Evidence for multiple modes of neutrophil serine protease recognition by the EAP family of Staphylococcal innate immune evasion proteins

Abstract

Neutrophils contain high levels of chymotrypsin-like serine proteases (NSPs) within their azurophilic granules that have a multitude of functions within the immune system. In response, the pathogen Staphylococcus aureus has evolved three potent inhibitors (Eap, EapH1, and EapH2) that protect the bacterium as well as several of its secreted virulence factors from the degradative action of NSPs. We previously showed that these so-called EAP domain proteins represent a novel class of NSP inhibitors characterized by a non-covalent inhibitory mechanism and a distinct target specificity profile. Based upon high levels of structural homology amongst the EAP proteins and the NSPs, as well as supporting biochemical data, we predicted that the inhibited complex would be similar for all EAP/NSP pairs. However, we present here evidence that EapH1 and EapH2 bind the canonical NSP, Neutrophil Elastase (NE), in distinct orientations. We discovered that alteration of EapH1 residues at the EapH1/NE interface caused a dramatic loss of affinity and inhibition of NE, while mutation of equivalent positions in EapH2 had no effect on NE binding or inhibition. Surprisingly, mutation of residues in an altogether different region of EapH2 severely impacted both the NE binding and inhibitory properties of EapH2. Even though EapH1 and EapH2 bind and inhibit NE and a second NSP, Cathepsin G, equally well, neither of these proteins interacts with the structurally related, but non-proteolytic granule protein, azurocidin. These studies expand our understanding of EAP/NSP interactions and suggest that members of this immune evasion protein family are capable of diverse target recognition modes.

Local structural plasticity of the Staphylococcus aureus evasion protein EapH1 enables engagement with multiple neutrophil serine proteases

“…While the results presented here show that EapH1 uses globally similar ligand-binding mode to recognize two different NSPs (Fig. 4), we recently provided evidence suggesting an altogether different NE-binding mode by the EAP domain protein, EapH2 (25). Since no crystal structure was available for NE/EapH2, we used computational modeling to provide initial insight into this complex (25,27).…”

“…Although Staphylococcus aureus expresses three different EAP domain proteins that act as high-affinity NSP inhibitors (7,21), a majority of our molecular level understanding on how EAP proteins exhibit this function has come from studies on EapH1 (21,25,26). In this investigation, we expanded upon our previous work on EapH1 by characterizing the kinetics and structural basis for its inhibition of CG.…”

“…We believe this observation reflects more about the incredibly potent inhibition of NE by EapH1 (Ki ~21 ± 5 pM) rather than a deficiency of its inhibition of CG (Ki ~9.8 ± 4.7 nM), which is still quite potent in its own right. In the past, we have extensively used sitedirected mutagenesis to define the functional contributions of various residues within EAP domain proteins, particularly as it pertains to inhibition of NSPs (25)(26)(27). Unfortunately, we noted that nearly all the specificity-driving interactions between EapH1 and CG appear to directly involve backbone atoms from the inhibitor (Fig.…”

“…This structure revealed that EapH1 forms a highly complementary, but non-covalent complex with NE that blocks substrate access to the protease active site (21). Interestingly, while a loop spanning EapH1 residues V53 to Y63 is responsible for a large majority of the buried surface area at the NE/EapH1 interface, mutation of sidechains in this region did not result in a noteworthy loss of EapH1 activity (25). Loss of EapH1 R89, however, diminished EapH1 activity by nearly 15,000-fold (26).…”

“…Loss of EapH1 R89, however, diminished EapH1 activity by nearly 15,000-fold (26). Subsequent studies showed that a second family member, EapH2, achieves inhibition of NE through an entirely different binding mode (25,27), despite sharing ~50% sequence identity with EapH1. This series of results was most unexpected, and raised questions as to whether EapH1 relied on the same protease-binding mode to inhibit other NSPs.…”