Structures of C3b in Complex with Factors B and D Give Insight into Complement Convertase Formation

Forneris, Federico; Ricklin, Daniel; Wu, Jiawang; Τζέκου, Αποστολία; Wallace, Rachel S.; Lambris, John D.; Gros, Piet

doi:10.1126/science.1195821

Cited by 242 publications

(321 citation statements)

References 40 publications

(75 reference statements)

Supporting

Mentioning

304

Contrasting

Unclassified

Order By: Relevance

“…A previous study (33) documented that Mn 2ϩ stabilizes C3bB in a form susceptible to FD cleavage, but C3bBb(Mn 2ϩ ), once formed, is highly unstable and dissociates immediately. FB binding to C3b depends on elements in fragment Ba and on the metal ion-de- ) (38,54). By using the new microplate/WB methods with ion-based selective stabilization, we could observe specific formation over time of C3bB(Mn 2ϩ ) or C3bBb(Mg 2ϩ ) in the absence or in the presence of FD, respectively.…”

Section: Discussionmentioning

confidence: 90%

Insights into the Effects of Complement Factor H on the Assembly and Decay of the Alternative Pathway C3 Proconvertase and C3 Convertase

Bettoni

Bresin

Noris

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

The activated fragment of C3 (C3b) and factor B form the C3 proconvertase (C3bB), which is cleaved by factor D to C3 convertase ( Immunol. 122, 75-81) indicated that the complement alternative pathway regulator factor H (FH) competes with factor B for C3b binding; however, the capability of FH to prevent C3bB assembly has not been formally investigated. Moreover, in the few published studies FH did not favor C3bB dissociation. Whether FH may affect C3bBb formation from C3bB is unknown. We set up user-friendly assays based on combined microplate/Western blotting techniques that specifically detect either C3bB or C3bBb, with the aim of investigating the effect of FH on C3bB assembly and decay and C3bBb formation and decay. We document that FH does not affect C3bB assembly, indicating that FH does not efficiently compete with factor B for C3b binding. We also found that FH does not dissociate C3bB. FH showed a strong C3bBb decay-accelerating activity, as reported previously, and also exerted an apparent inhibitory effect on C3bBb formation. The latter effect was not fully attributable to a rapid FH-mediated dissociation of C3bBb complexes, because blocking decay with properdin and C3 nephritic factor did not restore C3bBb formation. FH almost completely prevented release of the smaller cleavage subunit of FB (Ba), without modifying the amount of C3bB complexes, suggesting that FH inhibits the conversion of C3bB to C3bBb. Thus, the inhibitory effect of FH on C3bBb formation is likely the sum of inhibition of C3bB conversion to C3bBb and of C3bBb decay acceleration. Further studies are required to confirm these findings in physiological cell-based settings.The complement system is a complex network of plasma proteins that cooperate in the defense against foreign microorganisms and in the maintenance of tissue homeostasis (1-3). Interest in the complement system was rekindled in the past decade by accumulating evidence that genetically determined complement dysregulation is involved in two rare devastating disorders of the kidney, atypical hemolytic uremic syndrome (aHUS) 3 and C3 glomerulopathy (C3G) (4 -8), as well as in age-related macular degeneration, the leading cause of blindness for older people (9). Finding that treatment with eculizumab (10), a monoclonal antibody that blocks the formation of terminal complement complex C5b-9, efficiently cured aHUS (11, 12) and C3G (13-15) further corroborated the role of complement in human disease (16).Complement can be triggered by three activation pathways, the classical, the lectin, and the alternative (AP) pathways, all leading to the formation of unstable protease complexes, named C3 convertases, that cleave the inactive central component C3 into C3a and C3b fragments. C3b molecules deposited on pathogens or damaged self-cells provide a molecular platform for the formation of an active AP C3 convertase, C3bBb, which enzymatically generates many more C3b molecules, resulting in a positive feedback loop. Notably, the AP C3 convertase is crucial within the complement ca...

show abstract

Section: Discussionmentioning

confidence: 90%

Insights into the Effects of Complement Factor H on the Assembly and Decay of the Alternative Pathway C3 Proconvertase and C3 Convertase

Bettoni

Bresin

Noris

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Acting on the upstream of AP, factor D cleaves complement factor B bound with C3b into the C3bBb complex. 11 The ubiquitous role of factor D in AP activation led to the implication that inhibition of factor D can be an attractive strategy in complement-targeted therapy. Factor D belongs to the serine protease family.…”

mentioning

confidence: 99%

Buried Hydrogen Bond Interactions Contribute to the High Potency of Complement Factor D Inhibitors

Yang¹,

Phillips

Stuckey³

et al. 2016

ACS Med. Chem. Lett.

View full text Add to dashboard Cite

Aberrant activation of the complement system is associated with diseases, including paroxysmal nocturnal hemoglobinuria and age-related macular degeneration. Complement factor D is the rate-limiting enzyme for activating the alternative pathway in the complement system. Recent development led to a class of potent amide containing pyrrolidine derived factor D inhibitors. Here, we used biochemical enzymatic and biolayer interferometry assays to demonstrate that the amide group improves the inhibitor potency by more than 80-fold. Our crystal structures revealed buried hydrogen bond interactions are important. Molecular orbital analysis from quantum chemistry calculations dissects the chemical groups participating in these interactions. Free energy calculation supports the differential contributions of the amide group to the binding affinities of these inhibitors. Cell-based hemolysis assay confirmed these compounds inhibit factor D mediated complement activation via the alternative pathway. Our study highlights the important interactions contributing to the high potency of factor D inhibitors reported recently.

show abstract

“…Factor B binds to C3b in the presence of Mg 2ϩ , forming a transient complex (t1 ⁄ 2 ϳ5 s) (10, 11) that is cleaved by factor D at a single site in the linker region to generate the AP C3 convertase, C3bBb(Mg 2ϩ ) (t1 ⁄ 2 ϳ90 s) (12, 13). During this process a metal ion-dependent adhesion site (MIDAS) is formed at the apex of the VWA domain and mediates Mg 2ϩ -dependent C3b binding, and an exosite is formed at the VWA-serine protease domain interface and mediates factor D binding (14).In free factor B, a pair of salt bridges connects the Arg 234 side chain to Glu 207 of the linker region and Glu 446 of the VWA domain in a "locked" conformation (Fig. 1, B and C) (15).…”

mentioning

confidence: 99%

Access to the Complement Factor B Scissile Bond Is Facilitated by Association of Factor B with C3b Protein

Hourcade

Mitchell

2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

The complement system plays major roles in innate and adaptive immunity. Complement marks microbial pathogens for opsonization and clearance or for lysis and plays a key role in the identification and removal of damaged tissue and other potentially dangerous biological debris (1). It modulates the antibody repertoire (2, 3), and it plays a pivotal role in the normal activity of regulatory T cells (4). Complement activity is also critical in the pathogenesis of many diseases and injury states (5). Therapeutic agents designed to inhibit harmful complement activity have begun to emerge in the clinical environment (6).Complement is activated by three pathways: the classical pathway, the lectin pathway, and the alternative pathway (AP) 2 (1, 7). Each pathway directs the assembly of the C3 convertases, serine proteases that cleave the abundant plasma protein C3 to form C3b, an opsonin that marks potential targets for clearance and/or lysis, and C3a, a potent anaphylactic agent that promotes multiple inflammatory reactions. The AP also serves to amplify the activity of all three pathways, promoting a rapid and robust response. Fluid phase and membrane-bound complement regulators protect host tissues from complement-mediated damage (8).Factor B is a single chain glycoprotein, composed of an amino-terminal region of three complement control protein domains, a 40-amino acid linker region, a single von Willebrand factor type A domain (VWA), and a carboxyl-terminal serine protease domain (see Fig. 1A) (9). Factor B binds to C3b in the presence of Mg 2ϩ , forming a transient complex (t1 ⁄ 2 ϳ5 s) (10, 11) that is cleaved by factor D at a single site in the linker region to generate the AP C3 convertase, C3bBb(Mg 2ϩ ) (t1 ⁄ 2 ϳ90 s) (12, 13). During this process a metal ion-dependent adhesion site (MIDAS) is formed at the apex of the VWA domain and mediates Mg 2ϩ -dependent C3b binding, and an exosite is formed at the VWA-serine protease domain interface and mediates factor D binding (14).In free factor B, a pair of salt bridges connects the Arg 234 side chain to Glu 207 of the linker region and Glu 446 of the VWA domain in a "locked" conformation (Fig. 1, B and C) (15). The salt bridges sequester the scissile bond (between Arg 234 and Lys 235 ) and prevent unproductive cleavage of factor B by circulating serine proteases (16). It is not known how the double latch structure is opened to permit factor D cleavage of the scissile bond in the short lived C3bB(Mg 2ϩ ) complex. Here, we characterize the properties of factor B mutations that preclude one or both of the Arg 234 salt bridges. We examine their effects on the assembly and stability of the pro-convertase C3bB(Mg 2ϩ ). Our analyses indicate that the release of the Arg 234 salt bridges partially stabilizes the C3bB(Mg 2ϩ ) complex and that this effect is due predominantly to loss of the Arg 234 -Glu 446 bond. This result was greatly diminished in the case of CVFB(Mg 2ϩ ), a pro-convertase that incorporates the C3b homolog cobra venom factor (CVF) in place of C3b and is 100...

show abstract

Structures of C3b in Complex with Factors B and D Give Insight into Complement Convertase Formation

Cited by 242 publications

References 40 publications

Insights into the Effects of Complement Factor H on the Assembly and Decay of the Alternative Pathway C3 Proconvertase and C3 Convertase

Insights into the Effects of Complement Factor H on the Assembly and Decay of the Alternative Pathway C3 Proconvertase and C3 Convertase

Buried Hydrogen Bond Interactions Contribute to the High Potency of Complement Factor D Inhibitors

Access to the Complement Factor B Scissile Bond Is Facilitated by Association of Factor B with C3b Protein

Contact Info

Product

Resources

About