Identification of Hydrophobic Interactions between Proteins and Lipids:  Free Fatty Acids Activate Phospholipase C δ1 via Allosterism

Abstract

Lipids are well recognized ligands that bind to proteins in a specific manner and regulate their function. Most attention has been placed on the headgroup of phospholipids, and little is known about the role of the acyl chains in mediating any effects of lipids on proteins. In this report, free fatty acids (FFA) were found to bind and activate phospholipase C delta1(PLC delta1). The unsaturated FFA arachidonic acid (AA) and oleic acid were able to stimulate PLC delta1 up to 30-fold in a dose-dependent manner. The saturated FFA stearic acid and palmitic acid were less efficacious than unsaturated FFA, activating the enzyme up to 8-fold. The mechanism of activation appears to be due to a change in K(m) for substrate; 50 microM arachidonate reduced the K(m) for the soluble PLC substrate diC(4)PI from 1.7 +/- 0.6 mM to 0.24 +/- 0.04 mM (7-fold reduction). V(max) was not significantly altered. PLC delta1 bound to sucrose-loaded vesicles that contained AA in a concentration-dependent manner. A fragment of PLC delta1 that encompasses the EF-hand domain also bound to micelles containing AA using nondenaturing PAGE. This same fragment also inhibited AA activation of PLC delta1 in a competition assay. These results suggest that the function of the EF-hand domain of PLC delta1 is to bind lipid and to allosterically regulate catalysis. These results also suggest that esterified and nonesterified fatty acids can bind to and regulate protein function, identifying a functional role for hydrophobic interactions between lipids and proteins.

Stimulation of phospholipase Cβ by membrane interactions, interdomain movement, and G protein binding — How many ways can you activate an enzyme?

“…Unlike the role of EF-hands in other host proteins, the EF-hands of PLCs do not undergo Ca 2+ -dependent conformational changes. There are data suggesting that the EF-hands of PLC-δ1 bind specifically to fatty acids and that this binding stimulates the catalytic activity of the enzyme, implying that PLCs may be sensitive to the nature of the hydrocarbon chains [44,45]. Although the crystal structure shows limited contact between the EF hands and the catalytic domain, their role in the regulation of the enzyme either directly or through interaction with other proteins or cofactors remains to be seen.…”

“…This peptide was therefore defined as the signal transfer region of Gβ. Extensive studies with other peptides identified a second signal transfer region (Gβ [42][43][44][45][46][47][48][49][50][51][52][53][54] ) in the seventh blade of Gβ, and revealed segments in the second, fifth and seventh blade that hold the Gβγ/PLC-β2 complex together during activation ( Figure 5) [67,68]. In addition, it was found that the geranylgeranylated moiety attached to the C-terminal segment of the Gγ, which is proximal to the membrane and anchors Gβγ to lipids, binds to the PLC-β2 [69,70].…”

“…More striking is the identification of soluble fragments of Gβγ, Gβ [86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103][104][105] and Gβ [42][43][44][45][46][47][48][49][50][51][52][53][54] , that are able to convey full activation of PLC-β2 [66,67,79]. Additionally, Gβγ has been shown to stimulate the phosphodiesterase step of the PI(4,5)P 2 hydrolysis reaction which is not dependent on membrane surfaces, and further biochemical studies have shown that in the presence of Gβγ subunits, product inhibition does not occur suggesting that activation is through an enhanced release of product [19].…”

Ca2+-independent Binding of Anionic Phospholipids by Phospholipase C δ1 EF-hand Domain

“…The isolated EF-hand domain of PLC ␦1 has previously been expressed and shown to interact with free fatty acids (13,14). To further explore its binding partners, interaction of rEF with various phospholipid species was examined by a centrifugation-filtration vesicle binding assay.…”

“…Based on the structural similarities between the PLC ␦1 EF-hand and other calcium-binding EF-hand proteins such as calmodulin, it has been suggested that the EF-hand domain of PLC ␦1 might serve a regulatory role through calcium binding. Elsewhere, interactions of the EF-hand domain with free fatty acids have also been suggested (13,14). Additionally, numerous EF-hand motif-containing proteins have been reported to be involved in lipid binding.…”