Unlatching of the stem domains in the Staphylococcus aureus pore-forming leukocidin LukAB influences toxin oligomerization.

Staphylococcus aureus (S. aureus) is a serious global pathogen that causes a diverse range of invasive diseases. S. aureus utilizes a family of pore-forming toxins, known as bi-component leukocidins, to evade the host immune response and promote infection. Among these is LukAB (leukocidin A/leukocidin B), a toxin that assembles into an octameric ß-barrel pore in the target cell membrane, resulting in host cell death. The established cellular receptor for LukAB is CD11b of the Mac-1 complex. Here, we show that hydrogen voltage-gated channel 1 is also required for the cytotoxicity of all major LukAB variants. We demonstrate that while each receptor is sufficient to recruit LukAB to the plasma membrane, both receptors are required for maximal lytic activity. Why LukAB requires two receptors, and how each of these receptors contributes to pore-formation remains unknown. To begin to resolve this, we performed an alanine scanning mutagenesis screen to identify mutations that allow LukAB to maintain cytotoxicity without CD11b. We discovered 30 mutations primarily localized in the stem domains of LukA and LukB that enable LukAB to exhibit full cytotoxicity in the absence of CD11b. Using crosslinking, electron microscopy, and hydroxyl radical protein footprinting, we show these mutations increase the solvent accessibility of the stem domain, priming LukAB for oligomerization. Together, our data support a model in which CD11b binding unlatches the membrane penetrating stem domains of LukAB, and this change in flexibility promotes toxin oligomerization.

Improved Sequence Analysis of Intact Proteins by Parallel Ion Parking during Electron Transfer Dissociation.

Electron transfer dissociation (ETD) is an analytically useful tool for primary structure interrogation of intact proteins, but its utility is limited by higher-order reactions with the products. To inhibit these higher-order reactions, first-generation fragment ions are kinetically excited by applying an experimentally tailored parallel ion parking waveform during ETD (ETD-PIP). In combination with subsequent ion/ion proton transfer reactions, precursor-to-product conversion was maximized as evidenced by the consumption of more than 90% of the 21 kDa Protein G precursor to form ETD product ions. The employment of ETD-PIP increased sequence coverage to 90% from 80% with standard ETD. Additionally, the inhibition of sequential electron transfers was reflected in the high number of complementary ion pairs from ETD-PIP (90%) compared to standard ETD (39%).

Nitrogen-Containing Aromatic Radical Anions Perform Multiple Proton and Electron Transfers Near-Simultaneously with Multiply Protonated Cations.

Gas phase ion/ion reactions between singly charged radical reagent anions and multiply charged cation precursors primarily result in either proton or electron transfer. These ion/ion reactions have been extensively studied for bioanalysis, and many reagent anions have been tested and reported. Here, nitrogen-containing aromatic radical anions were tested for the ability to conduct proton or electron transfer by their reaction with the ubiquitin [M + 13H]+13 precursor. The singly charged anion of 2,2'-biquinoline was found to undergo charge inversion to singly protonated cations via near-simultaneous proton and electron transfers while reactants were bound in a single ion/ion reaction complex. Although the focus of this paper was 2,2'-biquinoline, all three nitrogen-containing aromatic compounds tested produced similar results.

Sequencing a Bispecific Antibody by Controlling Chain Concentration Effects When Using an Immobilized Nonspecific Protease.

Complete sequence coverage of monospecific antibodies was previously achieved using immobilized aspergillopepsin I in a single LC-MS/MS analysis. Bispecific antibodies are asymmetrical compared to their monospecific antibody counterparts, resulting in a decrease in the concentration of individual subunits. Four standard proteins were used to characterize the effect of a decrease in concentration when using this immobilized enzyme reactor. Low concentration samples resulted in the elimination of large peptide products due to a greater number of enzymatic cleavages. A competitive inhibitor rich in arginine residues reduced the number of enzymatic cleavages to the protein and retained large molecular weight products. The digestion of a bispecific antibody with competitive inhibition of aspergillopepsin I maintained large peptide products better suited for sequence reconstruction, resulting in complete sequence coverage from a single LC-MS/MS analysis.

Unambiguous Sequence Characterization of a Monoclonal Antibody in a Single Analysis Using a Nonspecific Immobilized Enzyme Reactor.

Accurate sequence characterization is essential for the development of therapeutic antibodies by the pharmaceutical industry. Presented here is a methodology to obtain comprehensive sequence analysis of a monoclonal antibody. An enzyme reactor of immobilized Aspergillopepsin I, a highly stable nonspecific protease, was used to cleave reduced antibody subunits into a peptide profile ranging from 1 to 20 kDa. Utilizing the Thermo Orbitrap Fusion's unique instrument architecture combined with state-of-the-art instrument control software allowed for dynamic instrument methods that optimally characterize eluting peptides based on their size and charge density. Using a data-dependent instrument method, both collisional dissociation and electron transfer dissociation were used to fragment the appropriate charge state of analyte peptides. The instrument layout also allowed for scans to be taken in parallel using both the ion trap and Orbitrap concurrently, thus allowing larger peptides to be analyzed in high resolution using the Orbitrap while simultaneously analyzing tryptic-like peptides using the ion trap. We harnessed these capabilities to develop a custom method to optimally fragment the eluting peptides based on their mass and charge density. Using this approach, we obtained 100% sequence coverage of the total antibody in a single chromatographic analysis, enabling unambiguous sequence assignment of all residues.