Persechini A, Stemmer P M, Ohashi I
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.
J Biol Chem. 1996 Dec 13;271(50):32217-25. doi: 10.1074/jbc.271.50.32217.
We have investigated the functional interchangeability of EF hands I and III or II and IV, which occupy structurally analogous positions in the native I-II and III-IV EF hand pairs of calmodulin. Our approach was to functionally characterize four engineered proteins, made by replacing in turn each EF hand in one pair by a duplicate of its structural analog in the other. In this way functional determinants we define as unique were localized to the component EF hands in each pair. Replacement of EF hand I by III reduces calmodulin-dependent activation of cerebellar nitric oxide synthase activity by 50%. Replacement of EF hand IV by II reduces by 60% activation of skeletal muscle myosin light chain kinase activity. There appear to be no major unique determinants for activation of these enzyme activities in the other EF hands. Replacement of EF hand III by I or IV by II reduces by 50-80% activation of smooth muscle myosin light chain kinase activity, and replacement of EF hand I by III or II by IV reduces by 90% activation of this enzyme activity. Thus, calmodulin-dependent activation of each of the enzyme activities examined, even the closely related kinases, is dependent upon a distinct pattern of unique determinants in the four EF hands of calmodulin. All the engineered proteins examined bind four Ca2+ ions with high affinity. Comparison of the Ca2+-binding properties of native and engineered CaMs indicates that the Ca2+-binding affinity of an engineered I-IV EF hand pair and a native I-II pair are similar, but an engineered III-II EF hand pair is intermediate in affinity to the native III-IV and I-II pairs, minimally suggesting that EF hands I and III contain unique determinants for the formation and function of EF hand pairs. The residues directly coordinating Ca2+ ion appear to play little or no role in establishing the different Ca2+-binding properties of the EF hand pairs in calmodulin.
我们研究了钙调蛋白天然I-II和III-IV EF手对中占据结构类似位置的EF手I和III或II和IV的功能互换性。我们的方法是对四种工程蛋白进行功能表征，这些蛋白是通过依次将一对中的每个EF手替换为另一对中其结构类似物的复制品而制成的。通过这种方式，我们定义为独特的功能决定因素被定位到每对中的组成EF手。用III替换EF手I可使小脑一氧化氮合酶活性的钙调蛋白依赖性激活降低50%。用II替换EF手IV可使骨骼肌肌球蛋白轻链激酶活性的激活降低60%。在其他EF手中似乎没有激活这些酶活性的主要独特决定因素。用I替换EF手III或用II替换EF手IV可使平滑肌肌球蛋白轻链激酶活性的激活降低50-80%，而用III替换EF手I或用IV替换EF手II可使该酶活性的激活降低90%。因此，所检测的每种酶活性的钙调蛋白依赖性激活，即使是密切相关的激酶，也依赖于钙调蛋白四个EF手中独特决定因素的不同模式。所有检测的工程蛋白都以高亲和力结合四个Ca2+离子。天然和工程化钙调蛋白的Ca2+结合特性比较表明，工程化I-IV EF手对和天然I-II对的Ca2+结合亲和力相似，但工程化III-II EF手对的亲和力介于天然III-IV和I-II对之间，这至少表明EF手I和III包含EF手对形成和功能的独特决定因素。直接配位Ca2+离子的残基在建立钙调蛋白中EF手对不同的Ca2+结合特性方面似乎几乎没有作用或没有作用。
