Conformational Fluctuations as an Intrinsic Mechanism of Action of Lipase Foldase Studied by the High-Precision FRET Toolkit and MD Simulations

Abstract

The lipase foldase (Lif) of P. aeruginosa is a steric chaperon, which acts as a conformational switch for the activation of lipase A (LipA). In the Lif:LipA complex, Lif forms flexible α-helical scaffold embracing LipA in headphone-like structure. In the unbound form, Lif does not stay in the hollow “headphone” conformation but rather exhibits large-scale conformational dynamics, where this α-helical structure undergoes reversible collapses and extensions on the microsecond to sub-microsecond timescale. This process allows Lif to bind LipA despite the fickleness of structure. Structural details of the process are studied by the fluorescence spectroscopic toolkit: a FRET distance landscape is investigated by ensemble lifetime-resolved FRET (eTCSPC), the timescales of dynamics by correlation techniques (filtered FCS) and overall FRET populations and dynamics by single-molecule MFD studies. The combination of these techniques allows drawing a kinetic scheme within the limiting states of the conformational ensemble of Lif, unraveling the mechanism of Lif action. The study is supported by all-atom MD simulations. The combination of accurate FRET, MFD, FRET positioning and screening (FPS) and fFCS with MD simulations in a hybrid approach provides in-depth insight in the complexity of the conformational dynamics of flexible proteins and protein complexes.