Harry J. Sterling

The Protective Antigen Component of Anthrax Toxin Forms Functional Octameric Complexes

The assembly of bacterial toxins and virulence factors is critical to their function, but the regulation of assembly during infection has not been studied. We begin to address this question using anthrax toxin as a model. The protective antigen (PA) component of the toxin assembles into ring-shaped homooligomers that bind the two other enzyme components of the toxin, lethal factor (LF) and edema factor (EF), to form toxic complexes. To disrupt the host, these toxic complexes are endocytosed, such that the PA oligomer forms a membrane-spanning channel that LF and EF translocate through to enter the cytosol. Using single-channel electrophysiology, we show that PA channels contain two populations of conductance states, which correspond to two different PA pre-channel oligomers observed by electron microscopy-the well-described heptamer and a novel octamer. Mass spectrometry demonstrates that the PA octamer binds four LFs, and assembly routes leading to the octamer are populated with even-numbered, dimeric and tetrameric, PA intermediates. Both heptameric and octameric PA complexes can translocate LF and EF with similar rates and efficiencies. Here, we report a 3.2-A crystal structure of the PA octamer. The octamer comprises approximately 20-30% of the oligomers on cells, but outside of the cell, the octamer is more stable than the heptamer under physiological pH. Thus, the PA octamer is a physiological, stable, and active assembly state capable of forming lethal toxins that may withstand the hostile conditions encountered in the bloodstream. This assembly mechanism may provide a novel means to control cytotoxicity.

Tpr directly binds to Mad1 and Mad2 and is important for the Mad1–Mad2-mediated mitotic spindle checkpoint

The mitotic arrest-deficient protein Mad1 forms a complex with Mad2, which is required for imposing mitotic arrest on cells in which the spindle assembly is perturbed. By mass spectrometry of affinity-purified Mad2-associated factors, we identified the translocated promoter region (Tpr), a component of the nuclear pore complex (NPC), as a novel Mad2-interacting protein. Tpr directly binds to Mad1 and Mad2. Depletion of Tpr in HeLa cells disrupts the NPC localization of Mad1 and Mad2 during interphase and decreases the levels of Mad1-bound Mad2. Furthermore, depletion of Tpr decreases the levels of Mad1 at kinetochores during prometaphase, correlating with the inability of Mad1 to activate Mad2, which is required for inhibiting APC(Cdc20). These findings reveal an important role for Tpr in which Mad1-Mad2 proteins are regulated during the cell cycle and mitotic spindle checkpoint signaling.

Effects of supercharging reagents on noncovalent complex structure in electrospray ionization from aqueous solutions.

The effects of two supercharging reagents, m-nitrobenzyl alcohol (m-NBA) and sulfolane, on the charge-state distributions and conformations of myoglobin ions formed by electrospray ionization were investigated. Addition of 0.4% m-NBA to aqueous ammonium acetate solutions of myoglobin results in an increase in the maximum charge state from 9+ to 19+, and an increase in the average charge state from 7.9+ to 11.7+, compared with solutions without m-NBA. The extent of supercharging with sulfolane on a per mole basis is lower than that with m-NBA, but comparable charging was obtained at higher concentration. Arrival time distributions obtained from traveling wave ion mobility spectrometry show that the higher charge state ions that are formed with these supercharging reagents are significantly more unfolded than lower charge state ions. Results from circular dichroism spectroscopy show that sulfolane can act as chemical denaturant, destabilizing myoglobin by ∼1.5 kcal/mol/M at 25 °C. Because these supercharging reagents have low vapor pressures, aqueous droplets are preferentially enriched in these reagents as evaporation occurs. Less evaporative cooling will occur after the droplets are substantially enriched in the low volatility supercharging reagent, and the droplet temperature should be higher compared with when these reagents are not present. Protein unfolding induced by chemical and/or thermal denaturation in the electrospray droplet appears to be the primary origin of the enhanced charging observed for noncovalent protein complexes formed from aqueous solutions that contain these supercharging reagents, although other factors almost certainly influence the extent of charging as well.

Protein Conformation and Supercharging with DMSO from Aqueous Solution

The efficacy of dimethyl sulfoxide (DMSO) as a supercharging reagent for protein ions formed by electrospray ionization from aqueous solution and the mechanism for supercharging were investigated. Addition of small amounts of DMSO to aqueous solutions containing hen egg white lysozyme or equine myoglobin results in a lowering of charge, whereas a significant increase in charge occurs at higher concentrations. Results from both near-UV circular dichroism spectroscopy and solution-phase hydrogen/deuterium exchange mass spectrometry indicate that DMSO causes a compaction of the native structure of these proteins at low concentration, but significant unfolding occurs at ~63% and ~43% DMSO for lysozyme and myoglobin, respectively. The DMSO concentrations required to denature these two proteins in bulk solution are ~3–5 times higher than the concentrations required for the onset of supercharging, consistent with a significantly increased concentration of this high boiling point supercharging reagent in the ESI droplet as preferential evaporation of water occurs. DMSO is slightly more basic than m-nitrobenzyl alcohol and sulfolane, two other supercharging reagents, based on calculated proton affinity and gas-phase basicity values both at the B3LYP and MP2 levels of theory, and all three of these supercharging reagents are significantly more basic than water. These results provide additional evidence that the origin of supercharging from aqueous solution is the result of chemical and/or thermal denaturation that occurs in the ESI droplet as the concentration of these supercharging reagents increases, and that proton transfer reactivity does not play a significant role in the charge enhancement observed.

Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers

The protein transporter anthrax lethal toxin is composed of protective antigen (PA), a transmembrane translocase, and lethal factor (LF), a cytotoxic enzyme. After its assembly into holotoxin complexes, PA forms an oligomeric channel that unfolds LF and translocates it into the host cell. We report the crystal structure of the core of a lethal toxin complex to 3.1-Å resolution; the structure contains a PA octamer bound to four LF PA-binding domains (LFN). The first α-helix and β-strand of each LFN unfold and dock into a deep amphipathic cleft on the surface of the PA octamer, which we call the α clamp. The α clamp possesses nonspecific polypeptide binding activity and is functionally relevant to efficient holotoxin assembly, PA octamer formation, and LF unfolding and translocation. This structure provides insight into the mechanism of translocation-coupled protein unfolding.

Origin of supercharging in electrospray ionization of noncovalent complexes from aqueous solution

The use of m-nitrobenzyl alcohol (m-NBA) to enhance charging of noncovalent complexes formed by electrospray ionization from aqueous solutions was investigated. Addition of up to 1% m-NBA can result in a significant increase in the average charging of complexes, ranging from ∼13% for the homo-heptamer of NtrC4-RC (317 kDa; maximum charge state increases from 42+ to 44+) to ∼49% for myoglobin (17.6 kDa; maximum charge state increases from 9+ to 16+). Charge state distributions of larger complexes obtained from heated solutions to which no m-NBA was added are remarkably similar to those containing small amounts of m-NBA. Dissociation of the complexes through identical channels both upon addition of higher concentrations of m-NBA and heating is observed. These results indicate that the enhanced charging upon addition of m-NBA to aqueous electrospray solutions is a result of droplet heating owing to the high boiling point of m-NBA, which results in a change in the higher-order structure and/or dissociation of the complexes. For monomeric proteins and small complexes, the enhancement of charging is lower for heated aqueous solutions than from solutions with m-NBA because rapid folding of proteins from heated solutions that do not contain m-NBA can occur after the electrospray droplet is formed and is evaporatively cooled.

Role of the Protective Antigen Octamer in the Molecular Mechanism of Anthrax Lethal Toxin Stabilization in Plasma

Anthrax is caused by strains of Bacillus anthracis that produce two key virulence factors, anthrax toxin (Atx) and a poly-gamma-D-glutamic acid capsule. Atx is comprised of three proteins: protective antigen (PA) and two enzymes, lethal factor (LF) and edema factor (EF). To disrupt cell function, these components must assemble into holotoxin complexes, which contain either a ring-shaped homooctameric or homoheptameric PA oligomer bound to multiple copies of LF and/or EF, producing lethal toxin (LT), edema toxin, or mixtures thereof. Once a host cell endocytoses these complexes, PA converts into a membrane-inserted channel that translocates LF and EF into the cytosol. LT can assemble on host cell surfaces or extracellularly in plasma. We show that, under physiological conditions in bovine plasma, LT complexes containing heptameric PA aggregate and inactivate more readily than LT complexes containing octameric PA. LT complexes containing octameric PA possess enhanced stability, channel-forming activity, and macrophage cytotoxicity relative to those containing heptameric PA. Under physiological conditions, multiple biophysical probes reveal that heptameric PA can prematurely adopt the channel conformation, but octameric PA complexes remain in their soluble prechannel configuration, which allows them to resist aggregation and inactivation. We conclude that PA may form an octameric oligomeric state as a means to produce a more stable and active LT complex that could circulate freely in the blood.

The role of conformational flexibility on protein supercharging in native electrospray ionization

Effects of covalent intramolecular bonds, either native disulfide bridges or chemical crosslinks, on ESI supercharging of proteins from aqueous solutions were investigated. Chemically modifying cytochrome c with up to seven crosslinks or ubiquitin with up to two crosslinks did not affect the average or maximum charge states of these proteins in the absence of m-nitrobenzyl alcohol (m-NBA), but the extent of supercharging induced by m-NBA increased with decreasing numbers of crosslinks. For the model random coil polypeptide reduced/alkylated RNase A, a decrease in charging with increasing m-NBA concentration attributable to reduced surface tension of the ESI droplet was observed, whereas native RNase A electrosprayed from these same solutions exhibited enhanced charging. The inverse relationship between the extent of supercharging and the number of intramolecular crosslinks for folded proteins, as well as the absence of supercharging for proteins that are random coils in aqueous solution, indicate that conformational restrictions induced by the crosslinks reduce the extent of supercharging. These results provide additional evidence that protein and protein complex supercharging from aqueous solution is primarily due to partial or significant unfolding that occurs as a result of chemical and/or thermal denaturation induced by the supercharging reagent late in the ESI droplet lifetime.

Electrothermal supercharging in mass spectrometry and tandem mass spectrometry of native proteins.

Electrothermal supercharging of protein ions formed by electrospray ionization from buffered aqueous solutions results in significant increases to both the maximum and average charge states compared to native mass spectrometry in which ions are formed from the same solutions but with lower spray potentials. For eight of the nine proteins investigated, the maximum charge states of protonated ions formed from native solutions with electrothermal supercharging is greater than those obtained from conventional denaturing solutions consisting of water/methanol/acid, although the average charging is slightly lower owing to contributions of small populations of more folded low charge-state structures. Under these conditions, electrothermal supercharging is slightly less effective for anions than for cations. Equivalent sequence coverage (80%) is obtained with electron transfer dissociation of the same high charge-state ion of cytochrome c formed by electrothermal supercharging from native solutions and from denaturing solutions. Electrothermal supercharging should be advantageous for combining structural studies of proteins in native environments with mass spectrometers that have limited high m/z capabilities and for significantly improving tandem mass spectrometry performance for protein ions formed from solutions in which the molecules have native structures and activities.

Effects of buffer loading for electrospray ionization mass spectrometry of a noncovalent protein complex that requires high concentrations of essential salts

Electrospray ionization (ESI) mass spectrometry (MS) is a powerful method for analyzing the active forms of macromolecular complexes of biomolecules. However, these solutions often contain high concentrations of salts and/or detergents that adversely effect ESI performance by making ion formation less reproducible, causing severe adduction or ion suppression. Many methods for separating complexes from nonvolatile additives are routinely used with ESI-MS, but these methods may not be appropriate for complexes that require such stabilizers for activity. Here, the effects of buffer loading using concentrations of ammonium acetate ranging from 0.22 to 1.41 M on the ESI mass spectra of a solution containing a domain truncation mutant of a σ54 activator from Aquifex aeolicus were studied. This 44.9 kDa protein requires the presence of millimolar concentrations of Mg2+, BeF3−, and ADP, (at ∼60 °C) to assemble into an active homo-hexamer. Addition of ammonium acetate can improve signal stability and reproducibility, and can significantly lower adduction and background signals. However, at higher concentrations, the relative ion abundance of the hexamer is diminished, while that of the constituent monomer is enhanced. These results are consistent with loss of enzymatic activity as measured by ATP hydrolysis and indicate that the high concentration of ammonium acetate interferes with assembly of the hexamer. This shows that buffer loading with ammonium acetate is effective for obtaining ESI signal for complexes that require high concentrations of essential salts, but can interfere with formation of, and/or destabilize complexes by disrupting crucial electrostatic interactions at high concentration.