Diversity in the C3b Convertase Contact Residues and Tertiary Structures of the Staphylococcal Complement Inhibitor (SCIN) Protein Family

Garcia, Brandon L.; Summers, Brady J.; Lin, Zhuoer; Ramyar, Kasra X.; Ricklin, Daniel; Kamath, Divya; Fu, Zhenfang; Lambris, John D.; Geisbrecht, Brian V.

doi:10.1074/jbc.m111.298984

Cited by 26 publications

(51 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We next utilized a bead-based AlphaScreen assay to explore both the affinity and specificity of the C4b/Eap interaction in greater detail (20, 28). Whereas untagged Eap itself could diminish the luminescence signal generated by interaction between myc-tagged Eap and C4b-biotin in a dose-dependent manner, neither EapH1 nor EapH2 had any competitive effect even at the highest concentrations tested (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…2B). To study the C4b/Eap interaction through an independent approach, we constructed an SPR biosensor wherein C4b-biotin was uniformly immobilized on a streptavidin-coated surface similarly to what we have previously reported for C3b-biotin (10, 20, 21, 28). Significantly, neither EapH1 nor EapH2 bound the C4b surface even at concentrations 10-fold higher than those which showed clear evidence of C4b binding by Eap (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This assay system is based upon modification of a previously published protocol (20) and is established via the following principle: a luminescence signal is generated by laser excitation of a streptavidin-coated donor bead, which recognizes C4b-biotin that binds directly to a second target protein (in this case myc-Eap), which itself can be adsorbed to an acceptor bead coated with anti-c-myc monoclonal IgG. C4b/Eap competition binding assays were carried out in a total volume of 25 μl by adding each assay component to the following final concentrations: 50 nM myc-Eap, 5 nM C4b-biotin, 20 μg/μl anti-c-myc AlphaScreen acceptor beads, and 20 μg/μl AlphaScreen donor beads.…”

Section: Methodsmentioning

confidence: 99%

“…Reactions were performed over 2.5 h and were begun by incubating the C4b-biotin, myc-Eap, and a given concentration of each competitor protein for 1 h. Following this, the acceptor beads were added, incubated for an hour, then the donor beads were added and incubated for an additional 0.5 h. At that point, the donor beads were excited at 680 nm and the evolving AlphaScreen signal (photon counts/s at 630 nm) for each data point was measured using an EnSpire multimode plate reader (Perkin Elmer Life Sciences). Data analysis and curve fitting was carried out as previously described (20). …”

Section: Methodsmentioning

confidence: 99%

“…To calculate residual C2 binding, the sensorgram of the corresponding Eap/Eap34 injection alone was subtracted from the Eap/Eap34+C2 injection series. The averaged response for the 5 s preceding the injection stop was plotted against the concentration of Eap/Eap34 and fit to a dose-response inhibition curve by non-linear regression as previously described (20). Regeneration of the surface was carried out with 2 M NaCl for 2 min followed by 0.2 M sodium citrate for 2 min.…”

Section: Methodsmentioning

confidence: 99%

See 4 more Smart Citations

The Extracellular Adherence Protein from Staphylococcus aureus Inhibits the Classical and Lectin Pathways of Complement by Blocking Formation of the C3 Proconvertase

Woehl

Stapels

Garcia

et al. 2014

The Journal of Immunology

Self Cite

View full text Add to dashboard Cite

The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. While the majority of staphylococcal complement inhibitors act on the alternative pathway (AP) to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical (CP) and lectin (LP) pathways. We screened a collection of recombinant, secreted staphylococcal proteins to determine if S. aureus produces other molecules that inhibit either the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 pro-convertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits the two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

The Extracellular Adherence Protein from Staphylococcus aureus Inhibits the Classical and Lectin Pathways of Complement by Blocking Formation of the C3 Proconvertase

Woehl

Stapels

Garcia

et al. 2014

The Journal of Immunology

Self Cite

View full text Add to dashboard Cite

show abstract

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

et al. 2017

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Staphylococcal Immune Evasion Proteins: Structure, Function, and Host Adaptation

Koymans

Vrieling

Gorham

et al. 2015

Current Topics in Microbiology and Immunology

View full text Add to dashboard Cite

Staphylococcus aureus is a successful human and animal pathogen. Its pathogenicity is linked to its ability to secrete a large amount of virulence factors. These secreted proteins interfere with many critical components of the immune system, both innate and adaptive, and hamper proper immune functioning. In recent years, numerous studies have been conducted in order to understand the molecular mechanism underlying the interaction of evasion molecules with the host immune system. Structural studies have fundamentally contributed to our understanding of the mechanisms of action of the individual factors. Furthermore, such studies revealed one of the most striking characteristics of the secreted immune evasion molecules: their conserved structure. Despite high-sequence variability, most immune evasion molecules belong to a small number of structural categories. Another remarkable characteristic is that S. aureus carries most of these virulence factors on mobile genetic elements (MGE) or ex-MGE in its accessory genome. Coevolution of pathogen and host has resulted in immune evasion molecules with a highly host-specific function and prevalence. In this review, we explore how these shared structures and genomic locations relate to function and host specificity. This is discussed in the context of therapeutic options for these immune evasion molecules in infectious as well as in inflammatory diseases.

show abstract

Diversity in the C3b Convertase Contact Residues and Tertiary Structures of the Staphylococcal Complement Inhibitor (SCIN) Protein Family

Cited by 26 publications

References 43 publications

The Extracellular Adherence Protein from Staphylococcus aureus Inhibits the Classical and Lectin Pathways of Complement by Blocking Formation of the C3 Proconvertase

The Extracellular Adherence Protein from Staphylococcus aureus Inhibits the Classical and Lectin Pathways of Complement by Blocking Formation of the C3 Proconvertase

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

Staphylococcal Immune Evasion Proteins: Structure, Function, and Host Adaptation

Contact Info

Product

Resources

About