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Structure of factor B in complex with cobra venom factor gives insights into convertase formation
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Abstracts / Molecular Immunology 45 (2008) 4095–4182
structure to make contact with CR2, also yields wild type binding in both cases. (3) Several mutations on the concave face of C3d significantly affect binding to both Sbi-IV and CR2, although there is divergence in which residues are most important in each case. The results with Sbi-IV strongly suggest that its mode of binding to C3d is highly similar to that visualized in the Efb-C/C3d complex. The results with CR2 confirm our earlier mapping studies, and cast even further doubt upon the physiologic relevance of the complex visualized in the C3d/CR2 co-crystal. References Hammel, M., Sfyroera, G., Ricklin, D., Magotti, P., Lambris, J.D., Geisbrecht, B.V., 2007. A structural basis for complement inhibition by Staphylococcus aureus. Nat. Immunol. 8, 430–437. Clemenza, L., Isenman, D.E., 2000. Structure-guided identification of C3d residues essential for its binding to complement receptor 2 (CD21). J. Immunol. 165, 3839–3848. Szakonyi, G., Guthridge, J.M., Li, D., Young, K., Holers, V.M., Chen, X.S., 2001. Structure of complement receptor 2 in complex with its C3d ligand. Science 292, 1725–1728. Hannan, J.P., Young, K.A., Guthridge, J.M., Asokan, R., Szakonyi, G., Chen, X.S., Holers, V.M., 2005. Mutational analysis of the complement receptor type 2 (CR2/CD21)–C3d interaction reveals a putative charged SCR1 binding site for C3d. J. Mol. Biol. 346, 845–858.
doi:10.1016/j.molimm.2008.08.003 O3 Structure of factor B in complex with cobra venom factor gives insights into convertase formation Bert Janssen a , Lucio Gomes a , David Fritzinger b , Carl-Wilhelm Vogel b , Hans Meeldijk a , Dmitri Svergun c , Roman Koning d , Abraham Koster d , Piet Gros a a
Utrecht University, Utrecht, The Netherlands Cancer Research Center of Hawaii, Honolulu, USA c EMBL, Hamburg, Germany d Leiden University Medical Center, Leiden, The Netherlands b
C3 convertases are bimolecular protease complexes that play a critical role in the complement activation pathways. These complexes are generated in a two-step assembly process. In the alternative pathway, the pro-enzyme factor B binds to its ligand C3b. Subsequently factor D cleaves factor B generating the active complex C3b-Bb. We have used both C3b and its homologue cobra venom factor from Naja kaouthia (monocled cobra) to study the pro-convertase and active convertase complexes. We crystallized the complex of CVF with factor B and solved the structure of the complex at 8 Å. resolution. This low-resolution model was validated by small-angle X-ray scattering and single-particle negative stain EM reconstruction. The resulting model reveals the critical contact sites in the CVF-B association, which are supported by various published data on C3b–factor B interactions. Furthermore, singleparticle negative stain EM reconstruction on the active convertase CVF-Bb reveals the rearrangement associated with convertase activation. We will discuss the biological activity of the C3 convertases in light of these structures. doi:10.1016/j.molimm.2008.08.004
O4 Use of NMR to define CR2:C3d interactions in solution reveals dual SCR1-2 interface with C3d: Confirmation using a novel ligand-selective inhibitory peptide James Kovacs, Jonathan Hannan, Elan Eisenmesser, Michael Holers University of Colorado, Denver, Aurora, USA Complement receptor 2 (CR2, CD21) is a cell membrane protein, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B cell responses and bridges innate and acquired immunity. The most distally located SCRs (SCR1-2) mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFN-␣, CD23). Inhibitory mAbs against SCR1-2 block binding of all ligands, and all ligands cross-compete for binding SCR1-2. To develop ligandspecific inhibitors that would also assist in identifying residues unique to each receptor–ligand interaction, phage were selected from randomly generated constrained and un-constrained libraries by panning with recombinant SCR1-2, followed by specific liganddriven elution. Derived peptides were tested by competition ELISA. Two peptides, C3dp1:APQHLSSQYSRT and gp350cp1:CSEGSLKGC, exhibited ligand-specific inhibition at 100 and 150 micromolar IC50 , respectively. C3d was titrated into N-15 labeled SCR1-2, which revealed chemical shift changes indicative of specific intermolecular interactions. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1-2. With regard to C3d, the binding surface includes regions of SCR1, SCR2 and the inter-SCR linker, specifically residues R13, Y16, R28, Y29, S32, T34, K48, D56, K57, Y68, R83, G84, N101, N105, S109 and R122. The CR2 binding surface incorporating SCR1 is inconsistent with a previous X-ray CR2-C3d co-crystal analysis, but consistent with mutagenesis, X-ray neutron scattering and inhibitory mAb epitope mapping. Titration with C3dp1 yielded chemical shift changes (R13, Y16, T34, K48, D56, K57, Y68, R83, G84, N105 and S109) overlapping with C3d, indicating that C3dp1 interacts at the same CR2 site as C3d. These peptides represent a novel method of selectively inhibiting single ligand binding to CR2 and have the potential to modulate CR2 function in vivo. R01 CA053615. doi:10.1016/j.molimm.2008.08.005 O5 The crystal structure of rat complement receptor 1-related protein Y (CrrY) helps dissecting decay-acceleration and fI-cofactor activities Pietro Roversi a , Kirstin Leath a , Natalie Harris b , B. Paul Morgan b , Susan Lea a a b
Hepburn b , Claire
L.
Oxford University, Oxford, UK Cardiff University, Cardiff, UK
Complement receptor 1-related protein Y (CrrY) is a membranebound rat complement regulator whose amino-terminal four complement control protein domains (CCP) are required for full regulatory and C3b-binding activities. Unlike other complement regulators that either only accelerate the irreversible decay of the C3 convertases (e.g. CD55) or only act as cofactors for factor I (fI) mediated cleavage of C3b (e.g. CD46), CrrY has both cofactor and decay acceleration activities. A soluble recombinant form of Crry, containing only the first four CCPs was expressed in bacteria and refolded successfully. The refolded protein has full complement regulatory activity in vitro. The crystal structure of this CrrY CCPs construct was determined by molecular replacement at 3.3 Å res-
Abstracts / Molecular Immunology 45 (2008) 4095–4182
structure to make contact with CR2, also yields wild type binding in both cases. (3) Several mutations on the concave face of C3d significantly affect binding to both Sbi-IV and CR2, although there is divergence in which residues are most important in each case. The results with Sbi-IV strongly suggest that its mode of binding to C3d is highly similar to that visualized in the Efb-C/C3d complex. The results with CR2 confirm our earlier mapping studies, and cast even further doubt upon the physiologic relevance of the complex visualized in the C3d/CR2 co-crystal. References Hammel, M., Sfyroera, G., Ricklin, D., Magotti, P., Lambris, J.D., Geisbrecht, B.V., 2007. A structural basis for complement inhibition by Staphylococcus aureus. Nat. Immunol. 8, 430–437. Clemenza, L., Isenman, D.E., 2000. Structure-guided identification of C3d residues essential for its binding to complement receptor 2 (CD21). J. Immunol. 165, 3839–3848. Szakonyi, G., Guthridge, J.M., Li, D., Young, K., Holers, V.M., Chen, X.S., 2001. Structure of complement receptor 2 in complex with its C3d ligand. Science 292, 1725–1728. Hannan, J.P., Young, K.A., Guthridge, J.M., Asokan, R., Szakonyi, G., Chen, X.S., Holers, V.M., 2005. Mutational analysis of the complement receptor type 2 (CR2/CD21)–C3d interaction reveals a putative charged SCR1 binding site for C3d. J. Mol. Biol. 346, 845–858.
doi:10.1016/j.molimm.2008.08.003 O3 Structure of factor B in complex with cobra venom factor gives insights into convertase formation Bert Janssen a , Lucio Gomes a , David Fritzinger b , Carl-Wilhelm Vogel b , Hans Meeldijk a , Dmitri Svergun c , Roman Koning d , Abraham Koster d , Piet Gros a a
Utrecht University, Utrecht, The Netherlands Cancer Research Center of Hawaii, Honolulu, USA c EMBL, Hamburg, Germany d Leiden University Medical Center, Leiden, The Netherlands b
C3 convertases are bimolecular protease complexes that play a critical role in the complement activation pathways. These complexes are generated in a two-step assembly process. In the alternative pathway, the pro-enzyme factor B binds to its ligand C3b. Subsequently factor D cleaves factor B generating the active complex C3b-Bb. We have used both C3b and its homologue cobra venom factor from Naja kaouthia (monocled cobra) to study the pro-convertase and active convertase complexes. We crystallized the complex of CVF with factor B and solved the structure of the complex at 8 Å. resolution. This low-resolution model was validated by small-angle X-ray scattering and single-particle negative stain EM reconstruction. The resulting model reveals the critical contact sites in the CVF-B association, which are supported by various published data on C3b–factor B interactions. Furthermore, singleparticle negative stain EM reconstruction on the active convertase CVF-Bb reveals the rearrangement associated with convertase activation. We will discuss the biological activity of the C3 convertases in light of these structures. doi:10.1016/j.molimm.2008.08.004
O4 Use of NMR to define CR2:C3d interactions in solution reveals dual SCR1-2 interface with C3d: Confirmation using a novel ligand-selective inhibitory peptide James Kovacs, Jonathan Hannan, Elan Eisenmesser, Michael Holers University of Colorado, Denver, Aurora, USA Complement receptor 2 (CR2, CD21) is a cell membrane protein, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B cell responses and bridges innate and acquired immunity. The most distally located SCRs (SCR1-2) mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFN-␣, CD23). Inhibitory mAbs against SCR1-2 block binding of all ligands, and all ligands cross-compete for binding SCR1-2. To develop ligandspecific inhibitors that would also assist in identifying residues unique to each receptor–ligand interaction, phage were selected from randomly generated constrained and un-constrained libraries by panning with recombinant SCR1-2, followed by specific liganddriven elution. Derived peptides were tested by competition ELISA. Two peptides, C3dp1:APQHLSSQYSRT and gp350cp1:CSEGSLKGC, exhibited ligand-specific inhibition at 100 and 150 micromolar IC50 , respectively. C3d was titrated into N-15 labeled SCR1-2, which revealed chemical shift changes indicative of specific intermolecular interactions. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1-2. With regard to C3d, the binding surface includes regions of SCR1, SCR2 and the inter-SCR linker, specifically residues R13, Y16, R28, Y29, S32, T34, K48, D56, K57, Y68, R83, G84, N101, N105, S109 and R122. The CR2 binding surface incorporating SCR1 is inconsistent with a previous X-ray CR2-C3d co-crystal analysis, but consistent with mutagenesis, X-ray neutron scattering and inhibitory mAb epitope mapping. Titration with C3dp1 yielded chemical shift changes (R13, Y16, T34, K48, D56, K57, Y68, R83, G84, N105 and S109) overlapping with C3d, indicating that C3dp1 interacts at the same CR2 site as C3d. These peptides represent a novel method of selectively inhibiting single ligand binding to CR2 and have the potential to modulate CR2 function in vivo. R01 CA053615. doi:10.1016/j.molimm.2008.08.005 O5 The crystal structure of rat complement receptor 1-related protein Y (CrrY) helps dissecting decay-acceleration and fI-cofactor activities Pietro Roversi a , Kirstin Leath a , Natalie Harris b , B. Paul Morgan b , Susan Lea a a b
Hepburn b , Claire
L.
Oxford University, Oxford, UK Cardiff University, Cardiff, UK
Complement receptor 1-related protein Y (CrrY) is a membranebound rat complement regulator whose amino-terminal four complement control protein domains (CCP) are required for full regulatory and C3b-binding activities. Unlike other complement regulators that either only accelerate the irreversible decay of the C3 convertases (e.g. CD55) or only act as cofactors for factor I (fI) mediated cleavage of C3b (e.g. CD46), CrrY has both cofactor and decay acceleration activities. A soluble recombinant form of Crry, containing only the first four CCPs was expressed in bacteria and refolded successfully. The refolded protein has full complement regulatory activity in vitro. The crystal structure of this CrrY CCPs construct was determined by molecular replacement at 3.3 Å res-