Bacterial killing by complement requires membrane attack complex formation via surface-bound C5 convertases.

The immune system kills bacteria by the formation of lytic membrane attack complexes (MACs), triggered when complement enzymes cleave C5. At present, it is not understood how the MAC perturbs the composite cell envelope of Gram-negative bacteria. Here, we show that the role of C5 convertase enzymes in MAC assembly extends beyond the cleavage of C5 into the MAC precursor C5b. Although purified MAC complexes generated from preassembled C5b6 perforate artificial lipid membranes and mammalian cells, these components lack bactericidal activity. In order to permeabilize both the bacterial outer and inner membrane and thus kill a bacterium, MACs need to be assembled locally by the C5 convertase enzymes. Our data indicate that C5b6 rapidly loses the capacity to form bactericidal pores; therefore, bacterial killing requires both in situ conversion of C5 and immediate insertion of C5b67 into the membrane. Using flow cytometry and atomic force microscopy, we show that local assembly of C5b6 at the bacterial surface is required for the efficient insertion of MAC pores into bacterial membranes. These studies provide basic molecular insights into MAC assembly and bacterial killing by the immune system.

Staphylococcal Immune Evasion Proteins: Structure, Function, and Host Adaptation.

Staphylococcus aureus is a successful human and animal pathogen. Its pathogenicity is linked to its ability to secrete a large amount of virulence factors. These secreted proteins interfere with many critical components of the immune system, both innate and adaptive, and hamper proper immune functioning. In recent years, numerous studies have been conducted in order to understand the molecular mechanism underlying the interaction of evasion molecules with the host immune system. Structural studies have fundamentally contributed to our understanding of the mechanisms of action of the individual factors. Furthermore, such studies revealed one of the most striking characteristics of the secreted immune evasion molecules: their conserved structure. Despite high-sequence variability, most immune evasion molecules belong to a small number of structural categories. Another remarkable characteristic is that S. aureus carries most of these virulence factors on mobile genetic elements (MGE) or ex-MGE in its accessory genome. Coevolution of pathogen and host has resulted in immune evasion molecules with a highly host-specific function and prevalence. In this review, we explore how these shared structures and genomic locations relate to function and host specificity. This is discussed in the context of therapeutic options for these immune evasion molecules in infectious as well as in inflammatory diseases.

AESOP: A Python Library for Investigating Electrostatics in Protein Interactions.

Electric fields often play a role in guiding the association of protein complexes. Such interactions can be further engineered to accelerate complex association, resulting in protein systems with increased productivity. This is especially true for enzymes where reaction rates are typically diffusion limited. To facilitate quantitative comparisons of electrostatics in protein families and to describe electrostatic contributions of individual amino acids, we previously developed a computational framework called AESOP. We now implement this computational tool in Python with increased usability and the capability of performing calculations in parallel. AESOP utilizes PDB2PQR and Adaptive Poisson-Boltzmann Solver to generate grid-based electrostatic potential files for protein structures provided by the end user. There are methods within AESOP for quantitatively comparing sets of grid-based electrostatic potentials in terms of similarity or generating ensembles of electrostatic potential files for a library of mutants to quantify the effects of perturbations in protein structure and protein-protein association.

Peptide redesign for inhibition of the complement system: Targeting age-related macular degeneration.

PURPOSE: To redesign a complement-inhibiting peptide with the potential to become a therapeutic for dry and wet age-related macular degeneration (AMD). METHODS: We present a new potent peptide (Peptide 2) of the compstatin family. The peptide is developed by rational design, based on a mechanistic binding hypothesis, and structural and physicochemical properties derived from molecular dynamics (MD) simulation. The inhibitory activity, efficacy, and solubility of Peptide 2 are evaluated using a hemolytic assay, a human RPE cell-based assay, and ultraviolet (UV) absorption properties, respectively, and compared to the respective properties of its parent peptide (Peptide 1). RESULTS: The sequence of Peptide 2 contains an arginine-serine N-terminal extension (a characteristic of parent Peptide 1) and a novel 8-polyethylene glycol (PEG) block C-terminal extension. Peptide 2 has significantly improved aqueous solubility compared to Peptide 1 and comparable complement inhibitory activity. In addition, Peptide 2 is more efficacious in inhibiting complement activation in a cell-based model that mimics the pathobiology of dry AMD. CONCLUSIONS: We have designed a new peptide analog of compstatin that combines N-terminal polar amino acid extensions and C-terminal PEGylation extensions. This peptide demonstrates significantly improved aqueous solubility and complement inhibitory efficacy, compared to the parent peptide. The new peptide overcomes the aggregation limitation for clinical translation of previous compstatin analogs and is a candidate to become a therapeutic for the treatment of AMD.

Molecular insights into the surface-specific arrangement of complement C5 convertase enzymes.

BACKGROUND: Complement is a large protein network in plasma that is crucial for human immune defenses and a major cause of aberrant inflammatory reactions. The C5 convertase is a multi-molecular protease complex that catalyses the cleavage of native C5 into its biologically important products. So far, it has been difficult to study the exact molecular arrangement of C5 convertases, because their non-catalytic subunits (C3b) are covalently linked to biological surfaces through a reactive thioester. Through development of a highly purified model system for C5 convertases, we here aim to provide insights into the surface-specific nature of these important protease complexes. RESULTS: Alternative pathway (AP) C5 convertases were generated on small streptavidin beads that were coated with purified C3b molecules. Site-specific biotinylation of C3b via the thioester allowed binding of C3b in the natural orientation on the surface. In the presence of factor B and factor D, these C3b beads could effectively convert C5. Conversion rates of surface-bound C3b were more than 100-fold higher than fluid-phase C3b, confirming the requirement of a surface. We determine that high surface densities of C3b, and its attachment via the thioester, are essential for C5 convertase formation. Combining our results with molecular modeling explains how high C3b densities may facilitate intermolecular interactions that only occur on target surfaces. Finally, we define two interfaces on C5 important for its recognition by surface-bound C5 convertases. CONCLUSIONS: We establish a highly purified model that mimics the natural arrangement of C5 convertases on a surface. The developed model and molecular insights are essential to understand the molecular basis of deregulated complement activity in human disease and will facilitate future design of therapeutic interventions against these critical enzymes in inflammation.

Hybrid flagellin as a T cell independent vaccine scaffold.

BACKGROUND: To extend the potency of vaccines against infectious diseases, vaccines should be able to exploit multiple arms of the immune system. One component of the immune system that is under-used in vaccine design is the subset of B cells known to be capable of responding to repetitive antigenic epitopes and differentiate into plasma cells even in the absence of T cell help (T-independent, TI). RESULTS: To target vaccine responses from T-independent B cells, we reengineered a bacterial Flagellin (FliC) by replacing its exposed D3 domain with a viral envelope protein from Dengue virus (DENV2). The resulting hybrid FliC protein (hFliC) was able to form stable filaments decorated with conformationally intact DENV2 envelope domains. These filaments were not only capable of inducing a T cell-dependent (TD) humoral antibody response, but also significant IgM and IgG3 antibody response in a helper T cell repertoire-restricted transgenic mouse model. CONCLUSIONS: Our results provide proof-of-principle demonstration that a reengineered hybrid FliC could be used as a platform for polymeric subunit vaccines, enhancing T cell-dependent and possibly inducing T-independent antibody responses from B-1 B cells as well.

Molecular analysis of the interaction between staphylococcal virulence factor Sbi-IV and complement C3d.

Staphylococcus aureus expresses numerous virulence factors that aid in immune evasion. The four-domain staphylococcal immunoglobulin binding (Sbi) protein interacts with complement component 3 (C3) and its thioester domain (C3d)-containing fragments. Recent structural data suggested two possible modes of binding of Sbi domain IV (Sbi-IV) to C3d, but the physiological binding mode remains unclear. We used a computational approach to provide insight into the C3d-Sbi-IV interaction. Molecular dynamics (MD) simulations showed that the first binding mode (PDB code 2WY8) is more robust than the second (PDB code 2WY7), with more persistent polar and nonpolar interactions, as well as conserved interfacial solvent accessible surface area. Brownian dynamics and steered MD simulations revealed that the first binding mode has faster association kinetics and maintains more stable intermolecular interactions compared to the second binding mode. In light of available experimental and structural data, our data confirm that the first binding mode represents Sbi-IV interaction with C3d (and C3) in a physiological context. Although the second binding mode is inherently less stable, we suggest a possible physiological role. Both binding sites may serve as a template for structure-based design of novel complement therapeutics.

Novel compstatin family peptides inhibit complement activation by drusen-like deposits in human retinal pigmented epithelial cell cultures.

We have used a novel human retinal pigmented epithelial (RPE) cell-based model that mimics drusen biogenesis and the pathobiology of age-related macular degeneration to evaluate the efficacy of newly designed peptide inhibitors of the complement system. The peptides belong to the compstatin family and, compared to existing compstatin analogs, have been optimized to promote binding to their target, complement protein C3, and to enhance solubility by improving their polarity/hydrophobicity ratios. Based on analysis of molecular dynamics simulation data of peptide-C3 complexes, novel binding features were designed by introducing intermolecular salt bridge-forming arginines at the N-terminus and at position -1 of N-terminal dipeptide extensions. Our study demonstrates that the RPE cell assay has discriminatory capability for measuring the efficacy and potency of inhibitory peptides in a macular disease environment.

Purified complement C3b triggers phagocytosis and activation of human neutrophils via complement receptor 1.

The complement system provides vital immune protection against infectious agents by labeling them with complement fragments that enhance phagocytosis by immune cells. Many details of complement-mediated phagocytosis remain elusive, partly because it is difficult to study the role of individual complement proteins on target surfaces. Here, we employ serum-free methods to couple purified complement C3b onto E. coli bacteria and beads and then expose human neutrophils to these C3b-coated targets. We examine the neutrophil response using a combination of flow cytometry, confocal microscopy, luminometry, single-live-cell/single-target manipulation, and dynamic analysis of neutrophil spreading on opsonin-coated surfaces. We show that purified C3b can potently trigger phagocytosis and killing of bacterial cells via Complement receptor 1. Comparison of neutrophil phagocytosis of C3b- versus antibody-coated beads with single-bead/single-target analysis exposes a similar cell morphology during engulfment. However, bulk phagocytosis assays of C3b-beads combined with DNA-based quenching reveal that these are poorly internalized compared to their IgG1 counterparts. Similarly, neutrophils spread slower on C3b-coated compared to IgG-coated surfaces. These observations support the requirement of multiple stimulations for efficient C3b-mediated uptake. Together, our results establish the existence of a direct pathway of phagocytic uptake of C3b-coated targets and present methodologies to study this process.

Role of Electrostatic Hotspots in the Selectivity of Complement Control Proteins Toward Human and Bovine Complement Inhibition.

Poxviruses are dangerous pathogens, which can cause fatal infection in unvaccinated individuals. The causative agent of smallpox in humans, variola virus, is closely related to the bovine vaccinia virus, yet the molecular basis of their selectivity is currently incompletely understood. Here, we examine the role of the electrostatics in the selectivity of the smallpox protein SPICE and vaccinia protein VCP toward the human and bovine complement protein C3b, a key component of the complement immune response. Electrostatic calculations, in-silico alanine-scan and electrostatic hotspot analysis, as introduced by Kieslich and Morikis (PLoS Comput. Biol. 2012), are used to assess the electrostatic complementarity and to identify sites resistant to local perturbation where the electrostatic potential is likely to be evolutionary conserved. The calculations suggest that the bovine C3b is electrostatically prone to selectively bind its VCP ligand. On the other hand, the human isoform of C3b exhibits a lower electrostatic complementarity toward its SPICE ligand. Yet, the human C3b displays a highly preserved electrostatic core, which suggests that this isoform could be less selective in binding different ligands like SPICE and the human Factor H. This is supported by experimental cofactor activity assays revealing that the human C3b is prone to bind both SPICE and Factor H, which exhibit diverse electrostatic properties. Additional investigations considering mutants of SPICE and VCP that revert their selectivity reveal an "electrostatic switch" into the central modules of the ligands, supporting the critical role of the electrostatics in the selectivity. Taken together, these evidences provide insights into the selectivity mechanism of the complement regulator proteins encoded by the variola and vaccinia viruses to circumvent the complement immunity and exert their pathogenic action. These fundamental aspects are valuable for the development of novel vaccines and therapeutic strategies.

Functional Characterization of Alternative and Classical Pathway C3/C5 Convertase Activity and Inhibition Using Purified Models.

Complement is essential for the protection against infections; however, dysregulation of complement activation can cause onset and progression of numerous inflammatory diseases. Convertase enzymes play a central role in complement activation and produce the key mediators of complement: C3 convertases cleave C3 to generate chemoattractant C3a and label target cells with C3b, which promotes phagocytosis; C5 convertases cleave C5 into chemoattractant C5a, and C5b, which drives formation of the membrane attack complex. Since convertases mediate nearly all complement effector functions, they are ideal targets for therapeutic complement inhibition. A unique feature of convertases is their covalent attachment to target cells, which effectively confines complement activation to the cell surface. However, surface localization precludes detailed analysis of convertase activation and inhibition. In our previous work, we developed a model system to form purified alternative pathway (AP) C5 convertases on C3b-coated beads and quantify C5 conversion via functional analysis of released C5a. Here, we developed a C3aR cell reporter system that enables functional discrimination between C3 and C5 convertases. By regulating the C3b density on the bead surface, we observe that high C3b densities are important for conversion of C5, but not C3, by AP convertases. Screening of well-characterized complement-binding molecules revealed that differential inhibition of AP C3 convertases (C3bBb) and C5 convertases [C3bBb(C3b)n] is possible. Although both convertases contain C3b, the C3b-binding molecules Efb-C/Ecb and FHR5 specifically inhibit C5 conversion. Furthermore, using a new classical pathway convertase model, we show that these C3b-binding proteins not only block AP C3/C5 convertases but also inhibit formation of a functional classical pathway C5 convertase under well-defined conditions. Our models enable functional characterization of purified convertase enzymes and provide a platform for the identification and development of specific convertase inhibitors for treatment of complement-mediated disorders.

Virtual Screening of Chemical Compounds for Discovery of Complement C3 Ligands.

The complement system is our first line of defense against foreign pathogens, but when it is not properly regulated, complement is implicated in the pathology of several autoimmune and inflammatory disorders. Compstatin is a peptidic complement inhibitor that acts by blocking the cleavage of complement protein C3 to the proinflammatory fragment C3a and opsonin fragment C3b. In this study, we aim to identify druglike small-molecule complement inhibitors with physicochemical, geometric, and binding properties similar to those of compstatin. We employed two approaches using various high-throughput virtual screening methods, which incorporate molecular dynamics (MD) simulations, pharmacophore model design, energy calculations, and molecular docking and scoring. We have generated a library of 274 chemical compounds with computationally predicted binding affinities for the compstatin binding site of C3. We have tested subsets of these chemical compounds experimentally for complement inhibitory activity, using hemolytic assays, and for binding affinity, using microscale thermophoresis. As a result, although none of the compounds showed inhibitory activity, compound 29 was identified to exhibit weak competitive binding against a potent compstatin analogue, therefore validating our computational approaches. Additional docking and MD simulation studies suggest that compound 29 interacts with C3 residues, which have been shown to be important in binding of compstatin to the C3c fragment of C3. Compound 29 is amenable to physicochemical optimization to acquire inhibitory properties. Additionally, it is possible that some of the untested compounds will demonstrate binding and inhibition in future experimental studies.

Let's Tie the Knot: Marriage of Complement and Adaptive Immunity in Pathogen Evasion, for Better or Worse.

The complement system is typically regarded as an effector arm of innate immunity, leading to recognition and killing of microbial invaders in body fluids. Consequently, pathogens have engaged in an arms race, evolving molecules that can interfere with proper complement responses. However, complement is no longer viewed as an isolated system, and links with other immune mechanisms are continually being discovered. Complement forms an important bridge between innate and adaptive immunity. While its roles in innate immunity are well-documented, its function in adaptive immunity is less characterized. Therefore, it is no surprise that the field of pathogenic complement evasion has focused on blockade of innate effector functions, while potential inhibition of adaptive immune responses (via complement) has been overlooked to a certain extent. In this review, we highlight past and recent developments on the involvement of complement in the adaptive immune response. We discuss the mechanisms by which complement aids in lymphocyte stimulation and regulation, as well as in antigen presentation. In addition, we discuss microbial complement evasion strategies, and highlight specific examples in the context of adaptive immune responses. These emerging ties between complement and adaptive immunity provide a catalyst for future discovery in not only the field of adaptive immune evasion but in elucidating new roles of complement.

Quantitative Modeling of the Alternative Pathway of the Complement System.

The complement system is an integral part of innate immunity that detects and eliminates invading pathogens through a cascade of reactions. The destructive effects of the complement activation on host cells are inhibited through versatile regulators that are present in plasma and bound to membranes. Impairment in the capacity of these regulators to function in the proper manner results in autoimmune diseases. To better understand the delicate balance between complement activation and regulation, we have developed a comprehensive quantitative model of the alternative pathway. Our model incorporates a system of ordinary differential equations that describes the dynamics of the four steps of the alternative pathway under physiological conditions: (i) initiation (fluid phase), (ii) amplification (surfaces), (iii) termination (pathogen), and (iv) regulation (host cell and fluid phase). We have examined complement activation and regulation on different surfaces, using the cellular dimensions of a characteristic bacterium (E. coli) and host cell (human erythrocyte). In addition, we have incorporated neutrophil-secreted properdin into the model highlighting the cross talk of neutrophils with the alternative pathway in coordinating innate immunity. Our study yields a series of time-dependent response data for all alternative pathway proteins, fragments, and complexes. We demonstrate the robustness of alternative pathway on the surface of pathogens in which complement components were able to saturate the entire region in about 54 minutes, while occupying less than one percent on host cells at the same time period. Our model reveals that tight regulation of complement starts in fluid phase in which propagation of the alternative pathway was inhibited through the dismantlement of fluid phase convertases. Our model also depicts the intricate role that properdin released from neutrophils plays in initiating and propagating the alternative pathway during bacterial infection.

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.

Electrostatic Interactions between Complement Regulator CD46(SCR1-2) and Adenovirus Ad11/Ad21 Fiber Protein Knob.

Adenoviruses bind to a variety of human cells to cause infection. Both the B2 adenovirus 11 and B1 adenovirus 21 use protein knobs to bind to complement regulator CD46(SCR1-2) in order to gain entry into host cells. In each complex, the two proteins are highly negatively charged but bind to each other at an interface with oppositely charged surface patches. We computationally generated single-alanine mutants of charged residues in the complexes CD46(SCR1-2)-Ad11k and CD46(SCR1-2)-Ad21k. We used electrostatic clustering and Poisson-Boltzmann free energy calculations to propose a hypothesis on the role of electrostatics in association. Our results delineate specific interfacial electrostatic interactions that are critical for association in both CD46(SCR1-2)-Ad11k and CD46(SCR1-2)-Ad21k. These results will serve as a predictive tool in the selection of mutants with desired binding affinity in experimental mutagenesis studies. This study will also serve as a foundation for the design of inhibitors to treat adenovirus infections.

Energetic evaluation of binding modes in the C3d and Factor H (CCP 19-20) complex.

As a part of innate immunity, the complement system relies on activation of the alternative pathway (AP). While feed-forward amplification generates an immune response towards foreign surfaces, the process requires regulation to prevent an immune response on the surface of host cells. Factor H (FH) is a complement protein secreted by native cells to negatively regulate the AP. In terms of structure, FH is composed of 20 complement-control protein (CCP) modules that are structurally homologous but vary in composition and function. Mutations in these CCPs have been linked to states of autoimmunity. In particular, several mutations in CCP 19-20 are correlated to atypical hemolytic uremic syndrome (aHUS). From crystallographic structures there are three putative binding sites of CCP 19-20 on C3d. Since there has been some controversy over the primary mode of binding from experimental studies, we approach characterization of binding using computational methods. Specifically, we compare each binding mode in terms of electrostatic character, structural stability, dissociative and associative properties, and predicted free energy of binding. After a detailed investigation, we found two of the three binding sites to be similarly stable while varying in the number of contacts to C3d and in the energetic barrier to complex dissociation. These sites are likely physiologically relevant and may facilitate multivalent binding of FH CCP 19-20 to C3b and either C3d or host glycosaminoglycans. We propose thermodynamically stable binding with modules 19 and 20, the latter driven by electrostatics, acting synergistically to increase the apparent affinity of FH for host surfaces.

Discovery of Small Molecules for Fluorescent Detection of Complement Activation Product C3d.

Complement activation plays a major role in many acute and chronic inflammatory conditions. C3d, a terminal product of complement activation, remains covalently attached to cells and is an excellent biomarker of complement-mediated inflammation. We employed a virtual high-throughput screening protocol to identify molecules with predicted binding to complement C3d and with intrinsic fluorescence properties to enable detection. Pharmacophore models were developed based on known C3d-ligand interactions and information from computational analysis of structural and molecular dynamics data. Iterative pharmacophore-based virtual screening was performed to identify druglike molecules with physicochemical similarity to the natural C3d ligand CR2. Hits from the pharmacophore screens were docked to C3d and ranked based on predicted binding free energies. Top-ranked molecules were selected for experimental validation of binding affinity to C3d, using microscale thermophoresis, and for their suitability to become molecular imaging agents, using fluorescence spectroscopy. This work serves as a foundation for identifying additional fluorescent molecules with high-affinity for C3d that will subsequently be explored as noninvasive in vivo diagnostics of complement-mediated inflammation, for spatiotemporal monitoring of disease progression, and for targeting therapeutics to sites of inflammation.

New compstatin peptides containing N-terminal extensions and non-natural amino acids exhibit potent complement inhibition and improved solubility characteristics.

Compstatin peptides are complement inhibitors that bind and inhibit cleavage of complement C3. Peptide binding is enhanced by hydrophobic interactions; however, poor solubility promotes aggregation in aqueous environments. We have designed new compstatin peptides derived from the W4A9 sequence (Ac-ICVWQDWGAHRCT-NH2, cyclized between C2 and C12), based on structural, computational, and experimental studies. Furthermore, we developed and utilized a computational framework for the design of peptides containing non-natural amino acids. These new compstatin peptides contain polar N-terminal extensions and non-natural amino acid substitutions at positions 4 and 9. Peptides with α-modified non-natural alanine analogs at position 9, as well as peptides containing only N-terminal polar extensions, exhibited similar activity compared to W4A9, as quantified via ELISA, hemolytic, and cell-based assays, and showed improved solubility, as measured by UV absorbance and reverse-phase HPLC experiments. Because of their potency and solubility, these peptides are promising candidates for therapeutic development in numerous complement-mediated diseases.

Viral regulators of complement activation: structure, function and evolution.

The complement system surveillance in the host is effective in controlling viral propagation. Consequently, to subvert this effector mechanism, viruses have developed a series of adaptations. One among these is encoding mimics of host regulators of complement activation (RCA) which help viruses to avoid being labeled as 'foreign' and protect them from complement-mediated neutralization and complement-enhanced antiviral adaptive immunity. In this review, we provide an overview on the structure, function and evolution of viral RCA proteins.