Diversity in the C3b 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.a111.298984

Cited by 8 publications

(37 citation statements)

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“…NlpC truncation A (NlpC-A) was also overexpressed from a modified version of the pT7HMT vector that encodes a cysteine residue as the first amino acid of the polypeptide of interest (13). Site-specifically biotinylated Cys-NlpC-A was produced using the EZ-Link maleimide-PEG 2 -biotin reagent (Thermo Fisher Scientific [Rockford, IL]) according to the manufacturer's suggestions.…”

Section: Methodsmentioning

confidence: 99%

“…Quantitative binding assays between truncated NlpC, its homolog (MAP1204c), and 14C5 were conducted by employing a luminescent microbead AlphaScreen technology (7,13). The principle of this assay relies upon a streptavidin donor bead, which recognizes a biotinylated ligand that binds a second target protein, which itself can be adsorbed to acceptor beads that recognize mouse monoclonal IgG.…”

Section: Methodsmentioning

confidence: 99%

“…The AlphaScreen signal was normalized to wells containing no inhibitor, and a dose-response curve was generated by plotting normalized signal versus log[competitor (M)]. Equilibrium dissociation constants (K D ) were calculated by nonlinear curve fitting to a four-parameter doseresponse inhibition curve (variable slope) using GraphPad Prism5 software (GraphPad, La Jolla, CA) as previously described (13).…”

Section: Methodsmentioning

confidence: 99%

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MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne's Disease

Bannantine

Lingle

Stabel

et al. 2012

Clin. Vaccine Immunol.

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The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that contains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne's disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were expressed and evaluated with sera from cattle with Johne's disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a distinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne's Disease

Bannantine

Lingle

Stabel

et al. 2012

Clin. Vaccine Immunol.

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“…Recent biochemical and structural studies have shed light on several mechanistic aspects of SCIN protein function (13)(14)(15). SCIN-A-containing co-crystal structures revealed that SCIN-A interacts directly with C3b in the absence of the Bb fragment and that the SCIN-A/C3b binding site is identical to the SCIN-A/C3c site (10,13).…”

Section: Complement Is a Network Of Interacting Circulatory And Cell mentioning

confidence: 99%

“…SCIN-A-containing co-crystal structures revealed that SCIN-A interacts directly with C3b in the absence of the Bb fragment and that the SCIN-A/C3b binding site is identical to the SCIN-A/C3c site (10,13). Subsequent studies using a protease-stable form of the SCIN-B protein showed that, like SCIN-A, SCIN-B binds C3b near the MG7 domain (14). Together, these structure/function studies identified residues on the second ␣-helix of SCINs that are required for high affinity interaction with C3b (14).…”

Section: Complement Is a Network Of Interacting Circulatory And Cell mentioning

confidence: 99%

A Structurally Dynamic N-terminal Helix Is a Key Functional Determinant in Staphylococcal Complement Inhibitor (SCIN) Proteins

Garcia¹,

Summers²,

Ramyar³

et al. 2013

Journal of Biological Chemistry

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

et al. 2017

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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.

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Diversity in the C3b contact residues and tertiary structures of the staphylococcal complement inhibitor (SCIN) protein family.

Abstract: Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.

Cited by 8 publications

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MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne's Disease

MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne's Disease

A Structurally Dynamic N-terminal Helix Is a Key Functional Determinant in Staphylococcal Complement Inhibitor (SCIN) Proteins

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

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