The structural basis for inhibition of the classical and lectin complement pathways by S. aureus extracellular adherence protein

Abstract

The extracellular adherence protein (Eap) plays a crucial role in pathogenesis and survival of Staphylococcus aureus by inhibiting the classical and lectin pathways of complement. We have previously shown that Eap binds with nanomolar affinity to complement C4b and disrupts the initial interaction between C4b and C2, thereby inhibiting formation of the classical and lectin pathway C3 pro-convertase. Although an underlying mechanism has been identified, the structural basis for Eap binding to C4b is poorly understood. Here, we show that Eap domains 3 and 4 each contain a low-affinity, but saturable binding site for C4b. Taking advantage of the high lysine content of Eap, we used a zero-length crosslinking approach to map the Eap binding site to both the α'- and γ-chains of C4b. We also probed the C4b/Eap interface through a chemical footprinting approach involving lysine modification, proteolytic digestion, and mass spectrometry. This identified seven lysines in Eap that undergo changes in solvent exposure upon C4b binding. We found that simultaneous mutation of these lysines to either alanine or glutamate diminished C4b binding and complement inhibition by Eap. Together, our results provide insight into Eap recognition of C4b, and suggest that the repeating domains that comprise Eap are capable of multiple ligand-binding modes.

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

“…These data, along with the mutagenesis and functional data presented herein demonstrate that EAP domains are capable of structurally divergent modes of ligand recognition. Further structure/function analyses of EAP domains' complexes with NSPs, and other known ligands such as complement component C4b, should only serve to broaden our understanding of the many interactions formed by this diverse family of immune evasion proteins.…”

1H, 15N, and 13C resonance assignments of the third domain from the S. aureus innate immune evasion protein Eap

“…The extracellular adherence protein (Eap) is a multidomain protein that participates in various protein-protein interactions that inhibit the innate immune response, including both the complement system (Woehl et al 2014) and Neutrophil Serine Proteases (NSPs) (Stapels et al 2014). The third domain of Eap, Eap3, is an ~11 kDa protein that was recently shown to bind complement component C4b (Woehl et al 2017) and therefore play an essential role in inhibiting the classical and lectin pathways of complement (Woehl et al 2014). Since structural characterization of Eap3 is still incomplete, we acquired a series of 2D and 3D NMR spectra of Eap3 in solution.…”

Intracellular Expression of Antifreeze Peptides in Food Grade Lactococcus lactis and Evaluation of Their Cryoprotective Activity

“…The cellular debris was removed by centrifuging at 12000 rpm for 10 min at 4 °C. The supernatant proteins were analyzed by Tricine‐sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and Western‐blot according to the same methods described by Woehl, Ramyar, Katz, Walker, and Geisbrecht (). The L. lactis strain NZ3900 with an empty pNZ8149 plasmid was cultivated under the same conditions to serve as a negative control.…”