PR3 Interacts Directly to Lipid Bilayers: Evidence from MD Simulations and SPR Experiments

Abstract

Proteinase 3 (PR3) is a serine protease of the neutrophils involved in inflammation processes. Its membrane expression is a risk factor for chronic inflammatory diseases. Experimental data led to divergent hypotheses on the binding mode of PR3 to the plasma membrane of neutrophils. While several studies performed on cell lines have identified a number of partner proteins, no direct interaction between hPR3 and any of these potential partners has been demonstrated (reviewed in Hajjar et al, FEBS J., 2010). Differential scanning calorimetry and spectrophotometric measurements, on the other hand, show a direct interaction of PR3 with DMPC vesicles (Goldman et al., Eur J Biochem, 1999).Using molecular dynamics simulations, we have characterized the membrane-binding site of PR3. Electrostatic surface potential calculations and simulations with an implicit membrane model (IMM1) have showed that PR3 possesses basic amino acids that provide the driving force to orient the protein at the membrane surface, so that a hydrophobic patch can anchor into the hydrophobic region of the membrane. In vitro mutagenesis experiments have confirmed the role of both types of amino acids. All-atom MD simulations and MM/PBSA energy decomposition identify three types of interactions contributing to the anchoring of PR3: hydrogen bonding and charge-pairing with lipid phosphate groups (R177, R186A, K187 and R222), hydrophobic anchoring into the lipid bilayer core (F165, F166 and L223) and cation-π interactions with the choline groups of DMPC (W218).Surface Plasmon Resonance experiments show a strong binding of Pr3 to DMPC and POPC bilayers.Altogether our results demonstrate that Pr3 has the ability to directly bind to lipid liposomes and most probably also to the plasma membrane of neutrophils.