Anthrax toxin translocation complex reveals insight into the lethal factor unfolding and refolding mechanism.

Translocation is essential to the anthrax toxin mechanism. Protective antigen (PA), the binding component of this AB toxin, forms an oligomeric pore that translocates lethal factor (LF) or edema factor, the active components of the toxin, into the cell. Structural details of the translocation process have remained elusive despite their biological importance. To overcome the technical challenges of studying translocation intermediates, we developed a method to immobilize, transition, and stabilize anthrax toxin to mimic important physiological steps in the intoxication process. Here, we report a cryoEM snapshot of PApore translocating the N-terminal domain of LF (LFN). The resulting 3.3 Å structure of the complex shows density of partially unfolded LFN near the canonical PApore binding site. Interestingly, we also observe density consistent with an α helix emerging from the 100 Å ß barrel channel suggesting LF secondary structural elements begin to refold in the pore channel. We conclude the anthrax toxin ß barrel aids in efficient folding of its enzymatic payload prior to channel exit. Our hypothesized refolding mechanism has broader implications for pore length of other protein translocating toxins.

A risk-stratified approach toward safely resuming OnabotulinumtoxinA injections based on dosing and ambulatory status in pediatric patients with cerebral palsy during the Coronavirus pandemic of 2019 (COVID-19).

PURPOSE: After the onset of the Coronavirus pandemic of 2019-2020 (COVID-19), physicians who inject OnabotulinumtoxinA (BoNT-A) were left with determining risks and benefits in pediatric patients with cerebral palsy. Many of these patients have pre-existing conditions that make them more prone to COVID-19 symptoms, and this susceptibility potentially increases after BoNT-A injections. METHODS: A retrospective chart review of 500 patients identified 256 pediatric patients with cerebral palsy who received an intramuscular BoNT-A injection to determine relative doses used for each Gross Motor Functional Classification Score (GMFCS). Data regarding age, weight, GMFCS, BoNT-A total body dosage, and inpatient hospitalizations for 6 months post-injection were collected. Differences between GMFCS levels were analyzed using one-way analysis of variance testing. Inpatient hospitalizations were recorded and assessed using relative risk to determine the population risk of hospitalization in the setting of initiating injections during the COVID-19 pandemic. RESULTS: Based on GMFCS level, patients who were GMFCS I or II received fewer units of BoNT-A medication per kilogram of body weight compared to GMFCS III-V (p< 0.0005, F= 25.38). There was no statistically significant difference in frequency or time to hospitalization when comparing patients receiving BoNT-A compared to a control group. CONCLUSIONS: Resumption of BoNT-A injections during the time of COVID-19 requires a systematic approach based on risks and potential benefits. Data from this analysis does not show increased risk for patients who received injections historically; however, recommendations for resumption of injections has not previously been proposed in the setting of a pandemic. In this manuscript, a tiered approach to considerations for injections was proposed. Botulinum toxin type A injections have a history of improving spasticity in the pediatric patient with cerebral palsy. Ensuring appropriate selection of patients for injection with BoNT-A during this pandemic is increasingly important.

Novel Use of Porcine Urinary Bladder Matrix in the Exenterated Socket.

The aim of exenteration reconstruction is to stabilize the postsurgical wound bed to promote expeditious healing particularly in patients who are undergoing adjuvant radiation and/or chemotherapy. Porcine urinary bladder matrix has previously been used successfully as a wound-healing scaffold in treatment of burns and in acute, chronic, and surgical wounds, but the use of these products has not previously been reported in the exenterated orbit. The authors present a case of the novel use of porcine urinary bladder matrix in a pediatric patient who underwent exenteration for recurrent embryonal rhabdomyosarcoma, subsequent split-thickness skin grafting, and adjuvant radiation.

Constructing Kinetically Controlled Denaturation Isotherms of Folded Proteins Using Denaturant-Pulse Chaperonin Binding.

Methods to assess the kinetic stability of proteins, particularly those that are aggregation prone, are very useful in establishing ligand induced stabilizing effects. Because aggregation prone proteins are by nature difficult to work with, most solution based methods are compromised by this inherent instability. Here, we describe a label-free method that examines the denaturation of immobilized proteins where the dynamic unfolded protein populations are captured and detected by chaperonin binding.

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy.

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

The Chaperonin GroEL: A Versatile Tool for Applied Biotechnology Platforms.

The nucleotide-free chaperonin GroEL is capable of capturing transient unfolded or partially unfolded states that flicker in and out of existence due to large-scale protein dynamic vibrational modes. In this work, three short vignettes are presented to highlight our continuing advances in the application of GroEL biosensor biolayer interferometry (BLI) technologies and includes expanded uses of GroEL as a molecular scaffold for electron microscopy determination. The first example presents an extension of the ability to detect dynamic pre-aggregate transients in therapeutic protein solutions where the assessment of the kinetic stability of any folded protein or, as shown herein, quantitative detection of mutant-type protein when mixed with wild-type native counterparts. Secondly, using a BLI denaturation pulse assay with GroEL, the comparison of kinetically controlled denaturation isotherms of various von Willebrand factor (vWF) triple A domain mutant-types is shown. These mutant-types are single point mutations that locally disorder the A1 platelet binding domain resulting in one gain of function and one loss of function phenotype. Clear, separate, and reproducible kinetic deviations in the mutant-type isotherms exist when compared with the wild-type curve. Finally, expanding on previous electron microscopy (EM) advances using GroEL as both a protein scaffold surface and a release platform, examples are presented where GroEL-protein complexes can be imaged using electron microscopy tilt series and the low-resolution structures of aggregation-prone proteins that have interacted with GroEL. The ability of GroEL to bind hydrophobic regions and transient partially folded states allows one to employ this unique molecular chaperone both as a versatile structural scaffold and as a sensor of a protein's folded states.

The Use of a GroEL-BLI Biosensor to Rapidly Assess Preaggregate Populations for Antibody Solutions Exhibiting Different Stability Profiles.

An automated method using biotinylated GroEL-streptavidin biosensors with biolayer interferometry (GroEL-BLI) was evaluated to detect the formation of transiently formed, preaggregate species in various pharmaceutically relevant monoclonal antibody (mAb) samples. The relative aggregation propensity of various IgG1 and IgG4 mAbs was rank ordered using the GroEL-BLI biosensor method, and the least stable IgG4 mAb was subjected to different stresses including elevated temperatures, acidic pH, and addition of guanidine HCl. The GroEL-BLI biosensor detects mAb preaggregate formation mostly before, or sometimes concomitantly with, observing soluble aggregates and subvisible particles using size-exclusion chromatography and microflow imaging, respectively. A relatively unstable bispecific antibody (Bis-3) was shown to bind the GroEL biosensor even at low temperatures (25°C). During thermal stress (50°C, 1 h), increased Bis-3 binding to GroEL-biosensors was observed prior to aggregation by size-exclusion chromatography or microflow imaging. Transmission electron microscopy analysis of Bis-3 preaggregate GroEL complexes revealed, in some cases, potential hydrophobic interaction sites between the Fc domain of the Bis-3 and GroEL protein. The automated BLI method not only enables detection of transiently formed preaggregate species that initiate protein aggregation pathways but also permits rapid mAb formulation stability assessments at low volumes and low protein concentrations.

Asymmetric Cryo-EM Structure of Anthrax Toxin Protective Antigen Pore with Lethal Factor N-Terminal Domain.

The anthrax lethal toxin consists of protective antigen (PA) and lethal factor (LF). Understanding both the PA pore formation and LF translocation through the PA pore is crucial to mitigating and perhaps preventing anthrax disease. To better understand the interactions of the LF-PA engagement complex, the structure of the LFN-bound PA pore solubilized by a lipid nanodisc was examined using cryo-EM. CryoSPARC was used to rapidly sort particle populations of a heterogeneous sample preparation without imposing symmetry, resulting in a refined 17 Å PA pore structure with 3 LFN bound. At pH 7.5, the contributions from the three unstructured LFN lysine-rich tail regions do not occlude the Phe clamp opening. The open Phe clamp suggests that, in this translocation-compromised pH environment, the lysine-rich tails remain flexible and do not interact with the pore lumen region.

Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins.

Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy for decreasing disease pathologies caused by protein folding defects or deleterious kinetic transitions. Current methods of examining binding of a ligand to these marginally stable native states are limited because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, and multidomain proteins) and metastable proteins (e.g., low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein, immobilized on a BLI biosensor, to increasing denaturant concentrations (urea or GuHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remains is detected by an increased level of GroEL binding. Because this kinetic denaturant pulse is brief, the amplitude of binding of GroEL to the immobilized protein depends on the duration of the exposure to the denaturant, the concentration of the denaturant, wash times, and the underlying protein unfolding-refolding kinetics; fixing all other parameters and plotting the GroEL binding amplitude versus denaturant pulse concentration result in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein manifests as a decreased level of GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be used to identify small molecules and/or solution conditions that can stabilize or destabilize thermally stable proteins, multidomain proteins, oligomeric proteins, and, most importantly, aggregation-prone metastable proteins.

Protein folding on biosensor tips: folding of maltodextrin glucosidase monitored by its interactions with GroEL.

Protein folding has been extensively studied for the past six decades by employing solution-based methods such as solubility, enzymatic activity, secondary structure analysis, and analytical methods like FRET, NMR, and HD exchange. However, for rapid analysis of the folding process, solution-based approaches are often plagued with aggregation side reactions resulting in poor yields. In this work, we demonstrate that a bio-layer interferometry (BLI) chaperonin detection system can identify superior refolding conditions for denatured proteins. The degree of immobilized protein folding as a function of time can be detected by monitoring the binding of the high-affinity nucleotide-free form of the chaperonin GroEL. GroEL preferentially interacts with proteins that have hydrophobic surfaces exposed in their unfolded or partially folded form, so a decrease in GroEL binding can be correlated with burial of hydrophobic surfaces as folding progresses. The magnitude of GroEL binding to the protein immobilized on bio-layer interferometry biosensor inversely reflects the extent of protein folding and hydrophobic residue burial. We demonstrate conditions where accelerated folding can be observed for the aggregation-prone protein maltodextrin glucosidase (MalZ). Superior immobilized folding conditions identified on the bio-layer interferometry biosensor surface were reproduced on Ni-NTA sepharose bead surfaces and resulted in significant improvement in folding yields of released MalZ (measured by enzymatic activity) compared to bulk refolding conditions in solution.

Following Natures Lead: On the Construction of Membrane-Inserted Toxins in Lipid Bilayer Nanodiscs.

Bacterial toxin or viral entry into the cell often requires cell surface binding and endocytosis. The endosomal acidification induces a limited unfolding/refolding and membrane insertion reaction of the soluble toxins or viral proteins into their translocation competent or membrane inserted states. At the molecular level, the specific orientation and immobilization of the pre-transitioned toxin on the cell surface is often an important prerequisite prior to cell entry. We propose that structures of some toxin membrane insertion complexes may be observed through procedures where one rationally immobilizes the soluble toxin so that potential unfolding â†” refolding transitions that occur prior to membrane insertion orientate away from the immobilization surface in the presence of lipid micelle pre-nanodisc structures. As a specific example, the immobilized prepore form of the anthrax toxin pore translocon or protective antigen can be transitioned, inserted into a model lipid membrane (nanodiscs), and released from the immobilized support in its membrane solubilized form. This particular strategy, although unconventional, is a useful procedure for generating pure membrane-inserted toxins in nanodiscs for electron microscopy structural analysis. In addition, generating a similar immobilized platform on label-free biosensor surfaces allows one to observe the kinetics of these acid-induced membrane insertion transitions. These platforms can facilitate the rational design of inhibitors that specifically target the toxin membrane insertion transitions that occur during endosomal acidification. This approach may lead to a new class of direct anti-toxin inhibitors.

Probing the kinetic stabilities of Friedreich's ataxia clinical variants using a solid phase GroEL chaperonin capture platform.

Numerous human diseases are caused by protein folding defects where the protein may become more susceptible to degradation or aggregation. Aberrant protein folding can affect the kinetic stability of the proteins even if these proteins appear to be soluble in vivo. Experimental discrimination between functional properly folded and misfolded nonfunctional conformers is not always straightforward at near physiological conditions. The differences in the kinetic behavior of two initially folded frataxin clinical variants were examined using a high affinity chaperonin kinetic trap approach at 25 °C. The kinetically stable wild type frataxin (FXN) shows no visible partitioning onto the chaperonin. In contrast, the clinical variants FXN-p.Asp122Tyr and FXN-p.Ile154Phe kinetically populate partial folded forms that tightly bind the GroEL chaperonin platform. The initially soluble FXN-p.Ile154Phe variant partitions onto GroEL more rapidly and is more kinetically liable. These differences in kinetic stability were confirmed using differential scanning fluorimetry. The kinetic and aggregation stability differences of these variants may lead to the distinct functional impairments described in Friedreich's ataxia, the neurodegenerative disease associated to frataxin functional deficiency. This chaperonin platform approach may be useful for identifying small molecule stabilizers since stabilizing ligands to frataxin variants should lead to a concomitant decrease in chaperonin binding.

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry.

The ability of a GroEL-based bio-layer interferometry (BLI) assay to detect structurally altered and/or aggregated species of pharmaceutically relevant proteins is demonstrated. Assay development included optimizing biotinylated-GroEL immobilization to streptavidin biosensors, combined with biophysical and activity measurements showing native and biotinylated GroEL are both stable and active. First, acidic fibroblast growth factor (FGF-1) was incubated under conditions known to promote (40°C) and inhibit (heparin addition) molten globule formation. Heat exposed (40°C) FGF-1 exhibited binding to GroEL-biosensors, which was significantly diminished in the presence of heparin. Second, a polyclonal human IgG solution containing 6-8% non-native dimer showed an increase in higher molecular weight aggregates upon heating by size exclusion chromatography (SEC). The poly IgG solution displayed binding to GroEL-biosensors initially with progressively increased binding upon heating. Enriched preparations of the IgG dimers or monomers showed significant binding to GroEL-biosensors. Finally, a thermally treated IgG1 monoclonal antibody (mAb) solution also demonstrated increased GroEL-biosensor binding, but with different kinetics. The bound complexes could be partially to fully dissociated after ATP addition (i.e., specific GroEL binding) depending on the protein, environmental stress, and the assay's experimental conditions. Transmission electron microscopy (TEM) images of GroEL-mAb complexes, released from the biosensor, also confirmed interaction of bound complexes at the GroEL binding site with heat-stressed mAb. Results indicate that the GroEL-biosensor-BLI method can detect conformationally altered and/or early aggregation states of proteins, and may potentially be useful as a rapid, stability-indicating biosensor assay for monitoring the structural integrity and physical stability of therapeutic protein candidates.

Monitoring the kinetics of the pH-driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance.

Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å ß barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH-dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor, from the endosome to the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance and biolayer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from 7.5 to 5.0, mirroring acidification of the endosome. Once it had undergone the transition, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto electron microscopy grids, where PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early (pH 5.5) or late (pH 5.0) endosomal pH conditions. Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and the soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions.

On the design of broad based screening assays to identify potential pharmacological chaperones of protein misfolding diseases.

Correcting aberrant folds that develop during protein folding disease states is now an active research endeavor that is attracting increasing attention from both academic and industrial circles. One particular approach focuses on developing or identifying small molecule correctors or pharmacological chaperones that specifically stabilize the native fold. Unfortunately, the limited screening platforms available to rapidly identify or validate potential drug candidates are usually inadequate or slow because the folding disease proteins in question are often transiently folded and/or aggregation-prone, complicating and/or interfering with the assay outcomes. In this review, we outline and discuss the numerous platform options currently being employed to identify small molecule therapeutics for folding diseases. Finally, we describe a new stability screening approach that is broad based and is easily applicable toward a very large number of both common and rare protein folding diseases. The label free screening method described herein couples the promiscuity of the GroEL binding to transient aggregation-prone hydrophobic folds with surface plasmon resonance enabling one to rapidly identify potential small molecule pharmacological chaperones.

A polymorphic position in electron transfer flavoprotein modulates kinetic stability as evidenced by thermal stress.

The electron transfer flavoprotein (ETF) is a hub interacting with at least 11 mitochondrial flavoenzymes and linking them to the respiratory chain. Here we report the effect of the ETFα-T/I171 polymorphism on protein conformation and kinetic stability under thermal stress. Although variants have comparable thermodynamic stabilities, kinetically their behavior is rather distinct as ETFα-T171 displays increased susceptibility to cofactor flavin adenine dinucleotide (FAD) loss and enhanced kinetics of inactivation during thermal stress. Mimicking a fever episode yields substantial activity loss. However, the presence of substoichiometric concentrations of GroEL is sufficient to act as an effective buffer against long-term thermal denaturation. Our investigations are compatible with the notion that the ETFα-T171 variant displays an altered conformational landscape that results in reduced protein function under thermal stress.

Identifying protein stabilizing ligands using GroEL.

Over the past 5 years, it has become increasingly apparent to researchers that the initial promise and excitement of using gene replacement therapies to ameliorate folding diseases are still far from being broadly or easily applicable. Because a large number of human diseases are protein folding diseases (approximately 30 to 50%), many researchers now realize that more directed approaches to target and reverse the fundamental misfolding reactions preceding disease are highly feasible and offer the potential of developing more targeted drug therapies. This is also true with a large number of so called orphan protein folding diseases. The development of a broad-based general screening array method using the chaperonin as a detection platform will enable us to screen large chemical combinatorial libraries for specific ligands against the elusive transient, primary reactions that often lead to protein misfolding. This development will provide a highly desirable tool for the pharmaceutical, academic, and medical professions.

Strategies for folding of affinity tagged proteins using GroEL and osmolytes.

Obtaining a proper fold of affinity tagged chimera proteins can be difficult. Frequently, the protein of interest aggregates after the chimeric affinity tag is cleaved off, even when the entire chimeric construct is initially soluble. If the attached protein is incorrectly folded, chaperone proteins such as GroEL bind to the misfolded construct and complicate both folding and affinity purification. Since chaperonin/osmolyte mixtures facilitate correct folding from the chaperonin, we explored the possibility that we could use this intrinsic binding reaction to advantage to refold two difficult-to-fold chimeric constructs. In one instance, we were able to recover activity from a properly folded construct after the construct was released from the chaperonin in the presence of osmolytes. As an added advantage, we have also found that this method involving chaperonins can enable researchers to decide (1) if further stabilization of the folded product is required and (2) if the protein construct in question will ever be competent to fold with osmolytes.

GroEL as a molecular scaffold for structural analysis of the anthrax toxin pore.

We analyzed the 440-kDa transmembrane pore formed by the protective antigen (PA) moiety of anthrax toxin in the presence of GroEL by negative-stain electron microscopy. GroEL binds both the heptameric PA prepore and the PA pore. The latter interaction retards aggregation of the pore, prolonging its insertion-competent state. Two populations of unaggregated pores were visible: GroEL-bound pores and unbound pores. This allowed two virtually identical structures to be reconstructed, at 25-A and 28-A resolution, respectively. The structures were mushroom-shaped objects with a 125-A-diameter cap and a 100-A-long stem, consistent with earlier biochemical data. Thus, GroEL provides a platform for obtaining initial glimpses of a membrane protein structure in the absence of lipids or detergents and can function as a scaffold for higher-resolution structural analysis of the PA pore.