for CaMNN and CaMCC are 10 and 40 nM, respectively. Cerebellar nitric oxide synthase activity (nNOS) is fully activated by CaMNN and CaMCN and is not significantly activated by CaMCC; the engineered CaMs have Kact or Ki values for this enzyme activity of 2-8 nM. These results indicate that the EF hand pairs contain distinct but overlapping sets of determinants for binding and activation of enzymes, with the greater degree of overlap in determinants for binding. Furthermore, while the structural changes associated with swapping the EF hand pairs do not affect activation of nNOS or gMLCK activities, they significantly reduce activation of skMLCK activity, indicating that this process requires specific determinants in CaM outside the EF hand pairs.
我们构建了三种工程化钙调蛋白（CaM），其中两个EF手性对相互替换或交换：CaMNN，其C末端EF手性对（第82 - 148位氨基酸残基）被N末端对（第9 - 75位氨基酸残基）的重复序列所取代；CaMCC，其N末端对被C末端对的重复序列所取代；CaMCN，两个EF手性对进行了交换。骨骼肌肌球蛋白轻链激酶（skMLCK）活性被CaMCC激活至最大水平的75%，被CaMCN激活至最大水平的45%，而CaMNN对其无显著激活作用；工程化CaM的Kact或Ki值为2 - 3.5 nM。平滑肌肌球蛋白轻链激酶活性（gMLCK）被CaMCN完全激活，而CaMNN和CaMCC对其均无显著激活作用；CaMCN的Kact值为2 nM，CaMNN和CaMCC的Ki值分别为10 nM和40 nM。小脑一氧化氮合酶活性（nNOS）被CaMNN和CaMCN完全激活，而CaMCC对其无显著激活作用；工程化CaM对该酶活性的Kact或Ki值为2 - 8 nM。这些结果表明，EF手性对包含用于酶结合和激活的不同但重叠的决定簇集，其中结合决定簇的重叠程度更大。此外，虽然与交换EF手性对相关的结构变化不影响nNOS或gMLCK活性的激活，但它们显著降低了skMLCK活性的激活，表明该过程需要CaM中EF手性对之外的特定决定簇。
