Spatially conserved motifs in complement control protein domains determine functionality in regulators of complement activation-family proteins

The complement system is highly efficient in targeting pathogens, but lack of its apposite regulation results in host-cell damage, which is linked to diseases. Thus, complement activation is tightly regulated by a series of proteins, which primarily belong to the regulators of complement activation (RCA) family. Structurally, these proteins are composed of repeating complement control protein (CCP) domains where two to four successive domains contribute to the regulatory functions termed decay-accelerating activity (DAA) and cofactor activity (CFA). However, the precise constitution of the functional units and whether these units can be joined to form a larger composition with dual function have not been demonstrated. Herein, we have parsed the functional units for DAA and CFA by constructing chimeras of the decay-accelerating factor (DAF) that exhibits DAA and membrane cofactor protein (MCP) that exhibits CFA. We show that in a four-CCP framework, a functional unit for each of the regulatory activities is formed by only two successive CCPs wherein each participates in the function, albeit CCP2 has a bipartite function. Additionally, optimal activity requires C-terminal domains that enhance the avidity of the molecule for C3b/C4b. Furthermore, by composing a four-CCP DAF-MCP chimera with robust CFA (for C3b and C4b) and DAA (for classical and alternative pathway C3 convertases), named decay cofactor protein, we show that CCP functional units can be linked to design a dual-activity regulator. These data indicate that the regulatory determinants for these two biological processes are distinct and modular in nature.

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Molecular Characterization of the Rhesus Rhadinovirus (RRV) ORF4 Gene and the RRV Complement Control Protein It Encodes

Journal of Virology ◽

10.1128/jvi.02069-06 ◽

2007 ◽

Vol 81 (8) ◽

pp. 4166-4176 ◽

Cited By ~ 13

Author(s):

Linda Mark ◽

O. Brad Spiller ◽

Marcin Okroj ◽

Simon Chanas ◽

Jim A. Aitken ◽

...

Keyword(s):

Complement Activation ◽

Biological Significance ◽

Full Length ◽

In Vivo Model ◽

Primate Model ◽

Complement Control Protein ◽

Functional Studies ◽

Alternatively Spliced ◽

Control Protein

ABSTRACT The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model.

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Retraction Notice to: Crystal Structure of a Complement Control Protein that Regulates Both Pathways of Complement Activation and Binds Heparan Sulfate Proteoglycans

Cell ◽

10.1016/j.cell.2018.11.047 ◽

2018 ◽

Vol 175 (7) ◽

pp. 1992

Author(s):

Krishna H.M. Murthy ◽

Scott A. Smith ◽

Vannakambadi K. Ganesh ◽

Ken W. Judge ◽

Nick Mullin ◽

...

Keyword(s):

Crystal Structure ◽

Heparan Sulfate ◽

Complement Activation ◽

Heparan Sulfate Proteoglycans ◽

Complement Control Protein ◽

Retraction Notice ◽

Control Protein

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Central modules of the vaccinia virus complement control protein are not in extensive contact

Biochemical Journal ◽

10.1042/bj3440167 ◽

1999 ◽

Vol 344 (1) ◽

pp. 167-175 ◽

Cited By ~ 12

Author(s):

Marina D. KIRKITADZE ◽

Colin HENDERSON ◽

Nicholas C. PRICE ◽

Sharon M. KELLY ◽

Nicholas P. MULLIN ◽

...

Keyword(s):

Vaccinia Virus ◽

Therapeutic Potential ◽

Chemical Shifts ◽

Axial Ratio ◽

Tryptophan Fluorescence ◽

Scanning Calorimetry ◽

Complement Control Protein ◽

Intrinsic Tryptophan Fluorescence ◽

Regulators Of Complement Activation ◽

Control Protein

The 28.6 kDa vaccinia virus complement control protein (VCP) is an inhibitor of the complement system and has therapeutic potential. It is composed of four domains or modules and is a homologue of complement receptor 1 (CR1) and other mammalian regulators of complement activation. A key aspect to structure-function relationships in these proteins is the extent of intramolecular module-module interactions, since these dictate the overall shape and flexibility of the molecules. A protein fragment (VCP∼ 2,3) encompassing modules 2 and 3 of VCP was over-expressed in Pichia pastoris. Ultracentrifugation showed that VCP∼ 2,3 is highly asymmetric with an axial ratio of 5.3:1, which is consistent with an end-to-end arrangement of the two modules. NMR spectroscopy, differential scanning calorimetry, CD and intrinsic tryptophan fluorescence were used to monitor unfolding of VCP∼ 2,3. Experiments performed over a range of temperatures and concentrations of guanidinium chloride revealed that module 2 unfolds under milder conditions than, and independently of, module 3. Unfolding of module 2 is not associated with extensive changes in amide 15N and 1H chemical shifts of module 3, implying that the modules do not form an extensive intermodular interface. Results obtained in this work for VCP∼ 2,3 are compared with those obtained in a study of CR1 modules 15-17 [Kirkitadze, Krych, Uhrin, Dryden, Smith, Cooper, Wang, Hauhart, Atkinson and Barlow (1999) Biochemistry 38, 7019-7031].

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