Abstract
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.
Highlights
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
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
In the CaM-nNOS complex, the C-terminal EF hand pair in CaM can be functionally replaced by the N-terminal EF hand pair, so it is not surprising to find that Kact and Fact values for x 2x2
Summary
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. Calmodulindependent 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. High resolution structures have been determined for the complexes between CaM and peptides representing the CaMbinding domains in either skeletal or smooth muscle myosin light chain kinase or in CaM-dependent protein kinase II [12, 13]. We localize unique functional determinants within the EF hand pairs to the individual EF hand domains
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