Identification of the calmodulin-binding domain of recombinant calcium-independent phospholipase A(2)beta - Implications for structure and function

Calcium-independent phospholipase A(2) (iPLA(2)) is the major phospholipase A, activity in many cell types, and at least one isoform of this enzyme cl ass is physically and functionally coupled to calmodulin (CaM) in a reversi ble calcium-dependent fashion. To identify the domain in recombinant iPLA(2 )beta (riPLA(2)beta) underlying this interaction, multiple techniques were employed. First, we identified calcium-activated CaM induced alterations in the kinetics of proteolytic fragment generation during limited trypsinolys is (i.e. CaM footprinting), Tryptic digests of riPLA(2)beta (83 kDa) in the presence of EGTA alone, Ca+2 alone, or EGTA and CaM together resulted in t he production of a major 68-kDa protein whose kinetic rate of formation was specifically attenuated in incubations containing CaM and Ca+2 together. W estern blotting utilizing antibodies directed against either the N- or C-te rminal regions of riPLA(2)beta indicated the specific protection of riPLA(2 )beta by calcium-activated CaM at a cleavage site approximate to 15 kDa fro m the C terminus. Moreover, calcium-activated calmodulin increased the kine tic rate of tryptic cleavage near the active site of riPLA(2)beta. Second, functional characterization of products from these partial tryptic digests demonstrated that approximate to 90% of the 68-kDa riPLA(2)beta tryptic pro duct (i.e. lacking the 18-kDa C-terminus) did not bind to a CaM affinity ma trix in the presence of Ca2+, although >95% of the noncleaved riPLA(2)beta as well as a 40-kDa C-terminal peptide bound tightly under these conditions . Third, when purified riPLA(2)beta was subjected to exhaustive trypsinolys is followed by ternary complex CaM affinity chromatography, a unique trypti c peptide ((694)AWSEM-VGIQYFR(705)) within the 15-kDa C-terminal fragment w as identified by RP-HPLC, which bound to CaM-agarose in the presence but no t the absence of calcium ion. Fourth, fluorescence energy transfer experime nts demonstrated that this peptide (694-705) bound to dansyl-calmodulin in a calcium-dependent fashion. Collectively, these results identify multiple contact points in the 15-kDa C terminus as being the major but not necessar ily the only binding site responsible for the calcium-dependent regulation of iPLA(2)beta by CaM.