Localization of Unique Functional Determinants in the Calmodulin Lobes to Individual EF Hands

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, 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. All the engineered proteins examined bind four Ca 2؉ ions with high affinity. The crystal structure of the multifunctional regulatory protein, calmodulin (CaM), 1 shows an elongated molecule in which two globular lobes are joined by an extended central helix (1). Each globular region is composed of a pair of EF hand Ca 2ϩ -binding domains, which include all amino acid residues in CaM apart from residues 1-8 at the N terminus and 76 -81 in the central helix (2). We shall refer to these regions as the Nterminal leader and the central helix linker, respectively. CaM appears to recognize in its enzyme targets basic amphiphilic helix-forming segments that are generally termed CaM-binding domains; there is no consensus amino acid sequence for these regions (3-5). The simplest model for CaM-dependent enzyme activation is one in which the CaM-binding domain also functions as an intrasteric inhibitor whose influence is relieved when it is bound by CaM. In a more complex model, occupancy of the CaM-binding domain is insufficient for enzyme activation and further, secondary, CaM-target interactions are required. Data for CaM-dependent activation of myosin light chain kinase activity suggest that this latter model may apply, while much of the available data for CaM-dependent activation of nitric oxide synthase and cyclic nucleotide phosphodiesterase activities are consistent with the former (6 -11). Comparison of the CaHigh 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). Given the considerable internal structural homology seen in CaM, it is perhaps not surprising that these complexes each exhibit ...

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