Computational prediction of the binding site of proteinase 3 to the plasma membrane

Abstract

Proteinase 3 (PR3) is a neutrophil-derived serine proteinase localized within cytoplasmic granules which can be released upon activation. PR3 is exposed at the neutrophil plasma membrane where it can mediate proinflammatory effects. Moreover, PR3 membrane expression is of special relevance in patients with Wegener's granulomatosis, a systemic vasculitis presenting anticytoplasmic neutrophil autoantibodies (ANCA) against PR3, which can bind to PR3 expressed at the surface of neutrophils and amplify their activation state. Therefore, it is of special relevance to unravel the molecular mechanisms governing its association with the membrane to be able to modulate it. To this end, we performed molecular dynamics (MD) simulations of PR3 with the implicit membrane model IMM1-GC to identify its interfacial binding site (IBS). Both the energies and structures resulting from the MD suggest that PR3 associates strongly with anionic membranes. We observe a unique IBS consisting of five basic (R177, R186A, R186B, K187, R222) and six hydrophobic (F165, F166, F224, L223, F184, W218) amino acids. The basic residues provide the driving force to orient PR3 at the membrane surface, so that the hydrophobic residues can anchor into the hydrocarbon region. Energy decomposition and in silico mutations show that only a few residues account for the membrane association. Similar calculations with HNE suggest a different membrane-binding mechanism. Our results agree with previous experimental observations and this work predicts, for the first time, the structural determinants of the binding of PR3 to membranes.

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

“…Membrane PR3 is not solubilized by high salt concentrations, which means that its membrane association is not charge dependant (Witko-Sarsat et al, 1999a;Korkmaz et al, 2009). Unlike HNE and CG, PR3 bears at its surface a hydrophobic patch formed by residues Phe166, Ile217, Trp218, Leu223, and Phe224 that is involved in membrane binding (Goldmann et al, 1999;Hajjar et al, 2008) (Fig. 3B).…”

A Hydrophobic Patch on Proteinase 3, the Target of Autoantibodies in Wegener Granulomatosis, Mediates Membrane Binding via NB1 Receptors

“…Direct interactions of PR3 to reconstituted lipid bilayers and lipid vesicles were recently studied, and a dissociation constant of 4.5 M relating to lipid vesicles with a mixed composition has been determined. A hydrophobic surface patch of PR3 consisting of Phe-166, Ile-217, Trp-218, Leu-223, and Phe-224 was suggested to insert into lipid bilayers (16,17).…”

New Selective Peptidyl Di(chlorophenyl) Phosphonate Esters for Visualizing and Blocking Neutrophil Proteinase 3 in Human Diseases

“…This results in increased resistance to inhibition by ␣-1-proteinase inhibitor, its main natural inhibitor in plasma (31). The resistance has been attributed to steric hindrance of interactions with high molecular mass (50 kDa) serpin (33,34). We examined this hypothesis using the cell-impermeable low M r compound 1 to inhibit the PR3 on the surface of activated neutrophils purified from control subjects and patients with GPA.…”