sPLA2 Wobbles on the Lipid Bilayer between Three Positions, Each Involved in the Hydrolysis Process

Abstract

Secreted phospholipases A2 (sPLA2s) are peripheral membrane enzymes that hydrolyze phospholipids in the sn-2 position. The action of sPLA2 is associated with the work of two active sites. One, the interface binding site (IBS), is needed to bind the enzyme to the membrane surface. The other one, the catalytic site, is needed to hydrolyze the substrate. The interplay between sites, how the substrate protrudes to, and how the hydrolysis products release from, the catalytic site remains in the focus of investigations. Here, we report that bee venom PLA2 has two additional interface binding modes and enzyme activity through constant switching between three different orientations (modes of binding), only one of which is responsible for substrate uptake from the bilayer. The finding was obtained independently using atomic force microscopy and molecular dynamics. Switching between modes has biological significance: modes are steps of the enzyme moving along the membrane, product release in biological milieu, and enzyme desorption from the bilayer surface.

Alternative Targets for sPLA2 Activity: Role of Membrane-Enzyme Interactions

“…In this case the catalytic site remains out of contact with the lipid bilayer, and thus the substrate cannot be bound. The possibility is based on the recent finding of multiple membrane-binding sites on the surface of sPLA2, only one of which is associated with the hydrolysis process [ 19 ]. The third possibility to prevent hydrolysis is to prevent a substrate from binding to the enzyme, keeping the catalytic site unaltered ( Figure 1 , process 3).…”

“…Recently, Kuzmina and co-authors [ 19 ] had reported that the bvPLA2 enzyme has three different membrane-binding modes ( Figure 3 ). This finding was obtained independently in “wet” experiments using atomic force microscopy and in silico experiments using molecular dynamics simulations.…”

“…The modification reduced lipid hydrolysis by 80%. In the case of bvPLA2, Trp128 is not involved in the IBS (mode 1) [ 30 ] but is involved in mode 2 (see also Figure 4 ) [ 19 ]. That is the reason that protein binding to the bilayer surface could be traced by tryptophan fluorescence [ 20 , 30 , 60 , 61 ] (upon binding to the bilayer, the tryptophan residue faces a change in the polarity of the environment, with a corresponding change in the fluorescence).…”

“…That is the reason that protein binding to the bilayer surface could be traced by tryptophan fluorescence [ 20 , 30 , 60 , 61 ] (upon binding to the bilayer, the tryptophan residue faces a change in the polarity of the environment, with a corresponding change in the fluorescence). It is then assumed [ 19 ] that formylkynurenine, being a reactive species, reacts with an amino group of the DOPE lipid (involved in the experiments by Nasri et al), thus stabilizing mode 2 and depopulating mode 1, preventing hydrolysis. In the experiment of Nasri et al, the enzyme was fixed in mode 2 using a chemical reaction between oxidized Trp128 residue and the DOPE lipid.…”

“…On the other hand, membrane composition could affect the enzyme distribution between modes. In the light of the works by Nasri et al [ 59 ] and Kuzmina et al [ 19 ], a change in the Trp128 fluorescence in bvPLA2 could be assigned to the change in protein distribution between modes. Thus, what is assigned to optimal binding (e.g., in [ 62 ]) is probably the biggest fraction of mode 2.…”