Allosteric modulation of the integral enzyme OmpLA by membrane asymmetry.

Abstract

Plasma membranes are well-known for their distinct lipid composition of their leaflets. Little is known about a potential coupling of membrane asymmetry to protein function. Protein dimerization, for example, is prone to be affected by bulk membrane properties. The outer membrane protein phospholipase A (OmpLA) is a well-studied integral enzyme that hydrolyses phospholipids upon dimerization. Thus, OmpLA appears as an ideal candidate for closer scrutiny. We therefore interrogated the activity of OmpLA in symmetric and asymmetric vesicles composed of palmitoyl oleoyl phosphatidylcholines and palmitoyl oleoyl phosphatidylethanolamines. The kinetics of hydrolysis of both lipids were determined using high-performance thin-layer chromatography and analyzed in terms of rate equations. Interestingly, we found that the hydrolysis of both lipids slowed down by about one order of magnitude upon imposing membrane asymmetry. Considering a mechanical coupling of the ensuing differential-stress changes to protein activation allowed us reproduce the essential features of this finding. We estimate that a differential stress translating to mechanical work of at least 4 kBT is needed for a significant decrease of phospholipid hydrolysis. We thus provide unambiguous evidence for a functional coupling between membrane asymmetry and OmpLA activity. It is likely that this extends in a related fashion also other integral membrane proteins.