LOCALIZATION OF UNIQUE FUNCTIONAL DETERMINANTS IN THE CALMODULIN LOBES TO INDIVIDUAL EF HANDS

We have investigated the functional interchangeability of EF hands I a nd 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 w as to functionally characterize four engineered proteins, made by repl acing in turn each EF hand in one pair by a duplicate of its structura l analog in the other. In this way functional determinants we define a s unique were localized to the component EF hands in each pair, Replac ement of EF hand I by III reduces calmodulin-dependent activation of c erebellar nitric oxide synthase activity by 50%, Replacement of EF han d IV by II reduces by 60% activation of skeletal muscle myosin light c hain kinase activity, There appear to be no major unique determinants for activation of these enzyme activities in the other EF hands. Repla cement 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 up on a distinct pattern of unique determinants in the four EF hands of c almodulin, All the engineered proteins examined bind four Ca2+ ions wi th high affinity, Comparison of the Ca2+-binding properties of native and engineered CaMs indicates that the Ca2+-binding affinity of an eng ineered I-IV EF hand pair and a native I-II pair are similar, but an e ngineered III-II EF hand pair is intermediate in affinity to the nativ e 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 litt le or no role in establishing the different Ca2+-binding properties of the EF hand pairs in calmodulin.