Lewy bodies (LBs), a pathological hallmark of synucleinopathies, are heterogeneous inclusions that contain α-Synuclein (αSyn) alongside numerous proteins, lipids, and damaged organelles. Current αSyn-fibrillization centric aggregation/phase separation models fail to explain how diverse cellular components are sequestered by disease-specific αSyn variants during LB formation. In the crowded intracellular milieu, proteins constantly encounter one another, but functional protein-protein interactions must outweigh disease-causing ‘hydrophobicity’ driven non-functional interactions. Although αSyn wild-type (WT) has a hydrophobic (NAC) core, it is shielded by long-range intramolecular interactions, rendering it “inert.” In contrast, Parkinson’s disease (PD)-specific αSyn variants—S129 phosphorylation and C-terminal truncations—aggregate and phase separate more rapidly, suggesting hydrophobic exposure. We hypothesize that exposed hydrophobic core in PD-specific αSyn variants not only drives aggregation and phase separation but also promotes promiscuous, non-functional binding to diverse proteins. Using various biochemical and biophysical approaches, we demonstrate that αSynWT engages in functional interactions, whereas C-terminal acidic tail truncated αSyn1–103 and S129-phosphomimicking (S129E) mutant are “reactive,” displaying broad, non-functional aberrant binding and impairing chaperone-mediated refolding. Based on our study, we propose a ‘Multifactorial Random Disorder Model’ outlining how PD-specific αSyn variants drive LB formation through non-functional heterotypic interactions. Parkinson’s Disease specific α-synuclein variants drive Lewy body (LB) formation by engaging in promiscuous and non-functional interactions. Here the authors propose the ‘Multifactorial Random Disorder Model’ describing LB formation.

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. ITC can determine binding affinities ranging from nM to low-mM. This chapter provides a detailed protocol to investigate the binding of FOXO3 peptide to 14-3-3ε using ITC.

α-Synuclein (αSyn) plays a critical role in the pathogenesis of 'Synucleinopathies'. Although increased nuclear αSyn localization induces neurotoxicity, its definitive physiological role remains elusive. Previous studies on nuclear αSyn are limited to its interactions with individual histones and dsDNA, leaving a significant gap in understanding its interactions with assembled histone H2a-H2b dimer and (H3-H4)2 tetramer, as well as its role in chromatin regulation. Here, we demonstrate that αSyn binds specifically to both H2a-H2b and (H3-H4)2 with high affinity. Truncation studies reveal that αSyn(1-103) region interacts with (H3-H4)2, while the acidic (121-140) C-terminal end is crucial for H2a-H2b binding and contains a conserved DEF/YxP motif present in other dimer-binding histone chaperones. High-resolution structure of αSyn(121-140) with H2a-H2b complex reveals that αSyn adopts two binding modes (BM-1 and BM-2). Nonetheless, the αSyn C-terminal end in both modes overlap but runs in opposite orientations, specifically interacting with the H2a-L2 and H2b-L1 loop regions of the dimer and cap the H2a-R78 residue. Mutational analysis confirms that αSyn-Y136 and P138 residues, part of the DEF/YxP motif, together with H2a-R78, are critical for αSyn-(H2a-H2b) interaction. The chaperoning assay supports αSyn's function as a histone chaperone, suggesting the potential role of αSyn in the nucleosome assembly/disassembly process.

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. With the advantages of lower sample consumption and faster read-outs, FP validates the interaction between the pairs and can also be used to develop a competitive assay to identify and validate the novel inhibitors for the interaction pair.

α-Synuclein (αSyn) plays a critical role in the pathogenesis of ′Synucleinopathies′. Although increased nuclear αSyn localization induces neurotoxicity, its definitive physiological role remains elusive. Previous studies on nuclear αSyn are limited to its interactions with individual histones and dsDNA, leaving a significant gap in understanding its interactions with assembled histone H2a-H2b dimer and (H3-H4)2 tetramer, as well as its role in chromatin regulation. Here, we demonstrated that αSyn binds specifically to both H2a-H2b and (H3-H4)2 with high affinity. Truncation studies revealed that αSyn(1-103) region interacts with (H3-H4)2, while the acidic (121-140) C-terminal end is crucial for H2a-H2b binding. Sequence analysis suggests αSyn-dimer binding region contains a conserved DEF/YxP motif present in other dimer-binding histone chaperones. High-resolution structure of αSyn-dimer binding region with H2a-H2b complex reveals that αSyn adopts two binding modes (BM1 and BM2). In BM-1, αSyn utilizes nucleosomal DNA-binding surface, while in BM-2, it engages with both DNA- and the H3-interaction interface. Additionally, dimer recognition by αSyn overlaps with other dimer-binding histone chaperones, suggesting αSyn′s potential role in the nucleosome assembly/disassembly process. ### Competing Interest Statement The authors have declared no competing interest.

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. Fluorescence-based techniques are routinely employed for accurate and robust measurements of in-vitro activity of molecular targets and for discovering novel chemical molecules that modulate the activity of molecular targets. In the current review, the authors focus on the applications of fluorescence-based high throughput screening (HTS) and fragment-based ligand discovery (FBLD) techniques such as fluorescence polarization (FP), Förster resonance energy transfer (FRET), fluorescence thermal shift assay (FTSA) and microscale thermophoresis (MST) for the discovery of chemical probe to exploring target’s role in disease biology and ultimately, serve as a foundation for drug discovery. Some recent advancements in these techniques for compound library screening against important classes of drug targets, such as G-protein-coupled receptors (GPCRs) and GTPases, as well as phosphorylation- and acetylation-mediated protein-protein interactions, are discussed. Overall, this review presents a landscape of how these techniques paved the way for the discovery of small-molecule modulators and biologics against these targets for therapeutic benefits.

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. Here, the crystal structure of T-state methemoglobin (T-metHb) from domestic duck (Anas platyrhynchos), a low oxygen affinity avian species, determined to 2.1Å resolution is presented. Duck T-metHb crystallized in the orthorhombic space group C2221 with unit cell parameters a = 59.89, b = 109.42 and c = 92.07Å. The final refined model with R-factor: 19.5

Background: The study aims to shed light on the mutations caused in Monkeypox virus genome which induced the sudden outbreak. The reason behind this induced mutation is identified as APOBEC enzyme which is an antiviral host protein. This protein has caused point mutation in viral DNA polymerase to evade the host immune system. Methods: In this review, 29 Research & review articles and 6 research magazine including World Health Organization were referred to construct this article. The articles were extracted from PubMed and Google Scholar. All sequences were extracted from NCBI and GISAID. The reference articles provided strong conceptual support. Phyre2 software was used in 3D modeling of DNA polymerase protein. It was helpful in identifying the mutation in the viral DNA polymerase. Results: Host protein APOBEC causes mutation in viral DNA polymerase. Analysis of 3D model of viral DNA polymerase revealed a point mutation in the enzyme at the beta sheet region caused by host protein APOBEC (Cytosine Deamination). Conclusion: The mutation in viral DNA polymerase has helped Monkeypox virus in effective immune evasion mechanism. The current outbreak exhibits heightened mutation rates, likely attributed to the involvement of the APOBEC3 enzyme. This enzyme induces point mutations and disrupts DNA proofreading, leading to rapid viral genetic changes. Further research is required to understand these mutations, aid in treatment development, and guide vaccine or therapeutic strategies.

α-Synuclein (αSyn) aggregation is associated with Parkinson's disease (PD). The region αSyn36-42 acts as the nucleation 'master controller' and αSyn1-12 as a 'secondary nucleation site'. They drive monomeric αSyn to aggregation. Small molecules targeting these motifs are promising for disease-modifying therapy. Using computational techniques, we screened thirty phytochemicals for αSyn binding. The top three compounds were experimentally validated for their binding affinity. Amongst them, celastrol showed high binding affinity. NMR analysis confirmed stable αSyn-celastrol interactions involving several residues in the N-terminus and NAC regions but not in the C-terminal tail. Importantly, celastrol interacted extensively with the key motifs that drive αSyn aggregation. Thioflavin-T assay indicated that celastrol reduced αSyn aggregation. Thus, celastrol holds promise as a potent drug candidate for PD.Communicated by Ramaswamy H. Sarma.

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. Structural integrity of the protein was validated with thermal stability, disulphide bond analysis and glutaraldehyde crosslinking experiments. To evaluate the functionality of 1269-Db, recombinant monomeric MBP-Pfs25 was produced from bacteria. Qualitative binding assays demonstrated that 1269-Db recognized the epitopes on Pfs25 in its native, but not the denatured state. An apparent KD of 2.6 nM was determined for 1269-Db with monomeric MBP-Pfs25, using isothermal titration calorimetry. 1269-Db recognized the periphery of zygotes/ookinetes, demonstrating recognition of Pfs25, expressed on the surface of the parasite. As the established refolding method resulted in a functional diabody, the optimized method pipeline for 1269-Db can potentially facilitate engineering of antibody fragments with desired properties.

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. Here, we report a comprehensive analysis using fluorescence polarization, isothermal titration calorimetry, small-angle X-ray scattering, X-ray crystallography, and molecular dynamics simulations to gain molecular-level insights into the interaction of FOXO3apS253 phosphopeptide with 14-3-3ε. A high-resolution structure of the fluorophore-labeled FOXO3apS253:14-3-3ε complex revealed a distinct mode of interaction compared to other 14-3-3 phosphopeptide complexes. FOXO3apS253 phosphopeptide showed significant structural difference in the positions of the -3 and -4 Arg residues relative to pSer, compared to that of a similar phosphopeptide, FOXO1pS256 bound to 14-3-3σ. Moreover, molecular dynamics studies show that the significant structural changes and molecular interactions noticed in the crystal structure of FOXO3apS253:14-3-3ε are preserved over the course of the simulation. Thus, this study reveals structural differences between the binding to 14-3-3 isoforms of FOXO1pS256 versus FOXO3apS253, providing a framework for the rational design of isoform-specific FOXO/14-3-3 protein-protein interaction inhibitors for therapy.

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 ( Kd ) 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. This study revealed that αS(wt) interacts specifically with N-terminal flexible tails of histone H3, H4, and flexible tails of H1. The αS(S129E) mutant recognizes histones similar to αS(wt) but binds with higher affinity. Intriguingly, αS(S129E) showed a binding affinity for control proteins (bovine serum albumin and lysozyme), while no interaction was seen for αS(wt). Based on our above observation, we contemplate that the physio-chemical properties of αS with S129-phosphorylation has changed compared to αS(wt), resulting in interaction for other proteins, which is the basis for Lewy body formation. Besides, this study showed αS binding to dsDNA is weak and nonspecific. Overall, αS specificity for histone binding suggests that its nuclear role is possibly driven through histone interaction.

SummaryHIV-1 causes diverse immunomodulatory responses in the host, including the down-regulation of co-stimulatory proteins CD80/86, mediated by HIV-1 protein Nef, blunting T-cell activation. Using a screening cascade of biochemical and cell-based assays, we identified potent small molecules representing three chemical scaffolds namely amino pyrimidine, phenoxy acetamide and bi-aryl heteroaryl carbamate which target the protein-protein interaction interface of CD80/86 and Nef with sub-micromolar potency. These molecules restore CD80/86 surface levels in HIV-1-Nef infected antigen presenting cells and T-cell activation. Nef-CD80 interface and small molecule binding sites were mapped by using computational docking and structural studies, followed by validation by mutational analysis. This analysis resulted in the identification of two key residues, K99 and R111, which were associated with down-modulation of CD80 surface levels by Nef and important for small molecule binding. Targeting these interacting residues disabled Nef-mediated down-modulation of CD80 surface levels, consequently restoring T-cell activation. Thus, we validate a new target, the Nef-CD80/86 protein-protein interaction interface, with a potential to develop new inhibitors to counteract the immunomodulatory consequences of HIV-1.

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. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify "protein interference," an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.

The ability ofMycobacteriato 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). Mycobacterial AhpC possesses a unique helical displacement when compared to its bacterial counterparts. Here,viamutagenesis studies, we demonstrate the importance of this helix for redox modulation of the catalytic activity of AhpC. Along with structural insights from crystallographic data, the impact of critical residues on the structure and flexibility of the helix and on AhpC oligomerization is described.

The Na+-translocating F1FO ATP synthase from Acetobacterium woodii (AwF-ATP synthase) with a subunit stoichiometry of α3:β3:γ:δ:ε:a:b2:(c2/3)9:c1 represents an evolutionary path between ATP-synthases and vacuolar ATPases, by containing a heteromeric rotor c-ring, composed of subunits c1, c2 and c3, and an extra loop (γ195-211) 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 F1 and FO domains distinctively bended to adopt multiple conformations. Moreover, both the F1 and FO domains tilted relative to each other to a maximum extent of 28° and 30°, respectively. The first 3D reconstruction of the AwF-ATP synthase was determined which accommodates well the modelled structure of the AwF-ATP synthase as well as the γ195-211-loop. Molecular simulations of the enzyme underlined the bending features and flexibility observed in the electron micrographs, and enabled assessment of the dynamics of the extra γ195-211-loop.

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. Here, using mutagenesis and enzymatic studies, we reveal the effect of an additional third cysteine (C503) in EfAhpF, which might optimize the functional adaptation of the E. faecalis enzyme under various physiological conditions. The crystal structure and solution NMR data of the engineered C503A mutant of the thioredoxin-like domain of EfAhpF were used to describe alterations in the environment of the additional cysteine residue during modulation of the redox-state. To glean insight into the epitope and mechanism of EfAhpF and -AhpC interaction as well as the electron transfer from the thioredoxin-like domain of EfAhpF to AhpC, NMR-titration experiments were performed, showing a coordinated disappearance of peaks in the thioredoxin-like domain of EfAhpF in the presence of full length EfAhpC, and indicating a stable EfAhpF-AhpC-complex. Combined with docking studies, the interacting residues of EfAhpF were identified and a mechanism of electron transfer of the EfAhpF donor to the electron acceptor EfAhpC is described.

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 F1FO-ATP synthase uses the proton motive force generated by the electron transport chain. This catalyst is essential in Mtb and inhibition by the diarylquinoline class of drugs like Bedaquilline, TBAJ-587, TBAJ-876 or squaramides demonstrated that this engine is an attractive target in TB drug discovery. A special feature of the mycobacterial F-ATP synthase is its inability to establish a significant proton gradient during ATP hydrolysis, and its latent ATPase activity, to prevent energy waste and to control the membrane potential. Recently, unique epitopes of mycobacterial F1FO-ATP synthase subunits absent in their prokaryotic or mitochondrial counterparts have been identified to contribute to the regulation of the low ATPase activity. Most recent structural insights into individual subunits, the F1 domain or the entire mycobacterial enzyme added to the understanding of mechanisms, regulation and differences of the mycobacterial F1FO-ATP synthase compared to other bacterial and eukaryotic engines. These novel insights provide the basis for the design of new compounds targeting this engine and even novel regimens for multidrug resistant TB.

In contrast to other prokaryotes, the Mycobacterial F1FO ATP synthase (α3:β3:γ:δ:ε:a:b:b':c9) 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 α3:β3:γ:ε-domain and c subunit ring were resolved. Here, we report the first purification protocol of the intact M. smegmatis F1FO ATP synthase including the F1-domain, the entire membrane-embedded FO sector, and the stator subunits b' and the fused b-δ. This enzyme purification enabled the determination of the first projected 2D- and 3D structure of the intact M. smegmatis F1FO ATP synthase by electron microscopy (EM) and single particle analysis. Expression and purification of the fused mycobacterial b-δ24-446 construct, excluding the membrane-embedded N-terminal amino acids, provided insight into its secondary structure. By combining these data with homology and ab-initio modeling techniques, a model of the mycobacterial peripheral stalk subunits b-δ and b' was generated. Superposition of the 3D M. smegmatis F-ATP synthase EM-structure, the α3:β3:γ:ε and c-ring, and the derived structural models of the peripheral stalk enabled a clear assignment of all F-ATP synthase subunits, in particular with respect to the unique mycobacterial peripheral stalk subunit b' and the elongated δ fused with subunit b. The arrangement of δ relative to the N-termini of the catalytic α3β3-headpiece and its potential as a drug target are discussed.

The Mycobacterium tuberculosis (Mtb) F1FO-ATP synthase (α3:β3:γ:δ:ε:a:b:b':c9) 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 αchi3:β3:γ we showed previously, that transfer of the C-terminal domain (CTD) of Mtb subunit α (Mtα514-549) to a standard F-ATP synthase α subunit suppresses ATPase activity. Here we determined the 3D reconstruction from electron micrographs of the αchi3:β3:γ complex reconstituted with the Mtb subunit ε (Mtε), which has been shown to crosstalk with the CTD of Mtα. Together with the first solution shape of Mtb subunit α (Mtα), derived from solution X-ray scattering, the structural data visualize the extended C-terminal stretch of the mycobacterial subunit α. In addition, Mtε mutants MtεR62L, MtεE87A, Mtε6-121, and Mtε1-120, reconstituted with αchi3:β3:γ provided insight into their role in coupling and in trapping inhibiting MgADP. NMR solution studies of MtεE87A gave insights into how this residue contributes to stability and crosstalk between the N-terminal domain (NTD) and the CTD of Mtε. Analyses of the N-terminal mutant Mtε6-121 highlight the differences of the NTD of mycobacterial subunit ε to the well described Geobacillus stearothermophilus or Escherichia coli counterparts. These data are discussed in context of a crosstalk between the very N-terminal amino acids of Mtε and the loop region of one c subunit of the c-ring turbine for coupling of proton-translocation and ATP synthesis activity.