The Experts below are selected from a list of 2022 Experts worldwide ranked by ideXlab platform
Daniel Ricklin - One of the best experts on this subject based on the ideXlab platform.
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Diversity in the C3b Convertase Contact Residues and Tertiary Structures of the Staphylococcal Complement Inhibitor (SCIN) Protein Family
Journal of Biological Chemistry, 2011Co-Authors: Brandon L. Garcia, Brady J. Summers, Zhuoer Lin, Kasra X. Ramyar, Daniel Ricklin, Divya V. Kamath, John D. Lambris, Brian V. GeisbrechtAbstract:To survive in immune-competent hosts, the pathogen Staphylococcus aureus expresses and secretes a sophisticated array of proteins that inhibit the complement system. Among these are the staphylococcal complement inhibitors (SCIN), which are composed of three active proteins (SCIN-A, -B, and -C) and one purportedly inactive member (SCIN-D or ORF-D). Because previous work has focused almost exclusively on SCIN-A, we sought to provide initial structure/function information on additional SCIN proteins. To this end we determined crystal structures of an active, N-terminal truncation mutant of SCIN-B (denoted SCIN-B18–85) both free and bound to the C3c fragment of complement component C3 at 1.5 and 3.4 Å resolution, respectively. Comparison of the C3c/SCIN-B18–85 structure with that of C3c/SCIN-A revealed that both proteins target the same functional hotspot on the C3b/C3c surface yet harbor diversity in both the type of residues and interactions formed at their C3b/C3c interfaces. Most importantly, these structures allowed identification of Arg44 and Tyr51 as residues key for SCIN-B binding to C3b and subsequent inhibition of the AP C3 convertase. In addition, we also solved several crystal structures of SCIN-D to 1.3 Å limiting resolution. This revealed an unexpected structural deviation in the N-terminal α helix relative to SCIN-A and SCIN-B. Comparative analysis of both electrostatic potentials and surface complementarity suggest a physical explanation for the inability of SCIN-D to bind C3b/C3c. Together, these studies provide a more thorough understanding of immune evasion by S. aureus and enhance potential use of SCIN proteins as templates for design of complement targeted therapeutics.
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Molecular Basis for Complement Recognition and Inhibition Determined by Crystallographic Studies of the Staphylococcal Complement Inhibitor (SCIN) Bound to C3c and C3b
2010Co-Authors: Brandon L. Garcia, Kasra X. Ramyar, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, William J. Mcwhorter, Brian V. GeisbrechtAbstract:The human complement system plays an essential role in innate and adaptive immunity by marking and eliminating microbial intruders. Activation of complement on foreign surfaces results in proteolytic cleavage of complement component 3 (C3) into the potent opsonin C3b, which triggers a variety of immune responses and participates in a self-amplification loop mediated by a multi-protein assembly known as the C3 convertase. The human pathogen Staphylococcus aureus has evolved a sophisticated and potent complement evasion strategy, which is predicated upon an arsenal of potent inhibitory proteins. One of these, the staphylococcal complement inhibitor (SCIN), acts at the level of the C3 convertase (C3bBb) and impairs downstream complement function by trapping the convertase in a stable but inactive state. Previously, we have shown that SCIN binds C3b directly and competitively inhibits binding of human factor H and, to a lesser degree, that of factor B to C3b. Here, we report the co-crystal structures of SCIN bound to C3b and C3c at 7.5 and 3.5 A limiting resolution, respectively, and show that SCIN binds a critical functional area on C3b. Most significantly, the SCIN binding site sterically occludes the binding sites of both factor H and factor B. Our results give insight into SCIN binding to activated derivatives of C3, explain how SCIN can recognize C3b in the absence of other complement components, and provide a structural basis for the competitive C3b-binding properties of SCIN. In the future, this may suggest templates for the design of novel complement inhibitors based upon the SCIN structure.
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A Molecular Insight into Complement Evasion by the Staphylococcal Complement Inhibitor Protein Family
The Journal of Immunology, 2009Co-Authors: Daniel Ricklin, Brandon L. Garcia, Apostolia Tzekou, William J. Mcwhorter, Michal Hammel, Georgia Sfyroera, V. Michael Holers, Andrew P. Herbert, Paul N. BarlowAbstract:Staphylococcus aureus possesses an impressive arsenal of complement evasion proteins that help the bacterium escape attack of the immune system. The staphylococcal complement inhibitor (SCIN) protein exhibits a particularly high potency and was previously shown to block complement by acting at the level of the C3 convertases. However, many details about the exact binding and inhibitory mechanism remained unclear. In this study, we demonstrate that SCIN directly binds with nanomolar affinity to a functionally important area of C3b that lies near the C terminus of its β-chain. Direct competition of SCIN with factor B for C3b slightly decreased the formation of surface-bound convertase. However, the main inhibitory effect can be attributed to an entrapment of the assembled convertase in an inactive state. Whereas native C3 is still able to bind to the blocked convertase, no generation and deposition of C3b could be detected in the presence of SCIN. Furthermore, SCIN strongly competes with the binding of factor H to C3b and influences its regulatory activities: the SCIN-stabilized convertase was essentially insensitive to decay acceleration by factor H and the factor I- and H-mediated conversion of surface-bound C3b to iC3b was significantly reduced. By targeting a key area on C3b, SCIN is able to block several essential functions within the alternative pathway, which explains the high potency of the inhibitor. Our findings provide an important insight into complement evasion strategies by S. aureus and may act as a base for further functional studies.
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Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor
Nature Immunology, 2009Co-Authors: Suzan H. M. Rooijakkers, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, Maartje Ruyken, Jin Wu, Robert Van Domselaar, Karel L Planken, Bert J C Janssen, Jos A G Van StrijpAbstract:Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg^2+-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics. Complement forms an ancient innate immune defense. Gros and colleagues provide new insight into the interactions between complement convertase C3b and its regulator factor H and with the staphylococcal inhibitor SCIN.
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Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor
Nature Immunology, 2009Co-Authors: Suzan H. M. Rooijakkers, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, Maartje Ruyken, Karel L Planken, Bert J C Janssen, Robert Van Domselaar, Jos A. G. Van StrijpAbstract:Complement forms an ancient innate immune defense. Gros and colleagues provide new insight into the interactions between complement convertase C3b and its regulator factor H and with the staphylococcal inhibitor SCIN.
Suzan H. M. Rooijakkers - One of the best experts on this subject based on the ideXlab platform.
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Staphylococcal Complement Inhibitor Modulates Phagocyte Responses by Dimerization of Convertases
The Journal of Immunology, 2009Co-Authors: Ilse Jongerius, Jos A. G. Van Strijp, Maartje Ruyken, Manon Puister, Suzan H. M. RooijakkersAbstract:The human pathogen Staphylococcus aureus produces several complement-evasion molecules that enable the bacterium to withstand the host immune response. The human-specific staphylococcal complement inhibitor (SCIN) blocks the central C3 convertase enzymes that trigger critical complement functions, such as C3b deposition, phagocytosis, and C5a generation. SCIN effectively blocks the conversion of C3 by alternative pathway C3 convertases (C3bBb), but also induces dimerization of these enzymes. In this study, we show that formation of dimeric convertases by SCIN is important for S. aureus immune evasion because it modulates complement recognition by phagocytic receptors. Dimeric, but not monomeric, SCIN convertases showed an impaired binding to complement receptor 1 and the complement receptor of the Ig superfamily. The dimerization site of SCIN is essential for its strong antiphagocytic properties. These studies provide critical insights into the unique immune-evasion strategies used by S. aureus.
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Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor
Nature Immunology, 2009Co-Authors: Suzan H. M. Rooijakkers, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, Maartje Ruyken, Jin Wu, Robert Van Domselaar, Karel L Planken, Bert J C Janssen, Jos A G Van StrijpAbstract:Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg^2+-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics. Complement forms an ancient innate immune defense. Gros and colleagues provide new insight into the interactions between complement convertase C3b and its regulator factor H and with the staphylococcal inhibitor SCIN.
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Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor
Nature Immunology, 2009Co-Authors: Suzan H. M. Rooijakkers, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, Maartje Ruyken, Karel L Planken, Bert J C Janssen, Robert Van Domselaar, Jos A. G. Van StrijpAbstract:Complement forms an ancient innate immune defense. Gros and colleagues provide new insight into the interactions between complement convertase C3b and its regulator factor H and with the staphylococcal inhibitor SCIN.
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Staphylococcal Complement Inhibitor: Structure and Active Sites
The Journal of Immunology, 2007Co-Authors: Suzan H. M. Rooijakkers, Fin J. Milder, Bart W. Bardoel, Maartje Ruyken, Jos A. G. Van Strijp, Piet GrosAbstract:The pathogenic bacterium Staphylococcus aureus counteracts the host immune defense by excretion of the 85 residue staphylococcal complement inhibitor (SCIN). SCIN inhibits the central complement convertases; thereby, it reduces phagocytosis following opsonization and efficiently blocks all downstream effector functions. In this study, we present the crystal structure of SCIN at 1.8 A resolution and the identification of its active site. Functional characterization of structure based chimeric proteins, consisting of SCIN and the structurally but nonfunctional homologue open reading frame-D, indicate an 18-residue segment (Leu-31-Gly-48) crucial for SCIN activity. In all complement activation pathways, chimeras lacking these SCIN residues completely fail to inhibit production of the potent mediator of inflammation C5a. Inhibition of alternative pathway-mediated opsonization (C3b deposition) and formation of the lytic membrane attack complex (C5b-9 deposition) are strongly reduced for these chimeras as well. For inhibition of the classical/lectin pathway-mediated C3b and C5b-9 deposition, the same residues are critical although additional sites are involved. These chimeras also display reduced capacity to stabilize the C3 convertases of both the alternative and the classical/lectin pathway indicating the stabilizing effect is pivotal for the complement inhibitory activity of SCIN. Because SCIN specifically and efficiently inhibits complement, it has a high potential in anti-inflammatory therapy. Our data are a first step toward the development of a second generation molecule suitable for such therapeutic complement intervention.
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Early expression of SCIN and CHIPS drives instant immune evasion by Staphylococcus aureus
Cellular Microbiology, 2006Co-Authors: Suzan H. M. Rooijakkers, Kok P. M. Van Kessel, Maartje Ruyken, Jos A. G. Van Strijp, Jos Van Roon, Willem J B Van WamelAbstract:Chemotaxis inhibitory protein of staphylococci (CHIPS) and Staphylococcal complement inhibitor (SCIN) are small, excreted molecules that play a crucial role in the staphylococcal defence against the human innate immune system. Here we show that they both counteract crucial acute responses of our immune system such as complement activation, neutrophil chemotaxis and neutrophil activation. By studying gene expression via promoter-green fluorescent protein fusions, Northern blots and protein expression analyses, we show that SCIN and CHIPS are produced during the early (exponential) growth stages. Although the SCIN and CHIPS genes are expressed simultaneously, they are differently regulated by various Staphylococcus aureus regulatory loci. However, the sae locus is crucial for upregulation of both SCIN and CHIPS. This is the first study that presents the expression of two extracellular S. aureus proteins early during growth. Because SCIN and CHIPS are both efficient modulators of neutrophil chemotaxis, phagocytosis and killing, their early expression is necessary for efficient modulation of the early immune response.
Apostolia Tzekou - One of the best experts on this subject based on the ideXlab platform.
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Molecular Basis for Complement Recognition and Inhibition Determined by Crystallographic Studies of the Staphylococcal Complement Inhibitor (SCIN) Bound to C3c and C3b
2010Co-Authors: Brandon L. Garcia, Kasra X. Ramyar, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, William J. Mcwhorter, Brian V. GeisbrechtAbstract:The human complement system plays an essential role in innate and adaptive immunity by marking and eliminating microbial intruders. Activation of complement on foreign surfaces results in proteolytic cleavage of complement component 3 (C3) into the potent opsonin C3b, which triggers a variety of immune responses and participates in a self-amplification loop mediated by a multi-protein assembly known as the C3 convertase. The human pathogen Staphylococcus aureus has evolved a sophisticated and potent complement evasion strategy, which is predicated upon an arsenal of potent inhibitory proteins. One of these, the staphylococcal complement inhibitor (SCIN), acts at the level of the C3 convertase (C3bBb) and impairs downstream complement function by trapping the convertase in a stable but inactive state. Previously, we have shown that SCIN binds C3b directly and competitively inhibits binding of human factor H and, to a lesser degree, that of factor B to C3b. Here, we report the co-crystal structures of SCIN bound to C3b and C3c at 7.5 and 3.5 A limiting resolution, respectively, and show that SCIN binds a critical functional area on C3b. Most significantly, the SCIN binding site sterically occludes the binding sites of both factor H and factor B. Our results give insight into SCIN binding to activated derivatives of C3, explain how SCIN can recognize C3b in the absence of other complement components, and provide a structural basis for the competitive C3b-binding properties of SCIN. In the future, this may suggest templates for the design of novel complement inhibitors based upon the SCIN structure.
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A Molecular Insight into Complement Evasion by the Staphylococcal Complement Inhibitor Protein Family
The Journal of Immunology, 2009Co-Authors: Daniel Ricklin, Brandon L. Garcia, Apostolia Tzekou, William J. Mcwhorter, Michal Hammel, Georgia Sfyroera, V. Michael Holers, Andrew P. Herbert, Paul N. BarlowAbstract:Staphylococcus aureus possesses an impressive arsenal of complement evasion proteins that help the bacterium escape attack of the immune system. The staphylococcal complement inhibitor (SCIN) protein exhibits a particularly high potency and was previously shown to block complement by acting at the level of the C3 convertases. However, many details about the exact binding and inhibitory mechanism remained unclear. In this study, we demonstrate that SCIN directly binds with nanomolar affinity to a functionally important area of C3b that lies near the C terminus of its β-chain. Direct competition of SCIN with factor B for C3b slightly decreased the formation of surface-bound convertase. However, the main inhibitory effect can be attributed to an entrapment of the assembled convertase in an inactive state. Whereas native C3 is still able to bind to the blocked convertase, no generation and deposition of C3b could be detected in the presence of SCIN. Furthermore, SCIN strongly competes with the binding of factor H to C3b and influences its regulatory activities: the SCIN-stabilized convertase was essentially insensitive to decay acceleration by factor H and the factor I- and H-mediated conversion of surface-bound C3b to iC3b was significantly reduced. By targeting a key area on C3b, SCIN is able to block several essential functions within the alternative pathway, which explains the high potency of the inhibitor. Our findings provide an important insight into complement evasion strategies by S. aureus and may act as a base for further functional studies.
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Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor
Nature Immunology, 2009Co-Authors: Suzan H. M. Rooijakkers, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, Maartje Ruyken, Jin Wu, Robert Van Domselaar, Karel L Planken, Bert J C Janssen, Jos A G Van StrijpAbstract:Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg^2+-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics. Complement forms an ancient innate immune defense. Gros and colleagues provide new insight into the interactions between complement convertase C3b and its regulator factor H and with the staphylococcal inhibitor SCIN.
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Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor
Nature Immunology, 2009Co-Authors: Suzan H. M. Rooijakkers, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, Maartje Ruyken, Karel L Planken, Bert J C Janssen, Robert Van Domselaar, Jos A. G. Van StrijpAbstract:Complement forms an ancient innate immune defense. Gros and colleagues provide new insight into the interactions between complement convertase C3b and its regulator factor H and with the staphylococcal inhibitor SCIN.
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Crystallization of human complement component C3b in the presence of a staphylococcal complement-inhibitor protein (SCIN).
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2009Co-Authors: Brandon L. Garcia, Kasra X. Ramyar, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, William J. Mcwhorter, Brian V. GeisbrechtAbstract:Staphylococcus aureus secretes a number of small proteins that effectively attenuate the human innate immune response. Among these, the staphylococcal complement-inhibitor protein (SCIN) disrupts the function of the complement component 3 (C3) convertase that is initiated through either the classical or the alternative pathway and thereby prevents amplification of the complement response on the bacterial surface. Recent studies have shown that SCIN may affect the activities of the C3 convertase by binding in an equimolar fashion to C3b, which is itself an integral although non-enzymatic component of the convertase. In order to better understand the nature of the C3b–SCIN interaction, the hanging-drop vapor-diffusion technique was used to crystallize human C3b in the presence of a recombinant form of SCIN. These crystals diffracted synchrotron X-rays to approximately 6 A Bragg spacing and grew in a primitive tetragonal space group (P41212 or P43212; unit-cell parameters a = b = 128.03, c = 468.59 A). Cell-content analysis of these crystals was consistent with the presence of either two 1:1 complexes or a single 2:2 assembly in the asymmetric unit, both of which correspond to a solvent content of 51.9%. By making use of these crystals, solution of the C3b–SCIN structure should further our understanding of complement inhibition and immune evasion by this pathogen.
Maartje Ruyken - One of the best experts on this subject based on the ideXlab platform.
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Identification of a staphylococcal complement inhibitor with broad host specificity in equid Staphylococcus aureus strains.
Journal of Biological Chemistry, 2018Co-Authors: Nienke W. M. De Jong, Brandon L. Garcia, Maartje Ruyken, Jos A. G. Van Strijp, Manouk Vrieling, G. Koop, Matt Brettmann, Piet C. Aerts, Mark A. Holmes, Ewan M. HarrisonAbstract:Staphylococcus aureus is a versatile pathogen capable of causing a broad range of diseases in many different hosts. S. aureus can adapt to its host through modification of its genome (e.g. by acquisition and exchange of mobile genetic elements that encode host-specific virulence factors). Recently, the prophage φSaeq1 was discovered in S. aureus strains from six different clonal lineages almost exclusively isolated from equids. Within this phage, we discovered a novel variant of staphylococcal complement inhibitor (SCIN), a secreted protein that interferes with activation of the human complement system, an important line of host defense. We here show that this equine variant of SCIN, eqSCIN, is a potent blocker of equine complement system activation and subsequent phagocytosis of bacteria by phagocytes. Mechanistic studies indicate that eqSCIN blocks equine complement activation by specific inhibition of the C3 convertase enzyme (C3bBb). Whereas SCIN-A from human S. aureus isolates exclusively inhibits human complement, eqSCIN represents the first animal-adapted SCIN variant that functions in a broader range of hosts (horses, humans, and pigs). Binding analyses suggest that the human-specific activity of SCIN-A is related to amino acid differences on both sides of the SCIN-C3b interface. These data suggest that modification of this phage-encoded complement inhibitor plays a role in the host adaptation of S. aureus and are important to understand how this pathogen transfers between different hosts.
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Staphylococcal Complement Inhibitor Modulates Phagocyte Responses by Dimerization of Convertases
The Journal of Immunology, 2009Co-Authors: Ilse Jongerius, Jos A. G. Van Strijp, Maartje Ruyken, Manon Puister, Suzan H. M. RooijakkersAbstract:The human pathogen Staphylococcus aureus produces several complement-evasion molecules that enable the bacterium to withstand the host immune response. The human-specific staphylococcal complement inhibitor (SCIN) blocks the central C3 convertase enzymes that trigger critical complement functions, such as C3b deposition, phagocytosis, and C5a generation. SCIN effectively blocks the conversion of C3 by alternative pathway C3 convertases (C3bBb), but also induces dimerization of these enzymes. In this study, we show that formation of dimeric convertases by SCIN is important for S. aureus immune evasion because it modulates complement recognition by phagocytic receptors. Dimeric, but not monomeric, SCIN convertases showed an impaired binding to complement receptor 1 and the complement receptor of the Ig superfamily. The dimerization site of SCIN is essential for its strong antiphagocytic properties. These studies provide critical insights into the unique immune-evasion strategies used by S. aureus.
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Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor
Nature Immunology, 2009Co-Authors: Suzan H. M. Rooijakkers, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, Maartje Ruyken, Jin Wu, Robert Van Domselaar, Karel L Planken, Bert J C Janssen, Jos A G Van StrijpAbstract:Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg^2+-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics. Complement forms an ancient innate immune defense. Gros and colleagues provide new insight into the interactions between complement convertase C3b and its regulator factor H and with the staphylococcal inhibitor SCIN.
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Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor
Nature Immunology, 2009Co-Authors: Suzan H. M. Rooijakkers, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, Maartje Ruyken, Karel L Planken, Bert J C Janssen, Robert Van Domselaar, Jos A. G. Van StrijpAbstract:Complement forms an ancient innate immune defense. Gros and colleagues provide new insight into the interactions between complement convertase C3b and its regulator factor H and with the staphylococcal inhibitor SCIN.
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Staphylococcal Complement Inhibitor: Structure and Active Sites
The Journal of Immunology, 2007Co-Authors: Suzan H. M. Rooijakkers, Fin J. Milder, Bart W. Bardoel, Maartje Ruyken, Jos A. G. Van Strijp, Piet GrosAbstract:The pathogenic bacterium Staphylococcus aureus counteracts the host immune defense by excretion of the 85 residue staphylococcal complement inhibitor (SCIN). SCIN inhibits the central complement convertases; thereby, it reduces phagocytosis following opsonization and efficiently blocks all downstream effector functions. In this study, we present the crystal structure of SCIN at 1.8 A resolution and the identification of its active site. Functional characterization of structure based chimeric proteins, consisting of SCIN and the structurally but nonfunctional homologue open reading frame-D, indicate an 18-residue segment (Leu-31-Gly-48) crucial for SCIN activity. In all complement activation pathways, chimeras lacking these SCIN residues completely fail to inhibit production of the potent mediator of inflammation C5a. Inhibition of alternative pathway-mediated opsonization (C3b deposition) and formation of the lytic membrane attack complex (C5b-9 deposition) are strongly reduced for these chimeras as well. For inhibition of the classical/lectin pathway-mediated C3b and C5b-9 deposition, the same residues are critical although additional sites are involved. These chimeras also display reduced capacity to stabilize the C3 convertases of both the alternative and the classical/lectin pathway indicating the stabilizing effect is pivotal for the complement inhibitory activity of SCIN. Because SCIN specifically and efficiently inhibits complement, it has a high potential in anti-inflammatory therapy. Our data are a first step toward the development of a second generation molecule suitable for such therapeutic complement intervention.
John D. Lambris - One of the best experts on this subject based on the ideXlab platform.
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Diversity in the C3b Convertase Contact Residues and Tertiary Structures of the Staphylococcal Complement Inhibitor (SCIN) Protein Family
Journal of Biological Chemistry, 2011Co-Authors: Brandon L. Garcia, Brady J. Summers, Zhuoer Lin, Kasra X. Ramyar, Daniel Ricklin, Divya V. Kamath, John D. Lambris, Brian V. GeisbrechtAbstract:To survive in immune-competent hosts, the pathogen Staphylococcus aureus expresses and secretes a sophisticated array of proteins that inhibit the complement system. Among these are the staphylococcal complement inhibitors (SCIN), which are composed of three active proteins (SCIN-A, -B, and -C) and one purportedly inactive member (SCIN-D or ORF-D). Because previous work has focused almost exclusively on SCIN-A, we sought to provide initial structure/function information on additional SCIN proteins. To this end we determined crystal structures of an active, N-terminal truncation mutant of SCIN-B (denoted SCIN-B18–85) both free and bound to the C3c fragment of complement component C3 at 1.5 and 3.4 Å resolution, respectively. Comparison of the C3c/SCIN-B18–85 structure with that of C3c/SCIN-A revealed that both proteins target the same functional hotspot on the C3b/C3c surface yet harbor diversity in both the type of residues and interactions formed at their C3b/C3c interfaces. Most importantly, these structures allowed identification of Arg44 and Tyr51 as residues key for SCIN-B binding to C3b and subsequent inhibition of the AP C3 convertase. In addition, we also solved several crystal structures of SCIN-D to 1.3 Å limiting resolution. This revealed an unexpected structural deviation in the N-terminal α helix relative to SCIN-A and SCIN-B. Comparative analysis of both electrostatic potentials and surface complementarity suggest a physical explanation for the inability of SCIN-D to bind C3b/C3c. Together, these studies provide a more thorough understanding of immune evasion by S. aureus and enhance potential use of SCIN proteins as templates for design of complement targeted therapeutics.
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Molecular Basis for Complement Recognition and Inhibition Determined by Crystallographic Studies of the Staphylococcal Complement Inhibitor (SCIN) Bound to C3c and C3b
2010Co-Authors: Brandon L. Garcia, Kasra X. Ramyar, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, William J. Mcwhorter, Brian V. GeisbrechtAbstract:The human complement system plays an essential role in innate and adaptive immunity by marking and eliminating microbial intruders. Activation of complement on foreign surfaces results in proteolytic cleavage of complement component 3 (C3) into the potent opsonin C3b, which triggers a variety of immune responses and participates in a self-amplification loop mediated by a multi-protein assembly known as the C3 convertase. The human pathogen Staphylococcus aureus has evolved a sophisticated and potent complement evasion strategy, which is predicated upon an arsenal of potent inhibitory proteins. One of these, the staphylococcal complement inhibitor (SCIN), acts at the level of the C3 convertase (C3bBb) and impairs downstream complement function by trapping the convertase in a stable but inactive state. Previously, we have shown that SCIN binds C3b directly and competitively inhibits binding of human factor H and, to a lesser degree, that of factor B to C3b. Here, we report the co-crystal structures of SCIN bound to C3b and C3c at 7.5 and 3.5 A limiting resolution, respectively, and show that SCIN binds a critical functional area on C3b. Most significantly, the SCIN binding site sterically occludes the binding sites of both factor H and factor B. Our results give insight into SCIN binding to activated derivatives of C3, explain how SCIN can recognize C3b in the absence of other complement components, and provide a structural basis for the competitive C3b-binding properties of SCIN. In the future, this may suggest templates for the design of novel complement inhibitors based upon the SCIN structure.
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Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor
Nature Immunology, 2009Co-Authors: Suzan H. M. Rooijakkers, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, Maartje Ruyken, Jin Wu, Robert Van Domselaar, Karel L Planken, Bert J C Janssen, Jos A G Van StrijpAbstract:Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg^2+-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics. Complement forms an ancient innate immune defense. Gros and colleagues provide new insight into the interactions between complement convertase C3b and its regulator factor H and with the staphylococcal inhibitor SCIN.
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Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor
Nature Immunology, 2009Co-Authors: Suzan H. M. Rooijakkers, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, Maartje Ruyken, Karel L Planken, Bert J C Janssen, Robert Van Domselaar, Jos A. G. Van StrijpAbstract:Complement forms an ancient innate immune defense. Gros and colleagues provide new insight into the interactions between complement convertase C3b and its regulator factor H and with the staphylococcal inhibitor SCIN.
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Crystallization of human complement component C3b in the presence of a staphylococcal complement-inhibitor protein (SCIN).
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2009Co-Authors: Brandon L. Garcia, Kasra X. Ramyar, Daniel Ricklin, John D. Lambris, Apostolia Tzekou, William J. Mcwhorter, Brian V. GeisbrechtAbstract:Staphylococcus aureus secretes a number of small proteins that effectively attenuate the human innate immune response. Among these, the staphylococcal complement-inhibitor protein (SCIN) disrupts the function of the complement component 3 (C3) convertase that is initiated through either the classical or the alternative pathway and thereby prevents amplification of the complement response on the bacterial surface. Recent studies have shown that SCIN may affect the activities of the C3 convertase by binding in an equimolar fashion to C3b, which is itself an integral although non-enzymatic component of the convertase. In order to better understand the nature of the C3b–SCIN interaction, the hanging-drop vapor-diffusion technique was used to crystallize human C3b in the presence of a recombinant form of SCIN. These crystals diffracted synchrotron X-rays to approximately 6 A Bragg spacing and grew in a primitive tetragonal space group (P41212 or P43212; unit-cell parameters a = b = 128.03, c = 468.59 A). Cell-content analysis of these crystals was consistent with the presence of either two 1:1 complexes or a single 2:2 assembly in the asymmetric unit, both of which correspond to a solvent content of 51.9%. By making use of these crystals, solution of the C3b–SCIN structure should further our understanding of complement inhibition and immune evasion by this pathogen.
