Antibodies targeting the shared cytokine receptor IL-1 receptor accessory protein invoke distinct mechanisms to block all cytokine signaling.

Interleukin-1 (IL-1)-family cytokines are potent modulators of inflammation, coordinating a vast array of immunological responses across innate and adaptive immune systems. Dysregulated IL-1-family cytokine signaling, however, is involved in a multitude of adverse health effects, such as chronic inflammatory conditions, autoimmune diseases, and cancer. Within the IL-1 family of cytokines, six-IL-1α, IL-1ß, IL-33, IL-36α, IL-36ß, and IL-36γ-require the IL-1 receptor accessory protein (IL-1RAcP) as their shared co-receptor. Common features of cytokine signaling include redundancy of signaling pathways, sharing of cytokines and receptors, pleiotropy of the cytokines themselves, and multifaceted immune responses. Accordingly, targeting multiple cytokines simultaneously is an emerging therapeutic strategy and can provide advantages over targeting a single cytokine pathway. Here, we show that two monoclonal antibodies, CAN10 and 3G5, which target IL-1RAcP for broad blockade of all associated cytokines, do so through distinct mechanisms and provide therapeutic opportunities for the treatment of inflammatory diseases.

Coupling of zinc and GTP binding drives G-domain folding in Acinetobacter baumannii ZigA.

COG0523 proteins, also known as nucleotide-dependent metallochaperones, are a poorly understood class of small P-loop G3E GTPases. Multiple family members play critical roles in bacterial pathogen survival during an infection as part of the adaptive response to host-mediated "nutritional immunity." Our understanding of the structure, dynamics, and molecular-level function of COG0523 proteins, apart from the eukaryotic homolog, Zng1, remains in its infancy. Here, we use X-ray absorption spectroscopy to establish that Acinetobacter baumannii (Ab) ZigA coordinates ZnII using all three cysteines derived from the invariant CXCC motif to form an S3(N/O) coordination complex, a feature inconsistent with the ZnII-bound crystal structure of a distantly related COG0523 protein of unknown function from Escherichia coli, EcYjiA. The binding of ZnII and guanine nucleotides is thermodynamically linked in AbZigA, and this linkage is more favorable for the substrate GTP relative to the product GDP. Part of this coupling originates with nucleotide-induced stabilization of the G-domain tertiary structure as revealed by global thermodynamics measurements and hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS also reveals that the HDX behavior of the G2 (switch 1) loop is highly sensitive to nucleotide status and becomes more exchange labile in the GDP (product)-bound state. Significant long-range perturbation of local stability in both the G-domain and the C-terminal domain define a candidate binding pocket for a client protein that appears sensitive to nucleotide status (GDP versus GTP). We place these new insights into the structure, dynamics, and energetics of intermolecular metal transfer into the context of a model for AbZigA metallochaperone function.

The structure of NAD+ consuming protein Acinetobacter baumannii TIR domain shows unique kinetics and conformations.

Toll-like and interleukin-1/18 receptor/resistance (TIR) domain-containing proteins function as important signaling and immune regulatory molecules. TIR domain-containing proteins identified in eukaryotic and prokaryotic species also exhibit NAD+ hydrolase activity in select bacteria, plants, and mammalian cells. We report the crystal structure of the Acinetobacter baumannii TIR domain protein (AbTir-TIR) with confirmed NAD+ hydrolysis and map the conformational effects of its interaction with NAD+ using hydrogen-deuterium exchange-mass spectrometry. NAD+ results in mild decreases in deuterium uptake at the dimeric interface. In addition, AbTir-TIR exhibits EX1 kinetics indicative of large cooperative conformational changes, which are slowed down upon substrate binding. Additionally, we have developed label-free imaging using the minimally invasive spectroscopic method 2-photon excitation with fluorescence lifetime imaging, which shows differences in bacteria expressing native and mutant NAD+ hydrolase-inactivated AbTir-TIRE208A protein. Our observations are consistent with substrate-induced conformational changes reported in other TIR model systems with NAD+ hydrolase activity. These studies provide further insight into bacterial TIR protein mechanisms and their varying roles in biology.

The conformational landscape of a serpin N-terminal subdomain facilitates folding and in-cell quality control.

Many multi-domain proteins including the serpin family of serine protease inhibitors contain non-sequential domains composed of regions that are far apart in sequence. Because proteins are translated vectorially from N- to C-terminus, such domains pose a particular challenge: how to balance the conformational lability necessary to form productive interactions between early and late translated regions while avoiding aggregation. This balance is mediated by the protein sequence properties and the interactions of the folding protein with the cellular quality control machinery. For serpins, particularly α1-antitrypsin (AAT), mutations often lead to polymer accumulation in cells and consequent disease suggesting that the lability/aggregation balance is especially precarious. Therefore, we investigated the properties of progressively longer AAT N-terminal fragments in solution and in cells. The N-terminal subdomain, residues 1-190 (AAT190), is monomeric in solution and efficiently degraded in cells. More ß-rich fragments, 1-290 and 1-323, form small oligomers in solution, but are still efficiently degraded, and even the polymerization promoting Siiyama (S53F) mutation did not significantly affect fragment degradation. In vitro, the AAT190 region is among the last regions incorporated into the final structure. Hydrogen-deuterium exchange mass spectrometry and enhanced sampling molecular dynamics simulations show that AAT190 has a broad, dynamic conformational ensemble that helps protect one particularly aggregation prone ß-strand from solvent. These AAT190 dynamics result in transient exposure of sequences that are buried in folded, full-length AAT, which may provide important recognition sites for the cellular quality control machinery and facilitate degradation and, under favorable conditions, reduce the likelihood of polymerization.

Mass Spectrometry-Based Methods to Determine the Substrate Specificities and Kinetics of N-Linked Glycan Hydrolysis by Endo-ß-N-Acetylglucosaminidases.

Glycosylation is a common posttranslational modification of proteins and refers to the covalent addition of glycans, chains of polysaccharides, onto proteins producing glycoproteins. The glycans influence the structure, function, and stability of proteins. They also play an integral role in the immune system, and aberrantly glycosylated proteins have wide ranging effects, including leading to diseases such as autoimmune conditions and cancer. Carbohydrate-active enzymes (CAZymes) are produced in bacteria, fungi, and humans and are enzymes which modify glycans via the addition or subtraction of individual or multiple saccharides from glycans. One of the hurdles in studying these enzymes is determining the types of substrates each enzyme is specific for and the kinetics of enzymatic activity. In this chapter, we discuss methods which are currently used to study the substrate specificity and kinetics of CAZymes and introduce a novel mass spectrometry-based technique which enables the specificity and kinetics of CAZymes to be determined accurately and efficiently.

Development and characterization of nanobodies that specifically target the oncogenic Phosphatase of Regenerating Liver-3 (PRL-3) and impact its interaction with a known binding partner, CNNM3.

Phosphatase of Regenerating Liver-3 (PRL-3) is associated with cancer progression and metastasis. The mechanisms that drive PRL-3's oncogenic functions are not well understood, partly due to a lack of research tools available to study this protein. We have begun to address these issues by developing alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3 with a KD of 30-300 nM and no activity towards highly homologous family members PRL-1 and PRL-2. We found that longer and charged N-terminal tags on PRL-3, such as GFP and FLAG, changed PRL-3 localization compared to untagged protein, indicating that the nanobodies may provide new insights into PRL-3 trafficking and function. The nanobodies perform equally, if not better, than commercially available antibodies in immunofluorescence and immunoprecipitation. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed that the nanobodies bind partially within the PRL-3 active site and can interfere with PRL-3 phosphatase activity. Co-immunoprecipitation with a known PRL-3 active site binding partner, the CBS domain of metal transporter CNNM3, showed that the nanobodies reduced the amount of PRL-3:CBS inter-action. The potential of blocking this interaction is highly relevant in cancer, as multiple research groups have shown that PRL-3 binding to CNNM proteins is sufficient to promote metastatic growth in mouse models. The anti-PRL-3 nanobodies represent an important expansion of the research tools available to study PRL-3 function and can be used to define the role of PRL-3 in cancer progression.

Mechanism of antibody-specific deglycosylation and immune evasion by Streptococcal IgG-specific endoglycosidases.

Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi-modular endo-ß-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component. Here, we present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. In combination with small-angle X-ray scattering, alanine scanning mutagenesis, hydrolytic activity measurements, enzyme kinetics, nuclear magnetic resonance and molecular dynamics analyses, we establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by EndoS and EndoS2. Our results provide a rational basis from which to engineer novel enzymes with antibody and glycan selectivity for clinical and biotechnological applications.

Characterization of interaction between blood coagulation factor VIII and LRP1 suggests dynamic binding by alternating complex contacts.

BACKGROUND: Deficiency in blood coagulation factor VIII (FVIII) results in life-threating bleeding (hemophilia A) treated by infusions of FVIII concentrates. To improve disease treatment, FVIII has been modified to increase its plasma half-life, which requires understanding mechanisms of FVIII catabolism. An important catabolic actor is hepatic low density lipoprotein receptor-related protein 1 (LRP1), which also regulates many other clinically significant processes. Previous studies showed complexity of FVIII site for binding LRP1. OBJECTIVES: To characterize binding sites between FVIII and LRP1 and suggest a model of the interaction. METHODS: A series of recombinant ligand-binding complement-type repeat (CR) fragments of LRP1 including mutated variants was generated in a baculovirus system and tested for FVIII interaction using surface plasmon resonance, tissue culture model, hydrogen-deuterium exchange mass spectrometry, and in silico. RESULTS: Multiple CR doublets within LRP1 clusters II and IV were identified as alternative FVIII-binding sites. These interactions follow the canonical binding mode providing major binding energy, and additional weak interactions are contributed by adjacent CR domains. A representative CR doublet was shown to have multiple contact sites on FVIII. CONCLUSIONS: FVIII and LRP1 interact via formation of multiple complex contacts involving both canonical and non-canonical binding combinations. We propose that FVIII-LRP1 interaction occurs via switching such alternative binding combinations in a dynamic mode, and that this mechanism is relevant to other ligand interactions of the low-density lipoprotein receptor family members including LRP1.

A fusion of the Bacteroides fragilis ferrous iron import proteins reveals a role for FeoA in stabilizing GTP-bound FeoB.

Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe2+ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system is the primary prokaryotic Fe2+ import machinery, and two constituent proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remains enigmatic. In this work, we explored the distribution of feoAB operons encoding a fusion of FeoA tethered to the N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion poises FeoA to interact with FeoB to affect function. To test this hypothesis, we characterized the soluble NFeoAB fusion protein from Bacteroides fragilis, a commensal organism implicated in drug-resistant infections. Using X-ray crystallography, we determined the 1.50-Å resolution structure of BfFeoA, which adopts an SH3-like fold implicated in protein-protein interactions. Using a combination of structural modeling, small-angle X-ray scattering, and hydrogen-deuterium exchange mass spectrometry, we show that FeoA and NFeoB interact in a nucleotide-dependent manner, and we mapped the protein-protein interaction interface. Finally, using guanosine triphosphate (GTP) hydrolysis assays, we demonstrate that BfNFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis that is not potassium-stimulated. Importantly, truncation of FeoA from this fusion demonstrates that FeoA-NFeoB interactions function to stabilize the GTP-bound form of FeoB. Taken together, our work reveals a role for FeoA function in the fused FeoAB system and suggests a function for FeoA among prokaryotes.

Modeling the native ensemble of PhuS using enhanced sampling MD and HDX-ensemble reweighting.

The cytoplasmic heme binding protein from Pseudomonas aeruginosa, PhuS, plays two essential roles in regulating heme uptake and iron homeostasis. First, PhuS shuttles exogenous heme to heme oxygenase (HemO) for degradation and iron release. Second, PhuS binds DNA and modulates the transcription of the prrF/H small RNAs (sRNAs) involved in the iron-sparing response. Heme binding to PhuS regulates this dual function, as the unliganded form binds DNA, whereas the heme-bound form binds HemO. Crystallographic studies revealed nearly identical structures for apo- and holo-PhuS, and yet numerous solution-based measurements indicate that heme binding is accompanied by large conformational rearrangements. In particular, hydrogen-deuterium exchange mass spectrometry (HDX-MS) of apo- versus holo-PhuS revealed large differences in deuterium uptake, notably in α-helices 6, 7, and 8 (α6,7,8), which contribute to the heme binding pocket. These helices were mostly labile in apo-PhuS but largely protected in holo-PhuS. In contrast, in silico-predicted deuterium uptake levels of α6,7,8 from molecular dynamics (MD) simulations of the apo- and holo-PhuS structures are highly similar, consistent only with the holo-PhuS HDX-MS data. To rationalize this discrepancy between crystal structures, simulations, and observed HDX-MS, we exploit a recently developed computational approach (HDXer) that fits the relative weights of conformational populations within an ensemble of structures to conform to a target set of HDX-MS data. Here, a combination of enhanced sampling MD, HDXer, and dimensionality reduction analysis reveals an apo-PhuS conformational landscape in which α6, 7, and 8 are significantly rearranged compared to the crystal structure, including a loss of secondary structure in α6 and the displacement of α7 toward the HemO binding interface. Circular dichroism analysis confirms the loss of secondary structure, and the extracted ensembles of apo-PhuS and of heme-transfer-impaired H212R mutant, are consistent with known heme binding and transfer properties. The proposed conformational landscape provides structural insights into the modulation by heme of the dual function of PhuS.

Current Trends and Limitations in Dengue Antiviral Research.

Dengue is the most prevalent arthropod-borne viral disease worldwide and affects approximately 2.5 billion people living in over 100 countries. Increasing geographic expansion of Aedes aegypti mosquitoes (which transmit the virus) has made dengue a global health concern. There are currently no approved antivirals available to treat dengue, and the only approved vaccine used in some countries is limited to seropositive patients. Treatment of dengue, therefore, remains largely supportive to date; hence, research efforts are being intensified for the development of antivirals. The nonstructural proteins, 3 and 5 (NS3 and NS5), have been the major targets for dengue antiviral development due to their indispensable enzymatic and biological functions in the viral replication process. NS5 is the largest and most conserved nonstructural protein encoded by flaviviruses. Its multifunctionality makes it an attractive target for antiviral development, but research efforts have, this far, not resulted in the successful development of an antiviral targeting NS5. Increase in structural insights into the dengue NS5 protein will accelerate drug discovery efforts focused on NS5 as an antiviral target. In this review, we will give an overview of the current state of therapeutic development, with a focus on NS5 as a therapeutic target against dengue.

A structure and function relationship study to identify the impact of the R721G mutation in the human mitochondrial lon protease.

Lon is an ATP-dependent protease belonging to the "ATPase associated with diverse cellular activities" (AAA+) protein family. In humans, Lon is translated as a precursor and imported into the mitochondria matrix through deletion of the first 114 amino acid residues. In mice, embryonic knockout of lon is lethal. In humans, some dysfunctional lon mutations are tolerated but they cause a developmental disorder known as the CODAS syndrome. To gain a better understanding on the enzymology of human mitochondrial Lon, this study compares the structure-function relationship of the WT versus one of the CODAS mutants R721G to identify the mechanistic features in Lon catalysis that are affected. To this end, steady-state kinetics were used to quantify the difference in ATPase and ATP-dependent peptidase activities between WT and R721G. The Km values for the intrinsic as well as protein-stimulated ATPase were increased whereas the kcat value for ATP-dependent peptidase activity was decreased in the R721G mutant. The mutant protease also displayed substrate inhibition kinetics. In vitro studies revealed that R721G did not degrade the endogenous mitochondrial Lon substrate pyruvate dehydrogenase kinase isoform 4 (PDK4) effectively like WT hLon. Furthermore, the pyruvate dehydrogenase complex (PDH) protected PDK4 from hLon degradation. Using hydrogen deuterium exchange/mass spectrometry and negative stain electron microscopy, structural perturbations associated with the R721G mutation were identified. To validate the in vitro findings under a physiologically relevant condition, the intrinsic stability as well as proteolytic activity of WT versus R721G mutant towards PDK 4 were compared in cell lysates prepared from immortalized B lymphocytes expressing the respective protease. The lifetime of PDK4 is longer in the mutant cells, but the lifetime of Lon protein is longer in the WT cells, which corroborate the in vitro structure-functional relationship findings.

Insights into substrate recognition and specificity for IgG by Endoglycosidase S2.

Antibodies bind foreign antigens with high affinity and specificity leading to their neutralization and/or clearance by the immune system. The conserved N-glycan on IgG has significant impact on antibody effector function, with the endoglycosidases of Streptococcus pyogenes deglycosylating the IgG to evade the immune system, a process catalyzed by the endoglycosidase EndoS2. Studies have shown that two of the four domains of EndoS2, the carbohydrate binding module (CBM) and the glycoside hydrolase (GH) domain are critical for catalytic activity. To yield structural insights into contributions of the CBM and the GH domains as well as the overall flexibility of EndoS2 to the proteins' catalytic activity, models of EndoS2-Fc complexes were generated through enhanced-sampling molecular-dynamics (MD) simulations and site-identification by ligand competitive saturation (SILCS) docking followed by reconstruction and multi-microsecond MD simulations. Modeling results predict that EndoS2 initially interacts with the IgG through its CBM followed by interactions with the GH yielding catalytically competent states. These may involve the CBM and GH of EndoS2 simultaneously interacting with either the same Fc CH2/CH3 domain or individually with the two Fc CH2/CH3 domains, with EndoS2 predicted to assume closed conformations in the former case and open conformations in the latter. Apo EndoS2 is predicted to sample both the open and closed states, suggesting that either complex can directly form following initial IgG-EndoS2 encounter. Interactions of the CBM and GH domains with the IgG are predicted to occur through both its glycan and protein regions. Simulations also predict that the Fc glycan can directly transfer from the CBM to the GH, facilitating formation of catalytically competent complexes and how the 734 to 751 loop on the CBM can facilitate extraction of the glycan away from the Fc CH2/CH3 domain. The predicted models are compared and consistent with Hydrogen/Deuterium Exchange data. In addition, the complex models are consistent with the high specificity of EndoS2 for the glycans on IgG supporting the validity of the predicted models.

Molecular Basis of Selective Cytokine Signaling Inhibition by Antibodies Targeting a Shared Receptor.

Interleukin-1 (IL-1) family cytokines are potent mediators of inflammation, acting to coordinate local and systemic immune responses to a wide range of stimuli. Aberrant signaling by IL-1 family cytokine members, however, is linked to myriad inflammatory syndromes, autoimmune conditions and cancers. As such, blocking the inflammatory signals inherent to IL-1 family signaling is an established and expanding therapeutic strategy. While several FDA-approved IL-1 inhibitors exist, including an Fc fusion protein, a neutralizing antibody, and an antagonist cytokine, none specifically targets the co-receptor IL-1 receptor accessory protein (IL-1RAcP). Most IL-1 family cytokines form productive signaling complexes by binding first to their cognate receptors - IL-1RI for IL-1α and IL-1ß; ST2 for IL-33; and IL-36R for IL-36α, IL-36ß and IL-36γ - after which they recruit the shared secondary receptor IL-1RAcP to form a ternary cytokine/receptor/co-receptor complex. Recently, IL-1RAcP was identified as a biomarker for both AML and CML. IL-1RAcP has also been implicated in tumor progression in solid tumors and an anti-IL1RAP antibody (nadunolimab, CAN04) is in phase II clinical studies in pancreatic cancer and non-small cell lung cancer (NCT03267316). As IL-1RAcP is common to all of the abovementioned IL-1 family cytokines, targeting this co-receptor raises the possibility of selective signaling inhibition for different IL-1 family cytokines. Indeed, previous studies of IL-1ß and IL-33 signaling complexes have revealed that these cytokines employ distinct mechanisms of IL-1RAcP recruitment even though their overall cytokine/receptor/co-receptor complexes are structurally similar. Here, using functional, biophysical, and structural analyses, we show that antibodies specific for IL-1RAcP can differentially block signaling by IL-1 family cytokines depending on the distinct IL-1RAcP epitopes that they engage. Our results indicate that targeting a shared cytokine receptor is a viable therapeutic strategy for selective cytokine signaling inhibition.

Interpreting Hydrogen-Deuterium Exchange Experiments with Molecular Simulations: Tutorials and Applications of the HDXer Ensemble Reweighting Software [Article v1.0].

Hydrogen-deuterium exchange (HDX) is a comprehensive yet detailed probe of protein structure and dynamics and, coupled to mass spectrometry, has become a powerful tool for investigating an increasingly large array of systems. Computer simulations are often used to help rationalize experimental observations of exchange, but interpretations have frequently been limited to simple, subjective correlations between microscopic dynamical fluctuations and the observed macroscopic exchange behavior. With this in mind, we previously developed the HDX ensemble reweighting approach and associated software, HDXer, to aid the objective interpretation of HDX data using molecular simulations. HDXer has two main functions; first, to compute H-D exchange rates that describe each structure in a candidate ensemble of protein structures, for example from molecular simulations, and second, to objectively reweight the conformational populations present in a candidate ensemble to conform to experimental exchange data. In this article, we first describe the HDXer approach, theory, and implementation. We then guide users through a suite of tutorials that demonstrate the practical aspects of preparing experimental data, computing HDX levels from molecular simulations, and performing ensemble reweighting analyses. Finally we provide a practical discussion of the capabilities and limitations of the HDXer methods including recommendations for a user's own analyses. Overall, this article is intended to provide an up-to-date, pedagogical counterpart to the software, which is freely available at https://github.com/Lucy-Forrest-Lab/HDXer.

Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation.

Fucosylation is important for the function of many proteins with biotechnical and medical applications. Alpha-fucosidases comprise a large enzyme family that recognizes fucosylated substrates with diverse α-linkages on these proteins. Lactobacillus casei produces an α-fucosidase, called AlfC, with specificity towards α(1,6)-fucose, the only linkage found in human N-glycan core fucosylation. AlfC and certain point mutants thereof have been used to add and remove fucose from monoclonal antibody N-glycans, with significant impacts on their effector functions. Despite the potential uses for AlfC, little is known about its mechanism. Here, we present crystal structures of AlfC, combined with mutational and kinetic analyses, hydrogen-deuterium exchange mass spectrometry, molecular dynamic simulations, and transfucosylation experiments to define the molecular mechanisms of the activities of AlfC and its transfucosidase mutants. Our results indicate that AlfC creates an aromatic subsite adjacent to the active site that specifically accommodates GlcNAc in α(1,6)-linkages, suggest that enzymatic activity is controlled by distinct open and closed conformations of an active-site loop, with certain mutations shifting the equilibrium towards open conformations to promote transfucosylation over hydrolysis, and provide a potentially generalizable framework for the rational creation of AlfC transfucosidase mutants.

Gallium(III)-Salophen as a Dual Inhibitor of Pseudomonas aeruginosa Heme Sensing and Iron Acquisition.

Pseudomonas aeruginosa is an opportunistic bacterium that causes life-threatening infections in immunocompromised patients. In infection, it uses heme as a primary iron source and senses the availability of exogenous heme through the heme assimilation system (Has), an extra cytoplasmic function σ-factor system. A secreted hemophore HasAp scavenges heme and, upon interaction with the outer-membrane receptor HasR, activates a signaling cascade, which in turn creates a positive feedback loop critical for sensing and adaptation within the host. The ability to sense and respond to heme as an iron source contributes to virulence. Consequently, the inhibition of this system will lead to a disruption in iron homeostasis, decreasing virulence. We have identified a salophen scaffold that successfully inhibits the activation of the Has signaling system while simultaneously targeting iron uptake via xenosiderophore receptors. We propose this dual mechanism wherein free Ga3+-salophen reduces growth through uptake and iron mimicry. A dual mechanism targeting extracellular heme signaling and uptake together with Ga3+-induced toxicity following active Ga3+salophen uptake provides a significant therapeutic advantage while reducing the propensity to develop resistance.

Antigen-Induced Allosteric Changes in a Human IgG1 Fc Increase Low-Affinity Fcγ Receptor Binding.

Antibody structure couples adaptive and innate immunity via Fab (antigen binding) and Fc (effector) domains that are connected by unique hinge regions. Because antibodies harbor two or more Fab domains, they are capable of crosslinking multi-determinant antigens, which is required for Fc-dependent functions through associative interactions with effector ligands, including C1q and cell surface Fc receptors. The modular nature of antibodies, with distal ligand binding sites for antigen and Fc-ligands, is reminiscent of allosteric proteins, suggesting that allosteric interactions might contribute to Fc-mediated effector functions. This hypothesis has been pursued for over 40 years and remains unresolved. Here, we provide evidence that allosteric interactions between Fab and Fc triggered by antigen binding modulate binding of Fc to low-affinity Fc receptors (FcγR) for a human IgG1. This work opens the path to further dissection of the relative roles of allosteric and associative interactions in Fc-mediated effector functions.

Insights on the Conformational Ensemble of Cyt C Reveal a Compact State during Peroxidase Activity.

Cytochrome c (cyt c) is known for its role in the electron transport chain but transitions to a peroxidase-active state upon exposure to oxidative species. The peroxidase activity ultimately results in the release of cyt c into the cytosol for the engagement of apoptosis. The accumulation of oxidative modifications that accompany the onset of the peroxidase function are well-characterized. However, the concurrent structural and conformational transitions of cyt c remain undercharacterized. Fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry is a protein footprinting technique used to structurally characterize proteins. FPOP coupled with native ion mobility separation shows that exposure to H2O2 results in the accumulation of a compact state of cyt c. Subsequent top-down fragmentation to localize FPOP modifications reveals changes in heme coordination between conformers. A time-resolved functional assay suggests that this compact conformer is peroxidase active. Altogether, combining FPOP, ion mobility separation, and top-down and bottom-up mass spectrometry allows us to discern individual conformations in solution and obtain a better understanding of the conformational ensemble and structural transitions of cyt c as it transitions from a respiratory role to a proapoptotic role.

A temperature-dependent conformational shift in p38α MAPK substrate-binding region associated with changes in substrate phosphorylation profile.

Febrile-range hyperthermia worsens and hypothermia mitigates lung injury, and temperature dependence of lung injury is blunted by inhibitors of p38 mitogen-activated protein kinase (MAPK). Of the two predominant p38 isoforms, p38α is proinflammatory and p38ß is cytoprotective. Here, we analyzed the temperature dependence of p38 MAPK activation, substrate interaction, and tertiary structure. Incubating HeLa cells at 39.5 °C stimulated modest p38 activation, but did not alter tumor necrosis factor-α (TNFα)-induced p38 activation. In in vitro kinase assays containing activated p38α and MAPK-activated kinase-2 (MK2), MK2 phosphorylation was 14.5-fold greater at 39.5 °C than at 33 °C. By comparison, we observed only 3.1- and 1.9-fold differences for activating transcription factor-2 (ATF2) and signal transducer and activator of transcription-1α (STAT1α) and a 7.7-fold difference for p38ß phosphorylation of MK2. The temperature dependence of p38α:substrate binding affinity, as measured by surface plasmon resonance, paralleled substrate phosphorylation. Hydrogen-deuterium exchange MS (HDX-MS) of p38α performed at 33, 37, and 39.5 °C indicated temperature-dependent conformational changes in an α helix near the common docking and glutamate:aspartate substrate-binding domains at the known binding site for MK2. In contrast, HDX-MS analysis of p38ß did not detect significant temperature-dependent conformational changes in this region. We observed no conformational changes in the catalytic domain of either isoform and no corresponding temperature dependence in the C-terminal p38α-interacting region of MK2. Because MK2 participates in the pathogenesis of lung injury, the observed changes in the structure and function of proinflammatory p38α may contribute to the temperature dependence of acute lung injury.