John H. Carra

Improved stability of a protein vaccine through elimination of a partially unfolded state

Ricin is a potent toxin presenting a threat as a biological weapon. The holotoxin consists of two disulfide‐linked polypeptides: an enzymatically active A chain (RTA) and a galactose/N‐acetylgalactosamine‐binding B chain. Efforts to develop an inactivated version of the A chain as a vaccine have been hampered by limitations of stability and solubility. Previously, recombinant truncated versions of the 267‐amino‐acid A chain consisting of residues 1–33/44–198 or 1–198 were designed by protein engineering to overcome these limits and were shown to be effective and nontoxic as vaccines in mice. Herein we used CD, dynamic light scattering, fluorescence, and Fourier‐transform infrared spectroscopy to examine the biophysical properties of these proteins. Although others have found that recombinant RTA (rRTA) adopts a partially unfolded, molten globule–like state at 45°C, rRTA 1–33/44–198 and 1–198 are significantly more thermostable, remaining completely folded at temperatures up to 53°C and 51°C, respectively. Deleting both an exposed loop region (amino acids 34–43) and the C‐terminal domain (199–267) contributed to increased thermostability. We found that chemically induced denaturation of rRTA, but not the truncated variants, proceeds through at least a three‐state mechanism. The intermediate state in rRTA unfolding has a hydrophobic core accessible to ANS and an unfolded C‐terminal domain. Removing the C‐terminal domain changed the mechanism of rRTA unfolding, eliminating a tendency to adopt a partially unfolded state. Our results support the conclusion that these derivatives are superior candidates for development as vaccines against ricin and suggest an approach of reduction to minimum essential domains for design of more thermostable recombinant antigens.

Association of Ebola Virus Matrix Protein VP40 with Microtubules

ABSTRACT Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.

Lethal Shock Induced by Streptococcal Pyrogenic Exotoxin A in Mice Transgenic for Human Leukocyte Antigen–DQ8 and Human CD4 Receptors: Implications for Development of Vaccines and Therapeutics

Streptococcal and staphylococcal infections result in significant human morbidity and mortality. This study used a transgenic murine model expressing human major histocompatibility complex (MHC) class II and human CD4 in which, without additional toxic sensitization, human-like responses to the bacterial superantigen (SAg) streptococcal pyrogenic exotoxin A (SpeA) could be simulated, as determined by studying multiple biologic effects of the SAgs in vivo. Expression of human leukocyte antigen (HLA)-DQ8 rendered the mice susceptible to SpeA-induced lethal shock that was accompanied by massive cytokine production and marked elevation of serum alanine and aspartate aminotransferase levels. Of importance, this model enabled examination of the efficacy of an engineered non-SAg vaccine candidate against SpeA in the context of HLA. This report is thought to be the first of a lethal shock triggered in mice by bacterial SAgs without prior sensitization and examination of a vaccine against streptococcal SAg in the context of human MHC receptors.

Machupo Virus Glycoprotein Determinants for Human Transferrin Receptor 1 Binding and Cell Entry

Machupo virus (MACV) is a highly pathogenic New World arenavirus that causes hemorrhagic fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to their cellular receptor, transferrin receptor 1 (TfR1). TfR1 residues essential for this interaction have been described, and a co-crystal of MACV GP1 bound to TfR1 suggests GP1 residues important for this association. We created MACV GP1 variants and tested their effect on TfR1 binding and virus entry to evaluate the functional significance of some of these and additional residues in human and simian cells. We found residues R111, D123, Y122, and F226 to be essential, D155, and P160 important, and D114, S116, D140, and K169 expendable for the GP1-TfR1 interaction and MACV entry. Several MACV GP1 residues that are critical for the interaction with TfR1 are conserved among other New World arenaviruses, indicating a common basis of receptor interaction. Our findings also open avenues for the rational development of viral entry inhibitors.

Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

BackgroundRicin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA) acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions.ResultsWe found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a ΔH of -13 ± 2 kJ/mol and a ΔS of -0.04 ± 0.01 kJ/(K*mol). The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site.ConclusionWe found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.

Improvement of a Potential Anthrax Therapeutic by Computational Protein Design

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.

Mutational Effects on Protein Folding Stability and Antigenicity: The Case of Streptococcal Pyrogenic Exotoxin A

The influence of mutationally induced changes in protein folding on development of effective neutralizing antibodies during vaccination remains largely unexplored. In this study, we probed how mutational substitutions of streptococcal pyrogenic exotoxin A (SPEA), a model bacterial superantigen, affect native conformational stability and antigenicity. Stability changes for the toxin variants were determined using circular dichroism and fluorescence measurements, and scanning calorimetry. Self-association was assayed by dynamic light scattering. Inactivated SPEA proteins containing particular combinations of mutations elicited antibodies in HLA-DQ8 transgenic mice that neutralized SPEA superantigenicity in vitro, and protected animals from lethal toxin challenge. However, a highly destabilized cysteine-free mutant of SPEA did not provide effective immunity, nor did an irreversibly denatured version of an otherwise effective mutant protein. These results suggest that protein conformation plays a significant role in generating effective neutralizing antibodies to this toxin, and may be an important factor to consider in vaccine design.