The Membrane‐Integrated Steric Chaperone Lif Facilitates Active Site Opening of Pseudomonas aeruginosa Lipase A

Abstract

Lipases are essential and widely used biocatalysts. Hence, the production of lipases requires a detailed understanding of the molecular mechanism of its folding and secretion. Lipase A from Pseudomonas aeruginosa, PaLipA, constitutes a prominent example that has additional relevance because of its role as a virulence factor in many diseases. PaLipA requires the assistance of a membrane-integrated steric chaperone, the lipasespecific foldase Lif, to achieve its enzymatically active state. However, the molecular mechanism of how Lif activates its cognate lipase has remained elusive. Here, we show by molecular dynamics simulations at the atomistic level and potential of mean force computations that Lif catalyzes the activation process of PaLipA by structurally stabilizing an intermediate PaLipA conformation, particularly a β-sheet in the region of residues 17-30, such that the opening of PaLipA's lid domain is facilitated. This opening allows substrate access to PaLipA's catalytic site. A surprising and so far not fully understood aspect of our study is that the open state of PaLipA is unstable compared to the closed one according to our computational and in vitro biochemical results. We thus speculate that further interactions of PaLipA with the Xcp secretion machinery and/or components of the extracellular matrix contribute to the remaining activity of secreted PaLipA.

Pseudomonas aeruginosa: An Audacious Pathogen with an Adaptable Arsenal of Virulence Factors

“…Lipases are secreted by the T2SS and cleave lipids, yielding free fatty acids and glycerol as final products [103]. LipA is the major lipase produced by P. aeruginosa and needs the chaperon lif to be activated [162]. It is encoded in the lipA/lipH operon, along with its cognate foldase LipH, which is also required for the expression of LipC [64].…”

Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation

“…We thus propose a mechanism of LipA activation based on the formation and stabilization of β-sheet 17-30 in LipA through interactions with Lif. We recently showed by molecular dynamics simulations and potential of mean force computations that stabilization of β-sheet 17-30 affects the dynamics of LipA's lid and thereby LipA activation 27 . Such small structural changes upon LipA activation are expected given the previously observed similarity of pre-active and native LipA 7,14 .…”

“…LipA (2 µM) was incubated with sLif (2 µM), sLif Y99A (2 µM), MD1 (2 µM) or MD1 Y99A (2 µM) overnight at 4 °C in TG buffer. The protein samples loaded into the measuring capillaries (Prometheus NT.Plex nanoDSF Grade Standard Capillary Chips) were heated from 15 °C to 95 °C (heating rate of 0.2 °C/min) and the intrinsic protein fluorescence was recorded at 330 nm and 350 nm using the Prometheus NT.Plex nanoDSF device (Nano Temper, Munich, Germany) 27 . The ratio of F 350nm and F 330nm and its first derivative were calculated by the PR.ThermControl software provided by the company.…”

“…The three-dimensional structure of P. aeruginosa Lif is currently unknown. Thus, a homology model of the P. aeruginosa sLif:LipA complex was constructed using the structure of the B. glumae lipase:foldase complex (PDB code 2ES4) as a template (sequence identity/ similarity: 39%/52%) for Lif and 41%/73% for LipA) 27 . The Phyre2 web server 41 was used for homology modelling.…”

“…The model obtained was energy minimized with the GROMOS96 43B1 force field as implemented in Swiss-PdbViewer 42 . After ten rounds of energy minimization, the C α atoms of the models were superimposed on the template structure and the model with the lowest RMSD was taken for further studies 27 . The model obtained was evaluated by using our in-house model quality assessment program TopScore 24,25 .…”