Staphylococcus aureus Evasion Proteins EapH1 and EapH2: Residue‐Level Investigation of an Alternative Binding Motif for Human Neutrophil Elastase

Abstract

The Staphylococcus aureus Extracellular Adherence Protein (Eap) and its homologs, EapH1 and EapH2, are a family of secreted proteins that potently inhibit the neutrophil serine proteases Neutrophil Elastase (hNE), Cathepsin G, and Proteinase 3. Similarly to EapH1, inhibition of hNE by EapH2 is characterized by a rapid association rate (2.9×105 M−1s−1) coupled with a very slow dissociation rate (5.9×10−4 s−1), yielding an apparent inhibition constant of 2.11 nM. Due to this slow off‐rate, inhibition of hNE by EapH2 is time‐dependent. A phenylalanine in EapH2 replaces the leucine in EapH1 that sits over the hNE catalytic serine and creates a potential steric clash. Indeed, the EapH1 L59F mutant is severely decreased in its ability to inhibit hNE (~9,500‐fold). When compared to the EapH1:hNE co‐crystal structure, a model of the EapH2:hNE complex predicts an alternative binding motif comprised of EapH2 residues 120 – 127. These putative interfacing residues were individually mutated and kinetically interrogated. The EapH2 N127A mutant resulted in the largest decrease in hNE inhibition (~200‐fold) and loss of the time‐dependent characteristic. Surprisingly, the time‐dependent characteristic was still abolished in the EapH2 T125A mutant, even though it was less perturbed in hNE inhibition (~25‐fold). T125 forms an intra‐molecular hydrogen bond to the carbonyl oxygen of N127 in the EapH2 crystal structure. Given these observations, we conclude (i) that EapH2 has an altogether distinct hNE binding motif than EapH1, (ii) that N127 is the main functional determinant in EapH2, and (iii) that T125 serves an ancillary role aiding in the optimal orientation of N127.Support or Funding InformationFunding was provided by a grant from the National Institute of General Medical Sciences (R01‐GM121511) to B.V.G.Comparison of EapH1 and EapH2 binding motifs.(A) EapH2 (magenta) binding to hNE, based on the energy minimized model. (B) EapH2 residues involved in the interface with hNE. (C) EapH1(green) binding to hNE (PDB:4nzl). (D) EapH1 residues involved in the interface with hNE.Figure 1