Patricia M. Legler

Histidine affinity tags affect MSP142 structural stability and immunodominance in mice

Inclusion of affinity tags has greatly facilitated process development for protein antigens, primarily for their recovery from complex mixtures. Although generally viewed as supportive of product development, affinity tags may have unintended consequences on protein solubility, susceptibility to aggregation, and immunogenicity. Merozoite surface protein 1 (MSP1), an erythrocytic stage protein of Plasmodium falciparum and a candidate malaria vaccine, was used to evaluate the impact of a metal ion affinity‐tag on both protein structure and the induction of immunity. To this end, codon harmonized gene sequences from the P. falciparum MSP142 of FVO and 3D7 parasites were cloned and purified with and without a histidine (His) tag. We report on the influence of His‐affinity tags on protein expression levels, solubility, secondary structure, thermal denaturation, aggregation and the impact on humoral and cellular immune responses in mice. While the overall immunogenicity induced by His‐tagged MSP142 proteins is greater, the fine specificity of the humoral and cellular immune responses is altered relative to anti‐parasitic antibody activity and the breadth of T‐cell responses. Thus, the usefulness of protein tags may be outweighed by their potential impact on structure and function, stressing the need for caution in their use.

Cellular and humoral immune effector mechanisms required for sterile protection against sporozoite challenge induced with the novel malaria vaccine candidate CelTOS

The malarial protein CelTOS, for cell-traversal protein for ookinetes and sporozoites, from Plasmodium berghei has been shown to mediate malarial invasion of both vertebrate and insect host cells and is required for establishing their successful infections. In the vertebrate host, Plasmodium sporozoites traverse via a complex passage through cellular barriers in the skin and the liver sinusoid to infect hepatocytes. Induction of immunity targeted to molecules involved in sporozoite motility and migration into hepatocytes may lead to abrogation of hepatocyte infection. We have previously demonstrated the potential of CelTOS as a target antigen for a pre-erythrocytic vaccine. The objective of the current study was to determine the potency of different vaccine platforms to induce protective immunity and determine the mode of action in protective immune responses. To this end, inbred Balb/c and outbred ICR mice were immunized with either the recombinant protein adjuvanted with Montanide ISA-720 or with a pCI-TPA plasmid encoding the P. berghei CelTOS (epidermal delivery by gene-gun) and assessed for the induction of protective responses against a homologous P. berghei challenge. Humoral and cellular immune responses induced by the various immunization regimens were evaluated in an effort to establish immune correlates. The results confirm that the CelTOS antigen is a potentially interesting pre-erythrocytic vaccine candidate and demonstrate that both arms of the adaptive immune system are required to mediate complete sterile protection against sporozoite challenge.

Probing the Donor and Acceptor Substrate Specificity of the γ-Glutamyl Transpeptidase

γ-Glutamyl transpeptidase (GGT) is a two-substrate enzyme that plays a central role in glutathione metabolism and is a potential target for drug design. GGT catalyzes the cleavage of γ-glutamyl donor substrates and the transfer of the γ-glutamyl moiety to an amine of an acceptor substrate or water. Although structures of bacterial GGT have revealed details of the protein-ligand interactions at the donor site, the acceptor substrate site is relatively undefined. The recent identification of a species-specific acceptor site inhibitor, OU749, suggests that these inhibitors may be less toxic than glutamine analogues. Here we investigated the donor and acceptor substrate preferences of Bacillus anthracis GGT (CapD) and applied computational approaches in combination with kinetics to probe the structural basis of the enzymes substrate and inhibitor binding specificities and compare them with human GGT. Site-directed mutagenesis studies showed that the R432A and R520S variants exhibited 6- and 95-fold decreases in hydrolase activity, respectively, and that their activity was not stimulated by the addition of the l-Cys acceptor substrate, suggesting an additional role in acceptor binding and/or catalysis of transpeptidation. Rat GGT (and presumably HuGGT) has strict stereospecificity for L-amino acid acceptor substrates, while CapD can utilize both L- and D-acceptor substrates comparably. Modeling and kinetic analysis suggest that R520 and R432 allow two alternate acceptor substrate binding modes for L- and D-acceptors. R432 is conserved in Francisella tularensis, Yersinia pestis, Burkholderia mallei, Helicobacter pylori and Escherichia coli, but not in human GGT. Docking and MD simulations point toward key residues that contribute to inhibitor and acceptor substrate binding, providing a guide to designing novel and specific GGT inhibitors.

Structure of RiVax: a recombinant ricin vaccine

The X-ray crystal structure (at 2.1 Å resolution) of an immunogen under development as part of a ricin vaccine for humans is presented and structure-based analysis of the results was conducted with respect to related proteins and the known determinants for inducing or suppressing the protective immune response.

Introduction of a disulfide bond leads to stabilization and crystallization of a ricin immunogen.

RTA1‐33/44‐198 is a catalytically inactive, single‐domain derivative of the ricin toxin A‐chain (RTA) engineered to serve as a stable protein scaffold for presentation of native immunogenic epitopes (Olson et al., Protein Eng Des Sel 2004;17:391–397). To improve the stability and solubility of RTA1‐33/44‐198 further, we have undertaken the design challenge of introducing a disulfide (SS) bond. Nine pairs of residues were selected for placement of the SS‐bond based on molecular dynamics simulation studies of the modeled single‐domain chain. Disulfide formation at either of two positions (R48C/T77C or V49C/E99C) involving a specific surface loop (44–55) increased the protein melting temperature by ∼5°C compared with RTA1‐33/44‐198 and by ∼13°C compared with RTA. Prolonged stability studies of the R48C/T77C variant (>60 days at 37°C, pH 7.4) confirmed a >40% reduction in self‐aggregation compared with RTA1‐33/44‐198 lacking the SS‐bond. The R48C/T77C variant retained affinity for anti‐RTA antibodies capable of neutralizing ricin toxin, including a monoclonal that recognizes a human B‐cell epitope. Introduction of either R48C/T77C or V49C/E99C promoted crystallization of RTA1‐33/44‐198, and the X‐ray structures of the variants were solved to 2.3Å or 2.1 Å resolution, respectively. The structures confirm formation of an intramolecular SS‐bond, and reveal a single‐domain fold that is significantly reduced in volume compared with RTA. Loop 44 to 55 is partly disordered as predicted by simulations, and is positioned to form self‐self interactions between symmetry‐related molecules. We discuss the importance of RTA loop 34 to 55 as a nucleus for unfolding and aggregation, and draw conclusions for ongoing structure‐based minimalist design of RTA‐based immunogens. Proteins 2011. Published 2010 Wiley‐Liss, Inc.

Evaluation of Disulfide Bond Position to Enhance the Thermal Stability of a Highly Stable Single Domain Antibody

Single domain antibodies are the small recombinant variable domains derived from camelid heavy-chain-only antibodies. They are renowned for their stability, in large part due to their ability to refold following thermal or chemical denaturation. In addition to refolding after heat denaturation, A3, a high affinity anti-Staphylococcal Enterotoxin B single domain antibody, possesses a melting temperature of ∼84°C, among the highest reported for a single domain antibody. In this work we utilized the recently described crystal structure of A3 to select locations for the insertion of a second disulfide bond and evaluated the impact that the addition of this second bond had on the melting temperature. Four double-disulfide versions of A3 were constructed and each was found to improve the melting temperature relative to the native structure without reducing affinity. Placement of the disulfide bond at a previously published position between framework regions 2 and 3 yielded the largest improvement (>6°C), suggesting this location is optimal, and seemingly provides a universal route to raise the melting temperature of single domain antibodies. This study further demonstrates that even single domain antibodies with extremely high melting points can be further stabilized by addition of disulfide bonds.

Comparison of Immunoreactivity of Staphylococcal Enterotoxin B Mutants for Use as Toxin Surrogates

The development and testing of detection methodologies for biothreat agents are by their very nature complicated by the necessity to handle hazardous materials. Toxoids prepared by thermal or chemical inactivation are often used in place of the native toxin; however, the process of detoxification can decrease the agents ability to be detected at similar concentrations. One method to overcome this limitation is the use of toxin mutants which have altered amino acid sequences sufficient to abrogate or greatly reduce their toxic activity. While this method of toxoid preparation is much more controlled, there is still no guarantee that the resulting product will be equal in detectability to the native toxin. In this work, we have evaluated the utility of two recombinantly expressed Staphylococcal Enterotoxin B (SEB) mutants, a single point mutant (Y89A), and a mutant with three amino acids changed (L45R, Y89A, Y94A), to act as surrogates for SEB in immunoassays. We evaluated the affinity of a number of anti-SEB monoclonal antibodies (mAb) and an anti-SEB single domain antibody (sdAb) for SEB and its surrogates. One of the mAbs affinity was decreased by a factor of 3000 for the triple mutant, and another mAbs affinity for the triple mutant was decreased by 11-fold while the others bound the mutants nearly as well as they did the native toxin. MAGPIX sandwich immunoassays were used to evaluate the ability of all combinations of the recognition reagents to detect the SEB mutants in comparison to SEB and a chemically inactivated SEB. These results show that recombinant mutants of SEB can serve as much more useful surrogates for this hazardous material relative to the chemically inactivated toxin; however, even the point mutant impacted limits of detection, illustrating the need to evaluate the utility of toxin mutants on a case-by-case basis depending on the immunoreagents being employed.

Structural and mutational analysis of a monomeric and dimeric form of a single domain antibody with implications for protein misfolding.

Camelid single domain antibodies (sdAb) are known for their thermal stability and reversible refolding. We have characterized an unusually stable sdAb recognizing Staphylococcal enterotoxin B with one of the highest reported melting temperatures (Tm = 85°C). Unexpectedly, ∼10−20% of the protein formed a dimer in solution. Three other cases where <20% of the sdAb dimerized have been reported; however, this is the first report of both the monomeric and dimeric X‐ray crystal structures. Concentration of the monomer did not lead to the formation of new dimer suggesting a stable conformationally distinct species in a fraction of the cytoplasmically expressed protein. Comparison of periplasmic and cytoplasmic expression showed that the dimer was associated with cytoplasmic expression. The disulfide bond was partially reduced in the WT protein purified from the cytoplasm and the protein irreversibly unfolded. Periplasmic expression produced monomeric protein with a fully formed disulfide bond and mostly reversible refolding. Crystallization of a disulfide‐bond free variant, C22A/C99V, purified from the periplasm yielded a structure of a monomeric form, while crystallization of C22A/C99V from the cytoplasm produced an asymmetric dimer. In the dimer, a significant conformational asymmetry was found in the loop residues of the edge β‐strands (S50‐Y60) containing the highly variable complementarity determining region, CDR2. Two dimeric assemblies were predicted from the crystal packing. Mutation of a residue at one of the interfaces, Y98A, disrupted the dimer in solution. The pleomorphic homodimer may yield insight into the stability of misfolded states and the importance of the conserved disulfide bond in preventing their formation. Proteins 2014; 82:3101–3116.

Structural insight into exosite binding and discovery of novel exosite inhibitors of botulinum neurotoxin serotype A through in silico screening.

Botulinum neurotoxin serotype A (BoNT/A) is the most lethal toxin among the Tier 1 Select Agents. Development of potent and selective small molecule inhibitors against BoNT/A zinc metalloprotease remains a challenging problem due to its exceptionally large substrate binding surface and conformational plasticity. The exosites of the catalytic domain of BoNT/A are intriguing alternative sites for small molecule intervention, but their suitability for inhibitor design remains largely unexplored. In this study, we employed two recently identified exosite inhibitors, D-chicoric acid and lomofungin, to probe the structural features of the exosites and molecular mechanisms of synergistic inhibition. The results showed that D-chicoric acid favors binding at the α-exosite, whereas lomofungin preferentially binds at the β-exosite by mimicking the substrate β-sheet binding interaction. Molecular dynamics simulations and binding interaction analysis of the exosite inhibitors with BoNT/A revealed key elements and hotspots that likely contribute to the inhibitor binding and synergistic inhibition. Finally, we performed database virtual screening for novel inhibitors of BoNT/A targeting the exosites. Hits C1 and C2 showed non-competitive inhibition and likely target the α- and β-exosites, respectively. The identified exosite inhibitors may provide novel candidates for structure-based development of therapeutics against BoNT/A intoxication.

3-substituted indole inhibitors against Francisella tularensis FabI identified by structure-based virtual screening.

In this study, we describe novel inhibitors against Francisella tularensis SchuS4 FabI identified from structure-based in silico screening with integrated molecular dynamics simulations to account for induced fit of a flexible loop crucial for inhibitor binding. Two 3-substituted indoles, 54 and 57, preferentially bound the NAD(+) form of the enzyme and inhibited growth of F. tularensis SchuS4 at concentrations near that of their measured Ki. While 57 was species-specific, 54 showed a broader spectrum of growth inhibition against F. tularensis , Bacillus anthracis , and Staphylococcus aureus . Binding interaction analysis in conjunction with site-directed mutagenesis revealed key residues and elements that contribute to inhibitor binding and species specificity. Mutation of Arg-96, a poorly conserved residue opposite the loop, was unexpectedly found to enhance inhibitor binding in the R96G and R96M variants. This residue may affect the stability and closure of the flexible loop to enhance inhibitor (or substrate) binding.