Identification and structural analysis of dimeric chicken complement component 3d and its binding with chicken complement receptor 2.

Complement component 3d (C3d), the final cleavage product of complement component C3, interacts with CR2 and thus plays a crucial role in linking the innate and adaptive immune systems. Additionally, human C3d executes various functions in its dimeric form, which is more effective than its monomeric form. In this study, we aimed to explored whether chicken C3d (chC3d) exhibits similar characteristics, namely dimerization and binding of dimeric chC3d to chicken CR2 (chCR2). We investigated the interaction and co-localization of chC3d with itself using coimmunoprecipitation and confocal laser scanning microscopy, respectively. Then, dimeric chC3d was detected using native polyacrylamide gel electrophoresis and western blotting, and its equilibrium dissociation constant KD (827 nM) was determined using surface plasmon resonance. Finally, the interaction modes of dimeric chC3d were identified using molecular docking simulations, which revealed that dimeric chC3d could crosslink with chCR2 receptor. Overall, our findings will facilitate future explorations of the chicken complement system.

How Epstein-Barr virus envelope glycoprotein gp350 tricks the CR2? A molecular dynamics study.

The connection of Epstein Barr virus (EBV) with diseases such as Burkitt Lymphoma, Hodgkin disease, multiple sclerosis, systemic lupus erythematosus and various B-cell lymphomas made EBV glycoproteins one of the most popular vaccine immunogens. As a protein being encoded by EBV, the viral membrane envelope protein gp350 is studied extensively due to its abundancy on the surface and its interaction with complementary receptor, CR2. The binding of CR2 and gp350 not only leads to the entrance of the virus to the B-cells, but also prevents CR2 and C3d protein interactions that are required for immune response. Thus, understanding the inhibition of gp350 activity is crucial for vaccine development. Although, the active residues on gp350 structure were determined by several mutational studies, the exact mechanism of CR2 binding is still not clear. To this end, we have performed molecular docking followed by molecular dynamics simulations and MM-PBSA on wildtype and several mutated gp350 and CR2 structures. Apart from identifying crucial amino acids, the results of per-residue decomposition energy analysis clarified the individual energy contributions of amino acids and were also found to be accurate in differentiating the active site residues in CR2 binding. Here, we highlight the role of binding region residues (linker-1) but more interestingly, the dynamic relation between the distant sites of gp350 (linker-2 and D3 residues) and CR2. These findings can lead further vaccine development strategies by pointing to the importance of computationally found novel regions that can be potentially used to modulate gp350 activity.

The molecular mechanism of pH-regulating C3d-CR2 interactions: Insights from molecular dynamics simulation.

The interactions of complement receptor 2 (CR2) and the degradation fragment C3d of complement component C3 mediate the innate and adaptive immune systems. Due to the importance of C3d-CR2 interaction in the design of vaccines, many studies have indicated the interactions are pH-dependent. Moreover, C3d-CR2 interactions at pH 5.0 are unknown. To investigate the molecular mechanism of pH-regulating C3d-CR2 interaction, molecular dynamics simulations for C3d-CR2 complex in different pH are performed. Our results revealed that the protonation of His9 in C3d at pH 6.0 slightly weakens C3d-CR2 association as reducing pH from 7.4 to 6.0, initiated from a key hydrogen bond formed between Gly270 and His9 in C3d at pH 6.0. When reducing pH from 6.0 to 5.0, the protonation of His33 in C3d weakens C3d-SCR1 association by changing the hydrogen-bond network of Asp36, Glu37, and Glu39 in C3d with Arg13 in CR2. In addition, the protonation of His90 significantly enhances C3d-SCR2 association. This is because the enhanced hydrogen-bond interactions of His90 with Glu63 and Ser69 of the linker change the conformations of the linker, Cys112-Asn116 and Pro87-Gly91 regions. This study uncovers the molecular mechanism of the mediation of pH on C3d-CR2 interaction, which is valuable for vaccine design.

The solvent at antigen-binding site regulated C3d-CR2 interactions through the C-terminal tail of C3d at different ion strengths: insights from molecular dynamics simulation.

BACKGROUND: The interactions of complement receptor 2 (CR2) and the degradation fragment C3d of complement component C3 play important links between the innate and adaptive immune systems. Due to the importance of C3d-CR2 interaction in the design of vaccines and inhibitors, a number of studies have been performed to investigate C3d-CR2 interaction. Many studies have indicated C3d-CR2 interactions are ionic strength-dependent. METHODS: To investigate the molecular mechanism of C3d-CR2 interaction and the origin of effects of ionic strength, molecular dynamics simulations for C3d-CR2 complex together with the energetic and structural analysis were performed. RESULTS: Our results revealed the increased interactions between charged protein and ions weaken C3d-CR2 association, as ionic strengths increase. Moreover, ion strengths have similar effects on antigen-binding site and CR2 binding site. Meanwhile, Ala17 and Gln20 will transform between the activated and non-activated states mediated by His133 and Glu135 at different ion strengths. CONCLUSIONS: Our results reveal the origins of the effects of ionic strengths on C3d-CR2 interactions are due to the changes of water, ion occupancies and distributions. GENERAL SIGNIFICANCE: This study uncovers the origin of the effect of ionic strength on C3d-CR2 interaction and deepens the understanding of the molecular mechanism of their interaction, which is valuable for the design of vaccines and small molecule inhibitors.

Electrostatic Steering Accelerates C3d:CR2 Association.

Electrostatic effects are ubiquitous in protein interactions and are found to be pervasive in the complement system as well. The interaction between complement fragment C3d and complement receptor 2 (CR2) has evolved to become a link between innate and adaptive immunity. Electrostatic interactions have been suggested to be the driving factor for the association of the C3d:CR2 complex. In this study, we investigate the effects of ionic strength and mutagenesis on the association of C3d:CR2 through Brownian dynamics simulations. We demonstrate that the formation of the C3d:CR2 complex is ionic strength-dependent, suggesting the presence of long-range electrostatic steering that accelerates the complex formation. Electrostatic steering occurs through the interaction of an acidic surface patch in C3d and the positively charged CR2 and is supported by the effects of mutations within the acidic patch of C3d that slow or diminish association. Our data are in agreement with previous experimental mutagenesis and binding studies and computational studies. Although the C3d acidic patch may be locally destabilizing because of unfavorable Coulombic interactions of like charges, it contributes to the acceleration of association. Therefore, acceleration of function through electrostatic steering takes precedence to stability. The site of interaction between C3d and CR2 has been the target for delivery of CR2-bound nanoparticle, antibody, and small molecule biomarkers, as well as potential therapeutics. A detailed knowledge of the physicochemical basis of C3d:CR2 association may be necessary to accelerate biomarker and drug discovery efforts.

The Structure-Function Relationships of Complement Receptor Type 2 (CR2; CD21).

Human complement receptor type 2 (CR2; CD21) is a surface-associated glycoprotein which binds to a variety of endogenous ligands, including the complement component C3 fragments iC3b, C3dg and C3d, the low-affinity IgE receptor CD23, and the type I cytokine, interferon-alpha. CR2 links the innate complement-mediated immune response to pathogens and foreign antigens with the adaptive immune response by binding to C3d that is covalently attached to targets, and which results in a cell signalling phenomenon that lowers the threshold for B cell activation. Variations or deletions of the CR2 gene in humans, or the Cr2 gene in mice associate with a variety of autoimmune and inflammatory conditions. A number of infectious agents including Epstein-Barr virus (EBV), Human Immunodeficiency Virus (HIV) and prions also bind to CR2 either directly or indirectly by means of C3d-targeted immune complexes. In this review we discuss the interactions that CR2 undertakes with its best characterized ligands C3d, CD23 and the EBV gp350/220 envelope protein. To date only a single physiologically relevant complex of CR2 with one of its ligands, C3d, has been elucidated. By contrast, the interactions with CD23 and EBV gp350/220, while being important from physiologic and disease-associated standpoints, respectively, are only incompletely understood. A detailed knowledge of the structure-function relationships that CR2 undergoes with its ligands is necessary to understand the implications of using recombinant CR2 in therapeutic or imaging agents, or alternatively targeting CR2 to down-regulate the antibody mediated immune response in cases of autoimmunity.

Rational Design of an Epstein-Barr Virus Vaccine Targeting the Receptor-Binding Site.

Epstein-Barr virus (EBV) represents a major global health problem. Though it is associated with infectious mononucleosis and ∼200,000 cancers annually worldwide, a vaccine is not available. The major target of immunity is EBV glycoprotein 350/220 (gp350) that mediates attachment to B cells through complement receptor 2 (CR2/CD21). Here, we created self-assembling nanoparticles that displayed different domains of gp350 in a symmetric array. By focusing presentation of the CR2-binding domain on nanoparticles, potent neutralizing antibodies were elicited in mice and non-human primates. The structurally designed nanoparticle vaccine increased neutralization 10- to 100-fold compared to soluble gp350 by targeting a functionally conserved site of vulnerability, improving vaccine-induced protection in a mouse model. This rational approach to EBV vaccine design elicited potent neutralizing antibody responses by arrayed presentation of a conserved viral entry domain, a strategy that can be applied to other viruses.

A theoretical view of the C3d:CR2 binding controversy.

The C3d:CR2(SCR1-2) interaction plays an important role in bridging innate and adaptive immunity, leading to enhanced antibody production at sites of complement activation. Over the past decade, there has been much debate over the binding mode of this interaction. An initial cocrystal structure (PDB: 1GHQ) was published in 2001, in which the only interactions observed were between the SCR2 domain of CR2 and a side-face of C3d whereas a cocrystal structure (PDB: 3OED) published in 2011 showed both the SCR1 and SCR2 domains of CR2 interacting with an acidic patch on the concave surface of C3d. The initial 1GHQ structure is at odds with the majority of existing biochemical data and the publication of the 3OED structure renewed uncertainty regarding the physiological relevance of 1GHQ, suggesting that crystallization may have been influenced by the presence of zinc acetate in the crystallization process. In our study, we used a variety of computational approaches to gain insight into the binding mode between C3d and CR2 and demonstrate that the binding site at the acidic patch (3OED) is electrostatically more favorable, exhibits better structural and dissociative stability, specifically at the SCR1 domain, and has higher binding affinity than the 1GHQ binding mode. We also observe that nonphysiological zinc ions enhance the formation of the C3d:CR2 complex at the side face of C3d (1GHQ) through increases in electrostatic favorability, intermolecular interactions, dissociative character and overall energetic favorability. These results provide a theoretical basis for the association of C3d:CR2 at the acidic cavity of C3d and provide an explanation for binding of CR2 at the side face of C3d in the presence of nonphysiological zinc ions.

Selective NaNO2 recognition by a simple heteroditopic salt receptor based on L-ornithine molecular scaffold.

This paper describes the development of a simple heteroditopic salt receptor consisting of aza-18-crown-6 (a cation binding domain), nitrophenylurea (an anion binding domain) and an additional metacrylamide group appended to the carboxylic, α-amine and δ-amino groups of L-ornithine. Detailed binding studies showed that this receptor is capable of effectively and selectively binding NaNO2 salt.

Molecular dynamics simulations of wild type and mutants of human complement receptor 2 complexed with C3d.

The interaction between human complement receptor type 2 (CR2) and antigen-bound C3d can bridge the innate and adaptive immune systems. The recently determined structure of the CR2(SCR1-2):C3d complex has revealed the expected binding interface of CR2-C3d. In this article, wild type (WT) and three mutants of the new structure are studied by molecular dynamics (MD) simulations. The differently decreased structural stabilities of the mutants relative to WT are shown to be consistent with the experimental data, which can be explained by the different hydrogen bond patterns at the interfaces. It is also found that two clusters of residues (D36/E37/E39 and E160/D163/E166) in the acidic pocket of C3d are important for CR2-C3d interactions, which is in good agreement with previous mutagenesis study. In addition, functional dynamics and the conformational change of CR2 are explored by using domain cross-correlation map (DCCM), principal component analysis (PCA), and free energy landscape (FEL) methods. The conformational change mainly corresponds to the opening of a V-shaped structure of CR2, which is consistent with the previously reported high interdomain flexibility of CR2. We further suppose that the opening of a V-shaped structure of CR2 may favor the binding stability of CR2(SCR1-2):C3d. This study would provide some new insights into the understanding of the CR2-C3d interaction mechanism.

Regulation of humoral immunity by complement.

The complement system of innate immunity is important in regulating humoral immunity largely through the complement receptor CR2, which forms a coreceptor on B cells during antigen-induced activation. However, CR2 also retains antigens on follicular dendritic cells (FDCs). Display of antigen on FDCs is critical for clonal selection and affinity maturation of activated B cells. This review will discuss the role of complement in adaptive immunity in general with a focus on the interplay between CR2-associated antigen on B cells with CR2 expressed on FDCs. This latter interaction provides an opportunity for memory B cells to sample antigen over prolonged periods. The cocrystal structure of CR2 with its ligand C3d provides insight into how the complement system regulates access of antigen by B cells with implications for therapeutic manipulations to modulate aberrant B cell responses in the case of autoimmunity.

The complement receptor 2/factor H fusion protein TT30 protects paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and C3 fragment.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated intravascular hemolysis because of the lack from erythrocyte surface of the complement regulators CD55 and CD59, with subsequent uncontrolled continuous spontaneous activation of the complement alternative pathway (CAP), and at times of the complement classic pathway. Here we investigate in an in vitro model the effect on PNH erythrocytes of a novel therapeutic strategy for membrane-targeted delivery of a CAP inhibitor. TT30 is a 65 kDa recombinant human fusion protein consisting of the iC3b/C3d-binding region of complement receptor 2 (CR2) and the inhibitory domain of the CAP regulator factor H (fH). TT30 completely inhibits in a dose-dependent manner hemolysis of PNH erythrocytes in a modified extended acidified serum assay, and also prevents C3 fragment deposition on surviving PNH erythrocytes. The efficacy of TT30 derives from its direct binding to PNH erythrocytes; if binding to the erythrocytes is disrupted, only partial inhibition of hemolysis is mediated by TT30 in solution, which is similar to that produced by the fH moiety of TT30 alone, or by intact human fH. TT30 is a membrane-targeted selective CAP inhibitor that may prevent both intravascular and C3-mediated extravascular hemolysis of PNH erythrocytes and warrants consideration for the treatment of PNH patients.

A targeted complement-dependent strategy to improve the outcome of mAb therapy, and characterization in a murine model of metastatic cancer.

Complement inhibitors expressed on tumor cells provide an evasion mechanism against mAb therapy and may modulate the development of an acquired antitumor immune response. Here we investigate a strategy to amplify mAb-targeted complement activation on a tumor cell, independent of a requirement to target and block complement inhibitor expression or function, which is difficult to achieve in vivo. We constructed a murine fusion protein, CR2Fc, and demonstrated that the protein targets to C3 activation products deposited on a tumor cell by a specific mAb, and amplifies mAb-dependent complement activation and tumor cell lysis in vitro. In syngeneic models of metastatic lymphoma (EL4) and melanoma (B16), CR2Fc significantly enhanced the outcome of mAb therapy. Subsequent studies using the EL4 model with various genetically modified mice and macrophage-depleted mice revealed that CR2Fc enhanced the therapeutic effect of mAb therapy via both macrophage-dependent FcγR-mediated antibody-dependent cellular cytotoxicity, and by direct complement-mediated lysis. Complement activation products can also modulate adaptive immunity, but we found no evidence that either mAb or CR2Fc treatment had any effect on an antitumor humoral or cellular immune response. CR2Fc represents a potential adjuvant treatment to increase the effectiveness of mAb therapy of cancer.

Solution structure of TT30, a novel complement therapeutic agent, provides insight into its joint binding to complement C3b and C3d.

A novel therapeutic reagent TT30 was designed to be effective in diseases of the alternative pathway of complement such as paroxysmal nocturnal hemoglobinuria and other diseases. TT30 is constructed from the first four short complement regulator (SCR) domains of complement receptor type 2 (CR2) that bind to complement C3d, followed by the first five SCR domains of complement factor H that bind to complement C3b. In order to assess how TT30 binds to C3d and C3b, we determined the TT30 solution structure by a combination of analytical ultracentrifugation, X-ray scattering and constrained modeling. The sedimentation coefficients and radius of gyration of TT30 were unaffected by citrate or phosphate-buffered saline buffers and indicate an elongated monomeric structure with a sedimentation coefficient of 3.1 S and a radius of gyration R(G) of 6.9 nm. Molecular modeling starting from 3000 randomized TT30 conformations showed that high-quality X-ray curve fits were obtained with extended SCR arrangements, showing that TT30 has a limited degree of inter-SCR flexibility in its solution structure. The best-fit TT30 structural models are readily merged with the crystal structure of C3b to show that the four CR2 domains extend freely into solution when the five complement factor H domains are bound within C3b. We reevaluated the solution structure of the CR2-C3d complex that confirmed its recent crystal structure. This recent CR2-C3d crystal structure showed that TT30 is able to interact readily with C3d ligands in many orientations when TT30 is bound to C3b.

Comparative binding of soluble fragments (derCD23, sCD23, and exCD23) of recombinant human CD23 to CD21 (SCR 1-2) and native IgE, and their effect on IgE regulation.

IgE, responsible for type I hypersensitivities, is regulated by interactions between its receptor, CD23, and co-receptor CD21. To examine comparative binding of recombinant human CD21 SCR 1-2 and native human IgE to CD23 plus the effect of CD23 on IgE production, we engineered recombinant soluble human CD23 fragments; (1) derCD23, (2) sCD23 and (3) exCD23, formed in vivo by proteolysis. SPR analysis revealed a progressive increment in affinity of soluble fragments for IgE, upon increasing length of CD23 "stalk" domain, exCD23>sCD23>derCD23. Soluble CD23 fragments and their oligomeric state are shown to fine-tune the immune response. Oligomers appear more important in enhancing IgE synthesis and monomers lacking the tail residues fail to bind CD21 yet bind membrane IgE and down-regulate IgE synthesis. Co-ligation of membrane IgE and CD21 through soluble CD23 monomers is disturbed. This study supports anti-allergic therapies involving stabilizing membrane CD23, or preventing shedding of soluble CD23.

A crystal structure of the complex between human complement receptor 2 and its ligand C3d.

The interaction of complement receptor 2 (CR2)--which is present on B cells and follicular dendritic cells--with its antigen-bound ligand C3d results in an enhanced antibody response, thus providing an important link between the innate and adaptive immune systems. Although a cocrystal structure of a complex between C3d and the ligand-binding domains of CR2 has been published, several aspects of this structure, including the position in C3d of the binding interface, remained controversial because of disagreement with biochemical data. We now report a cocrystal structure of a CR2(SCR1-2):C3d complex at 3.2 angstrom resolution in which the interaction interfaces differ markedly from the previously published structure and are consistent with the biochemical data. It is likely that, in the previous structure, the interaction was influenced by the presence of zinc acetate additive in the crystallization buffer, leading to a nonphysiological complex. Detailed knowledge of the binding interface now at hand gives the potential to exploit the interaction in vaccine design or in therapeutics directed against autoreactive B cells.

Delineation of the complement receptor type 2-C3d complex by site-directed mutagenesis and molecular docking.

The interactions between the complement receptor type 2 (CR2) and the C3 complement fragments C3d, C3dg, and iC3b are essential for the initiation of a normal immune response. A crystal-derived structure of the two N-terminal short consensus repeat (SCR1-2) domains of CR2 in complex with C3d has previously been elucidated. However, a number of biochemical and biophysical studies targeting both CR2 and C3d appear to be in conflict with these structural data. Previous mutagenesis and heteronuclear NMR spectroscopy studies directed toward the C3d-binding site on CR2 have indicated that the CR2-C3d cocrystal structure may represent an encounter/intermediate or nonphysiological complex. With regard to the CR2-binding site on C3d, mutagenesis studies by Isenman and coworkers [Isenman, D. E., Leung, E., Mackay, J. D., Bagby, S. & van den Elsen, J. M. H. (2010). Mutational analyses reveal that the staphylococcal immune evasion molecule Sbi and complement receptor 2 (CR2) share overlapping contact residues on C3d: Implications for the controversy regarding the CR2/C3d cocrystal structure. J. Immunol. 184, 1946-1955] have implicated an electronegative "concave" surface on C3d in the binding process. This surface is discrete from the CR2-C3d interface identified in the crystal structure. We generated a total of 18 mutations targeting the two (X-ray crystallographic- and mutagenesis-based) proposed CR2 SCR1-2 binding sites on C3d. Using ELISA analyses, we were able to assess binding of mutant forms of C3d to CR2. Mutations directed toward the concave surface of C3d result in substantially compromised CR2 binding. By contrast, targeting the CR2-C3d interface identified in the cocrystal structure and the surrounding area results in significantly lower levels of disruption in binding. Molecular modeling approaches used to investigate disparities between the biochemical data and the X-ray structure of the CR2-C3d cocrystal result in highest-scoring solutions in which CR2 SCR1-2 is docked within the concave surface of C3d.

Biophysical investigations of complement receptor 2 (CD21 and CR2)-ligand interactions reveal amino acid contacts unique to each receptor-ligand pair.

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte 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, IFNalpha, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNalpha were titrated into (15)N-labeled SCR1-2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1-2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn(11), Arg(13), Ala(22), Arg(28), Ser(32), Arg(36), Lys(41), Lys(57), Tyr(64), Lys(67), Tyr(68), Arg(83), Gly(84), and Arg(89). With regard to IFNalpha, the binding is similar to the CR2-C3d interaction with specific residues being Arg(13), Tyr(16), Arg(28), Ser(42), Lys(48), Lys(50), Tyr(68), Arg(83), Gly(84), and Arg(89). We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with K(d) values of 0.13 and 160 microm, whereas the CR2-gp350 and CR2-IFNalpha interactions were characterized as single site binding events with affinities of 0.014 and 0.035 microm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.

Solvation effects in calculated electrostatic association free energies for the C3d-CR2 complex and comparison with experimental data.

The complement system is an integral part of the innate immune system that participates in the clearance of pathogens from the body. The association between complement protein fragment C3d and B or T cell-receptor complement receptor (CR) 2 represents a crucial link between innate and adaptive immunities. The goal of this study is to predict association abilities of C3d and CR2 mutants by theoretically calculating electrostatic free energies of association and to assess the importance of solvation effects in the calculations. We demonstrate that calculated solvation free energy differences and Coulombic free energies of association are more sensitive than electrostatic free energies of association in solution and, thus, more accurate in predicting previously published experimental data for the association abilities (relative to the parent proteins) of specific C3d and CR2 mutants. We show that a proportional relationship exists between the predicted solvation free energy differences and the experimental data, while an inversely proportional relationship exists between the predicted Coulombic free energies of association and the experimental data. Our results yield new insights into the physicochemical properties underlying C3d-CR2 association. We discuss the predictive validity of Coulombic, solvation, and solution electrostatic free energies of association and the generalization of our method for theoretical mutagenesis studies of other systems. This is a basic study, aimed toward improving our understanding of the theoretical basis of immune system regulation at the molecular level. Such insight can serve as the groundwork for the design of regulators with tailored properties, vaccines, and other biotechnology products.