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Abstracts / Molecular Imm

P discrimination, and especially its carboxyl-terminal domains9–20, is well exemplified by a severe systemic disease, atypicalemolytic uremic syndrome (aHUS), associated most often with autation in FH domains 19–20. We have previously reported X-

ay crystallographic structure of FH 19–20 and various functionaleficits caused by the aHUS associated mutations on binding ofhese domains to C3d/C3b, heparin, and endothelial cell surfaces.

e have now solved crystal structures of two FH 19–20 mutantsnd show that both the domains are involved in binding to C3d.sing new mutants of FH 19–20 and C3d as well as further X-rayrystallographic analyses, including a preliminary structure of a3d:FH 19–20 co-crystal, we have obtained novel structural and

unctional data on the effects of the aHUS-associated mutations onH 19–20 and the interaction of these domains with the C3d part of3b. On the basis of these results we propose a new possible mech-nism for target discrimination by AP. This mechanism fits to bothhe published structural data on C3b and a wide range of previouslyeported biochemical data on the alternative pathway activation.t also explains the various functional deficits caused by the aHUSssociated mutations.

oi:10.1016/j.molimm.2010.05.183

57

haracterization of the interactions between C3b and comple-ent regulators

in Wu a, Daniel Ricklin b, Georgia Sfyroera b, Apostolia Tzekou b,ohn P. Atkinson c, John D. Lambris b,1, Piet Gros a,1

Crystal and Structural Chemistry, Bijvoet Center for Biomolecularesearch, Utrecht University, Utrecht, The NetherlandsDepartment of Pathology and Laboratory Medicine, University ofennsylvania, Philadelphia, PA, USADepartment of Medicine, Division of Rheumatology, Washington Uni-ersity School of Medicine, St. Louis, MO, USA

The efficient machinery with which complement fights infec-ions and clears cellular debris requires tight regulation to avoidndesirable attacks on healthy host cells. As a consequence, mam-als express various cell-surface bound and soluble complement

egulators, which impair the initiation and amplification of com-lement response at the convertase level. In addition, certainiruses escape complement attack by secreting proteins that mimiche structure and function of these host regulators; well-studied

odels are vaccinia virus complement-control protein (VCP) andmallpox inhibitor of complement enzymes (SPICE). We recentlyublished the crystal structure of C3b in complex with the firstour complement-control protein (CCP1-4) repeats of host reg-lator factor H. Our structure showed in atomic detail how allour CCP domains bind C3b to support decay-accelerating activ-ty and cofactor activity. Here, we extended our structural studieso host complement regulators other than factor H and to viralomologues to address the generality of the observations and inter-retations derived from C3b-factor H (CCP1-4). For this purpose, werystallized several regulators in complex with C3b and exploredow these structurally similar regulators bind C3b and give riseo distinct and overlapping functional repertoires. Though generaleatures of C3b-regulator complexes were found to be conserved,

ll characterized regulators displayed unique binding patterns thatan be related to their diverse behavior in activity assays. We williscuss these structures together with the new mutagenesis andunctional data, which provide further evidence to support the

1 Both the authors shared supervision of the project.

gy 47 (2010) 2198–2294 2259

proposed factor I binding area. These results provide a fascinat-ing insight into complement regulation and a comprehensive viewof the molecular mechanisms underlying the regulatory activities.

doi:10.1016/j.molimm.2010.05.184

258

Potent inhibition of the classical pathway of complement by anovel C1q-binding peptide derived from the human astroviruscoat protein

Jenny Q. Gronemus, Pamela S. Hair, Katrina B. Crawford, Kenji M.Cunnion, Neel K. Krishna

Department of Microbiology and Molecular Cell Biology and Depart-ment of Pediatrics, Eastern Virginia Medical School and Children’sHospital of the Kings Daughters, Norfolk, VA 23507, United States

Previous work from our laboratories has demonstrated that thecoat protein (CP) purified from human astrovirus serotype 1 bindsC1q and MBL resulting in inhibition of the classical and lectin path-ways of complement, respectively. In order to identify the CP aminoacid residues which mediate C1q binding and suppress comple-ment activation, we performed a deletion analysis of the 787 aminoacid CP molecule and C1q binding was found to map to amino acidresidues 51–415. Further deletion analysis identified a 30 aminoacid peptide derived from this region of the CP molecule that wasshown to competitively inhibit the binding of wild-type CP toC1q. The parent peptide and various derivatives were subsequentlyassayed for C1q binding, inhibition of C1 and C4 activation as wellas suppression of complement activation in hemolytic assays. Theparent peptide and several derivatives inhibited complement acti-vation in these functional assays to varying degrees. One peptidederivative in particular displayed superior inhibition of comple-ment activation in the C4 activation assay and the hemolytic assaywith normal human serum and factor B-depleted serum, but didnot effect alternative pathway activation. Based upon these find-ings, we hypothesize that further rationale optimization of thisinhibitory peptide may result in a promising therapeutic inhibitorfor the treatment of inflammatory and autoimmune diseases inwhich dysregulated activation the classical and lectin pathways ofcomplement contribute to pathogenesis.

doi:10.1016/j.molimm.2010.05.185

259

Crystal structure of C3bB in complex with factor D

Federico Forneris a, Jin Wu a, Rachel S. Wallace a, ApostoliaTzekou b, Daniel Ricklin b, John D. Lambris b,1, Piet Gros a,1

a Crystal and Structural Chemistry, Bijvoet Center for BiomolecularResearch, Department of Chemistry, Utrecht University, Utrecht, TheNetherlandsb Department of Pathology and Laboratory Medicine, University ofPennsylvania, Philadelphia, PA, USA

The finely tuned activation of the complement cascade relieson multiple proteolytic reactions that are mediated by largemacromolecular complexes. Upon initiation, the three comple-

ment pathways converge into the critical step of the conversionof C3 to its activated form C3b by means of short-lived enzy-matic complexes called C3 convertases. These convertases amplifyC3b production near target surfaces, resulting in opsonization of

1 Both the authors shared supervision of the project.