Characterization of FOXO3-14-3-3 Interaction by Isothermal Titration Calorimetry.

Isothermal titration calorimetry (ITC) is a well-established and convenient label-free technique for measuring biomolecular interactions in aqueous solutions. ITC enables accurate measurement of the affinities and thermodynamic parameters, such as changes in Gibbs energy, enthalpy, and entropy, which help dissect the binding mechanisms. ITC is commonly used to study protein-protein, protein-peptide, protein-DNA, and small-molecule inhibitor binding to target proteins.

Fluorescence Polarization Method to Assess the FOXO Phosphopeptide Interaction with 14-3-3.

Fluorescence polarization (FP) is a reliable and straightforward mix-and-read assay to quantify the interaction of a protein-peptide pair. Labeling the peptide with a suitable fluorophore enables the distinction between the protein-bound and protein-unbound states of a peptide by a change in the emitted light's polarization. Hereby, we present the method to establish the binding affinity for FOXO3a phosphopeptide with 14-3-3ɛ protein using FP.

Molecular Insights of an Avian Species with Low Oxygen Affinity, the Crystal Structure of Duck T-State Methemoglobin.

Hemoglobin (Hb) is the key metalloprotein within red blood cells involved in oxygen transportation from lungs to body cells. The heme-iron atom inherent within Hb effectuates the mechanism of oxygen transportation and carbon dioxide removal. Structural investigations on avian Hb are limited when compared with the enormous work has been carried out on mammalian Hb.

Target-based drug discovery: Applications of fluorescence techniques in high throughput and fragment-based screening.

Target-based discovery of first-in-class therapeutics demands an in-depth understanding of the molecular mechanisms underlying human diseases. Precise measurements of cellular and biochemical activities are critical to gain mechanistic knowledge of biomolecules and their altered function in disease conditions. Such measurements enable the development of intervention strategies for preventing or treating diseases by modulation of desired molecular processes.

High-affinity binding of celastrol to monomeric α-synuclein mitigates in vitro aggregation.

α-Synuclein (αSyn) aggregation is associated with Parkinson's disease (PD). The region αSyn acts as the nucleation 'master controller' and αSyn as a 'secondary nucleation site'. They drive monomeric αSyn to aggregation.

Refolding and characterization of a diabody against Pfs25, a vaccine candidate of Plasmodium falciparum.

Pfs25, a vaccine candidate, expressed on the surface of the malarial parasite, plays an important role in the development of Plasmodium falciparum. 1269, a monoclonal antibody targeting the epidermal growth factor-like domain 1 and epidermal growth factor-like domain 3 of Pfs25, blocks the transmission of parasites in mosquitoes. In this study, we refolded 1269-Db, a dimeric antibody fragment referred as diabody, designed from 1269, with a yield of 3 mg/litre of bacterial culture.

Structure of a 14-3-3ε:FOXO3a Phosphopeptide Complex Reveals 14-3-3 Isoform-Specific Binding of Forkhead Box Class O Transcription Factor (FOXO) Phosphoproteins.

The transcriptional activity of Forkhead Box O3 (FOXO3a) is inactivated by AKT-mediated phosphorylation on Serine 253 (S253), which enables FOXO3a binding to 14-3-3. Phosphorylated FOXO3a binding to 14-3-3 facilitates the nuclear exclusion of FOXO3a, causing cancer cell proliferation. The FOXO3a/14-3-3 interaction has, therefore, emerged as an important therapeutic target.

Molecular insights into α-synuclein interaction with individual human core histones, linker histone, and dsDNA.

α-Synuclein (αS) plays a key role in Parkinson's disease (PD). The αS nuclear role, its binding affinity and specificity to histones and dsDNA remains unknown. Here, we have measured the binding affinity ( ) between αS wild-type (wt) and PD-specific αS S129-phosphorylation mimicking (S129E) mutant with full-length and flexible tail truncated individual core histones (H2a, H2b, H3, and H4), linker histone (H1), and carried out αS-dsDNA interaction studies.

Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library.

Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth.

Residues of helix ɑ2 are critical for catalytic efficiency of mycobacterial alkylhydroperoxide reductase subunit C.

The ability of Mycobacteria to overcome oxidative stress is of paramount importance for its survival within the host. One of the key enzymes that are involved in protecting the bacterium from reactive oxygen species is the catalase-peroxidase (KatG). However, in strains resistant to the antibiotic isoniazid, KatG is rendered ineffective, which is associated with an increased expression of alkylhydroperoxide reductase subunit C (AhpC).

3D reconstruction and flexibility of the hybrid engine Acetobacterium woodii F-ATP synthase.

The Na-translocating FF ATP synthase from Acetobacterium woodii (AwF-ATP synthase) with a subunit stoichiometry of α:β:γ:δ:ε:a:b:(c):c represents an evolutionary path between ATP-synthases and vacuolar ATPases, by containing a heteromeric rotor c-ring, composed of subunits c, c and c, and an extra loop (γ) within the rotary γ subunit. Here, the recombinant AwF-ATP synthase was subjected to negative stain electron microscopy and single particle analysis. The reference free 2D class averages revealed high flexibility of the enzyme, wherein the F and F domains distinctively bended to adopt multiple conformations.

Unique structural and mechanistic properties of mycobacterial F-ATP synthases: Implications for drug design.

The causative agent of Tuberculosis (TB) Mycobacterium tuberculosis (Mtb) encounters unfavourable environmental conditions in the lungs, including nutrient limitation, low oxygen tensions and/or low/high pH values. These harsh conditions in the host triggers Mtb to enter a dormant state in which the pathogen does not replicate and uses host-derived fatty acids instead of carbohydrates as an energy source. Independent to the energy source, the bacterium's energy currency ATP is generated by oxidative phosphorylation, in which the FF-ATP synthase uses the proton motive force generated by the electron transport chain.

Structure and subunit arrangement of Mycobacterial FF ATP synthase and novel features of the unique mycobacterial subunit δ.

In contrast to other prokaryotes, the Mycobacterial FF ATP synthase (α:β:γ:δ:ε:a:b:b':c) is essential for growth. The mycobacterial enzyme is also unique as a result of its 111 amino acids extended δ subunit, whose gene is fused to the peripheral stalk subunit b. Recently, the crystallographic structures of the mycobacterial α:β:γ:ε-domain and c subunit ring were resolved.

Effect of the additional cysteine 503 of vancomycin-resistant Enterococcus faecalis (V583) alkylhydroperoxide reductase subunit F (AhpF) and the mechanism of AhpF and subunit C assembling.

The vancomycin-resistant Enterococcus faecalis alkyl hydroperoxide reductase complex (AhpR) with its subunits AhpC (EfAhpC) and AhpF (EfAhpF) is of paramount importance to restore redox homeostasis. Therefore, knowledge about this defense system is essential to understand its antibiotic-resistance and survival in hosts. Recently, we described the crystallographic structures of EfAhpC, the two-fold thioredoxin-like domain of EfAhpF, the novel phenomenon of swapping of the catalytic domains of EfAhpF as well as the unique linker length, connecting the catalytically active N-and C-terminal domains of EfAhpF.

Structure and function of Mycobacterium-specific components of F-ATP synthase subunits α and ε.

The Mycobacterium tuberculosis (Mtb) FF-ATP synthase (α:β:γ:δ:ε:a:b:b':c) is an essential enzyme that supplies energy for both the aerobic growing and the hypoxic dormant stage of the mycobacterial life cycle. Employing the heterologous F-ATP synthase model system α:β:γ we showed previously, that transfer of the C-terminal domain (CTD) of Mtb subunit α (Mtα) to a standard F-ATP synthase α subunit suppresses ATPase activity. Here we determined the 3D reconstruction from electron micrographs of the α:β:γ complex reconstituted with the Mtb subunit ε (Mtε), which has been shown to crosstalk with the CTD of Mtα.

Molecular mechanism of the Escherichia coli AhpC in the function of a chaperone under heat-shock conditions.

Peroxiredoxins (Prxs) are ubiquitous antioxidants utilizing a reactive cysteine for peroxide reduction and acting as a molecular chaperone under various stress conditions. Besides other stimulating factors, oxidative- and heat stress conditions trigger their ATP-independent chaperoning function. So far, many studies were intended to reveal the chaperoning mechanisms of the so-called sensitive Prxs of eukaryotes, which are susceptible to inactivation by over-oxidation of its reactive cysteine during HO reduction.

Active site C-loop dynamics modulate substrate binding, catalysis, oligomerization, stability, over-oxidation and recycling of 2-Cys Peroxiredoxins.

Peroxiredoxins (Prxs) catalyse the rapid reduction of hydrogen peroxide, organic hydroperoxide and peroxynitrite, using a fully conserved peroxidatic cysteine (C) located in a conserved sequence Pxxx(T/S)xxC motif known as C-loop. In addition, Prxs are involved in cellular signaling pathways and regulate several redox-dependent process related disease. The effective catalysis of Prxs is associated with alterations in the C-loop between reduced, Fully Folded (FF), and oxidized, Locally Unfolded (LU) conformations, which are linked to dramatic changes in the oligomeric structure.

Essential role of the flexible linker on the conformational equilibrium of bacterial peroxiredoxin reductase for effective regeneration of peroxiredoxin.

Reactive oxygen species (ROS) can damage DNA, proteins, and lipids, so cells have antioxidant systems that regulate ROS. In many bacteria, a dedicated peroxiredoxin reductase, alkyl hydroperoxide reductase subunit F (AhpF), catalyzes the rapid reduction of the redox-active disulfide center of the antioxidant protein peroxiredoxin (AhpC) to detoxify ROS such as hydrogen peroxide, organic hydroperoxide, and peroxynitrite. AhpF is a flexible multidomain protein that enables a series of electron transfers among the redox centers by accepting reducing equivalents from NADH.

Transition steps in peroxide reduction and a molecular switch for peroxide robustness of prokaryotic peroxiredoxins.

In addition to their antioxidant function, the eukaryotic peroxiredoxins (Prxs) facilitate peroxide-mediated signaling by undergoing controlled inactivation by peroxide-driven over-oxidation. In general, the bacterial enzyme lacks this controlled inactivation mechanism, making it more resistant to high HO concentrations. During peroxide reduction, the active site alternates between reduced, fully folded (FF), and oxidized, locally unfolded (LU) conformations.

Low resolution solution structure of an enzymatic active AhpC10:AhpF2 ensemble of the Escherichia coli Alkyl hydroperoxide Reductase.

The ability of bacteria to combat oxidative stress is imperative for their survival. The Alkyl hydroperoxide Reductase (AhpR) system, composed of the AhpC and AhpF proteins, is one of the dominant antioxidant defense systems required for scavenging hydrogen peroxide and organic peroxide. Therefore, it is necessary to understand the mechanism of the AhpR ensemble formation.

NMR studies reveal a novel grab and release mechanism for efficient catalysis of the bacterial 2-Cys peroxiredoxin machinery.

In bacteria, an ensemble of alkyl hydroperoxide reductase subunits C (AhpC) and F (AhpF) is responsible for scavenging H2O2. AhpC donates electrons for the reduction of H2O2, which are provided after NADH oxidation by AhpF. The latter contains an N-terminal domain (NTD), catalyzing the electron transfer from NADH via a FAD of the C-terminal domain (CTD) into AhpC.

Crystallographic and solution studies of NAD(+)- and NADH-bound alkylhydroperoxide reductase subunit F (AhpF) from Escherichia coli provide insight into sequential enzymatic steps.

Redox homeostasis is significant for the survival of pro- and eukaryotic cells and is crucial for defense against reactive oxygen species like superoxide and hydrogen peroxide. In Escherichia coli, the reduction of peroxides occurs via the redox active disulfide center of the alkyl hydroperoxide reductase C subunit (AhpC), whose reduced state becomes restored by AhpF. The 57kDa EcAhpF contains an N-terminal domain (NTD), which catalyzes the electron transfer from NADH via an FAD of the C-terminal domain into EcAhpC.

Structural transition in Bcl-xL and its potential association with mitochondrial calcium ion transport.

Bcl-2 family proteins are key regulators for cellular homeostasis in response to apoptotic stimuli. Bcl-xL, an antiapoptotic Bcl-2 family member, undergoes conformational transitions, which leads to two conformational states: the cytoplasmic and membrane-bound. Here we present the crystal and small-angle X-ray scattering (SAXS) structures of Bcl-xL treated with the mild detergent n-Octyl β-D-Maltoside (OM).

Structure, mechanism and ensemble formation of the alkylhydroperoxide reductase subunits AhpC and AhpF from Escherichia coli.

Hydroperoxides are reactive oxygen species (ROS) that are toxic to all cells and must be converted into the corresponding alcohols to alleviate oxidative stress. In Escherichia coli, the enzyme primarily responsible for this reaction is alkylhydroperoxide reductase (AhpR). Here, the crystal structures of both of the subunits of EcAhpR, EcAhpF (57 kDa) and EcAhpC (21 kDa), have been solved.

Key roles of the Escherichia coli AhpC C-terminus in assembly and catalysis of alkylhydroperoxide reductase, an enzyme essential for the alleviation of oxidative stress.

2-Cys peroxiredoxins (Prxs) are a large family of peroxidases, responsible for antioxidant function and regulation in cell signaling, apoptosis and differentiation. The Escherichia coli alkylhydroperoxide reductase (AhpR) is a prototype of the Prxs-family, and is composed of an NADH-dependent AhpF reductase (57 kDa) and AhpC (21 kDa), catalyzing the reduction of H2O2. We show that the E.