Crosslinked flagella as a stabilized vaccine adjuvant scaffold.

BACKGROUND: Engineered vaccine proteins incorporating both antigen and adjuvant components are constructed with the aim of combining functions to induce effective protective immunity. Bacterial flagellin is a strong candidate for an engineered vaccine scaffold as it is known to provide adjuvant activity through its TLR5 and inflammasome activation. Moreover, polymerized flagellin filaments can elicit a more robust immunoglobulin response than monomeric flagellin, and the multimeric antigen form can also promote T cell-independent antibody responses. Here, we aim to produce and test a covalently stabilized polymerized flagellar filament, providing additional immune efficacy through stabilization of its polymeric filament structure, as well as stabilization for long-term storage. RESULTS: Computational modeling of monomer packing in flagellin filaments helped identify amino acids with proximity to neighboring flagella protofilaments. Paired cysteine substitutions were made at amino acids predicted to form inter-monomer disulfide cross-links, and these substitutions were capable of forming flagella when transfected into a flagellin-negative strain of Salmonella enterica subspecies Typhimurium. Interestingly, each paired substitution stabilized different helical conformational polymorphisms; the stabilized filaments lost the ability to transition between conformations, reducing bacterial motility. More importantly, the paired substitutions enabled extensive disulfide cross links and intra-filament multimer formation, and in one of the three variants, permitted filament stability in high acidic and temperature conditions where wild-type filaments would normally rapidly depolymerize. In addition, with regard to potential adjuvant activity, all crosslinked flagella filaments were able to induce wild-type levels of epithelial NF-κB in a cell reporter system. Finally, bacterial virulence was unimpaired in epithelial adherence and invasion, and the cysteine substitutions also appeared to increase bacterial resistance to oxidizing and reducing conditions. CONCLUSIONS: We identified amino acid pairs, with cysteine substitutions, were able to form intermolecular disulfide bonds that stabilized the resulting flagellar filaments in detergent, hydrochloric acid, and high temperatures while retaining its immunostimulatory function. Flagellar filaments with disulfide-stabilized protofilaments introduce new possibilities for the application of flagella as a vaccine adjuvant. Specifically, increased stability and heat tolerance permits long-term storage in a range of temperature environments, as well as delivery under a range of clinical conditions.

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.

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.

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.

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.