Identification of the calmodulin-binding domain of recombinant calcium-independent phospholipase A₂β. Implications for structure and function

Calcium-independent phospholipase A₂ (iPLA₂) is the major phospholipase A₂ activity in many cell types, and at least one isoform of this enzyme class is physically and functionally coupled to calmodulin (CaM) in a reversible calcium-dependent fashion. To identify the domain in recombinant iPLA₂β (riPLA₂β underlying this interaction, multiple techniques were employed. First, we identified calcium-activated CaM induced alterations in the kinetics of proteolytic fragment generation during limited trypsinolysis (i.e. CaM footprinting). Tryptic digests of riPLA₂β (83 kDa) in the presence of EGTA alone, Ca⁺² alone, or EGTA and CaM together resulted in the production of a major 68-kDa protein whose kinetic rate of formation was specifically attenuated in incubations containing CaM and Ca ⁺² together. Western blotting utilizing antibodies directed against either the N- or C-terminal regions of riPLA₂β indicated the specific protection of riPLA₂β by calcium-activated CaM at a cleavage site ≈15 kDa from the C terminus. Moreover, calcium-activated calmodulin increased the kinetic rate of tryptic cleavage near the active site of riPLA₂β. Second, functional characterization of products from these partial tryptic digests demonstrated that ≈90% of the 68-kDa riPLA₂β tryptic product (i.e. lacking the 15-kDa C-terminus) did not bind to a CaM affinity matrix in the presence of Ca²⁺, although > 95% of the noncleaved riPLA₂β as well as a 40-kDa C-terminal peptide bound tightly under these conditions. Third, when purified riPLA₂β was subjected to exhaustive trypsinolysis followed by ternary complex CaM affinity chromatography, a unique tryptic peptide ( ⁶⁹⁴AWSEM-VGIQYFR⁷⁰⁵) within the 15-kDa C-terminal fragment was identified by RP-HPLC, which bound to CaM-agarose in the presence but not the absence of calcium ion. Fourth, fluorescence energy transfer experiments demonstrated that this peptide (694-705) bound to dansylcalmodulin in a calcium-dependent fashion. Collectively, these results identify multiple contact points in the 15-kDa C terminus as being the major but not necessarily the only binding site responsible for the calcium-dependent regulation of iPLA₂β by CaM.