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The human protein Abelson kinase (Abl), a tyrosine kinase, plays a pivotal role in developing chronic myeloid leukemia (CML). Abl's involvement in various signaling pathways underscores its significance in regulating fundamental biological processes, including DNA damage responses, actin polymerization, and chromatin structural changes. The discovery of the Bcr-Abl oncoprotein, resulting from a chromosomal translocation in CML patients, revolutionized the understanding and treatment of the disease. The introduction of targeted therapies, starting with interferon-alpha and culminating in the development of tyrosine kinase inhibitors (TKIs) like imatinib, significantly improved patient outcomes. However, challenges such as drug resistance and side effects persist, indicating the necessity of research into novel therapeutic strategies. This review describes advancements in Abl kinase inhibitor development, emphasizing rational compound design from structural and regulatory information. Strategies, including bivalent inhibitors, PROTACs, and compounds targeting regulatory domains, promise to overcome resistance and minimize side effects. Additionally, leveraging the intricate structure and interactions of Bcr-Abl may provide insights into developing inhibitors for other kinases. Overall, this review highlights the importance of continued research into Abl kinase inhibition and its broader implications for therapeutic interventions targeting kinase-driven diseases. It provides valuable insights and strategies that may guide the development of next-generation therapies., (© 2024 Wiley-VCH GmbH.)

The ( S )-norcoclaurine synthase from Thalictrum flavum ( Tf NCS) stereoselectively catalyzes the Pictet-Spengler reaction between dopamine and 4-hydroxyphenylacetaldehyde to give ( S )-norcoclaurine. Tf NCS can catalyze the Pictet-Spengler reaction with various aldehydes and ketones, leading to diverse tetrahydroisoquinolines. This substrate promiscuity positions Tf NCS as a highly promising enzyme for synthesizing fine chemicals. Understanding carbonyl-containing substrates' structural and electronic signatures that influence Tf NCS activity can help expand its applications in the synthesis of different compounds and aid in protein optimization strategies. In this study, we investigated the influence of the molecular properties of aldehydes and ketones on their reactivity in the Tf NCS-catalyzed Pictet-Spengler reaction. Initially, we compiled a library of reactive and unreactive compounds from previous publications. We also performed enzymatic assays using nuclear magnetic resonance to identify some reactive and unreactive carbonyl compounds, which were then included in the library. Subsequently, we employed QSAR and DFT calculations to establish correlations between substrate-candidate structures and reactivity. Our findings highlight correlations of structural and stereoelectronic features, including the electrophilicity of the carbonyl group, to the reactivity of aldehydes and ketones toward the Tf NCS-catalyzed Pictet-Spengler reaction. Interestingly, experimental data of seven compounds out of fifty-three did not correlate with the electrophilicity of the carbonyl group. For these seven compounds, we identified unfavorable interactions between them and the Tf NCS. Our results demonstrate the applications of in silico techniques in understanding enzyme promiscuity and specificity, with a particular emphasis on machine learning methodologies, DFT electronic structure calculations, and molecular dynamic (MD) simulations.

Flaviviruses are among the most critical pathogens in tropical regions and cause a growing number of severe diseases in developing countries. The development of antiviral therapeutics is crucial for managing flavivirus outbreaks. Among the ten proteins encoded in the flavivirus RNA, non-structural protein 5, NS5, is a promising drug target. NS5 plays a fundamental role in flavivirus replication, viral RNA methylation, RNA polymerization, and host immune system evasion. Most of the NS5 inhibitor candidates target NS5 active sites. However, the similarity of NS5 activity sites with human enzymes can cause side effects. Identifying new allosteric sites in NS5 can contribute enormously to antiviral development. The NS5 structural characterization enabled exploring new regions, such as the residues involved in MTase-RdRp interaction, NS5 oligomerization, and NS5 interaction with other viral and host-cell proteins. Targeting NS5 critical interactions might lead to new compounds and overcomes the toxicity of current NS5-inhibitor candidates., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The N-terminal domain of Tetracenomycin aromatase/cyclase (TcmN), an enzyme derived from Streptomyces glaucescens, is involved in polyketide cyclization, aromatization, and folding. Polyketides are a diverse class of secondary metabolites produced by certain groups of bacteria, fungi, and plants with various pharmaceutical applications. Examples include antibiotics, such as tetracycline, and anticancer drugs, such as doxorubicin. Because TcmN is a promising enzyme for in vitro production of polyketides, it is important to identify conditions that enhance its thermal resistance and optimize its function., Methods: TcmN unfolding, stability, and dynamics were evaluated by fluorescence spectroscopy, circular dichroism, nuclear magnetic resonance 15 N relaxation experiments, and microsecond molecular dynamics (MD) simulations., Results: TcmN thermal resistance was enhanced at low protein and high salt concentrations, was pH-dependent, and denaturation was irreversible. Conformational dynamics on the μs-ms timescale were detected for residues in the substrate-binding cavity, and two predominant conformers representing opened and closed cavity states were observed in the MD simulations., Conclusion: Based on the results, a mechanism was proposed in which the thermodynamics and kinetics of the TcmN conformational equilibrium modulate enzyme function by favoring ligand binding and avoiding aggregation., General Significance: Understanding the principles underlying TcmN stability and dynamics may help in designing mutants with optimal properties for biotechnological applications., (Copyright © 2021. Published by Elsevier B.V.)

Cardiac TnC (cTnC) is highly conserved among mammals, and genetic variants can result in disease by perturbing Ca 2+ -regulation of myocardial contraction. Here, we report the molecular basis of a human mutation in cTnC's αD-helix ( TNNC1 -p.C84Y) that impacts conformational dynamics of the D/E central-linker and sampling of discrete states in the N-domain, favoring the "primed" state associated with Ca 2+ binding. We demonstrate cTnC's αD-helix normally functions as a central hub that controls minimally frustrated interactions, maintaining evolutionarily conserved rigidity of the N-domain. αD-helix perturbation remotely alters conformational dynamics of the N-domain, compromising its structural rigidity. Transgenic mice carrying this cTnC mutation exhibit altered dynamics of sarcomere function and hypertrophic cardiomyopathy. Together, our data suggest that disruption of evolutionary conserved molecular frustration networks by a myofilament protein mutation may ultimately compromise contractile performance and trigger hypertrophic cardiomyopathy., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Aberrant regulation of myocardial force production represents an early biomechanical defect associated with sarcomeric cardiomyopathies, but the molecular mechanisms remain poorly defined. Here, we evaluated the pathogenicity of a previously unreported sarcomeric gene variant identified in a pediatric patient with sporadic dilated cardiomyopathy, and we determined a molecular mechanism. Trio whole-exome sequencing revealed a de novo missense variant in TNNC1 that encodes a p.I4M substitution in the N-terminal helix of cardiac troponin C (cTnC). Reconstitution of this human cTnC variant into permeabilized porcine cardiac muscle preparations significantly decreases the magnitude and rate of isometric force generation at physiological Ca 2+ -activation levels. Computational modeling suggests that this inhibitory effect can be explained by a decrease in the rates of cross-bridge attachment and detachment. For the first time, we show that cardiac troponin T (cTnT), in part through its intrinsically disordered C terminus, directly binds to WT cTnC, and we find that this cardiomyopathic variant displays tighter binding to cTnT. Steady-state fluorescence and NMR spectroscopy studies suggest that this variant propagates perturbations in cTnC structural dynamics to distal regions of the molecule. We propose that the intrinsically disordered C terminus of cTnT directly interacts with the regulatory N-domain of cTnC to allosterically modulate Ca 2+ activation of force, perhaps by controlling the troponin I switching mechanism of striated muscle contraction. Alterations in cTnC-cTnT binding may compromise contractile performance and trigger pathological remodeling of the myocardium., (© 2019 Johnston et al.)

Two new complexes of Ru(II) with mixed ligands were prepared: [Ru(bpy) 2 smp](PF 6 ) ( 1 ) and [Ru(phen) 2 smp](PF 6 ) ( 2 ), in which smp = sulfamethoxypyridazine; bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline. The complexes have been characterized by elemental and conductivity analyses; infrared, NMR, and electrospray ionization mass spectroscopies; and X-ray diffraction of single crystal. Structural analyses reveal a distorted octahedral geometry around Ru(II) that is bound to two bpy (in 1 ) or two phen (in 2 ) via their two heterocyclic nitrogens and to two nitrogen atoms from sulfamethoxypyridazine-one of the methoxypyridazine ring and the sulfonamidic nitrogen, which is deprotonated. Both complexes inhibit the growth of chronic myelogenous leukemia cells. The interaction of the complexes with bovine serum albumin and DNA is described. DNA footprinting using an oligonucleotide as substrate showed the complexes' preference for thymine base rich sites. It is worth notifying that the complexes interact with the Src homology SH3 domain of the Abl tyrosine kinase protein. Abl protein is involved in signal transduction and implicated in the development of chronic myelogenous leukemia. Nuclear magnetic resonance (NMR) studies of the interaction of complex 2 with the Abl-SH3 domain showed that the most affected residues were T79, G97, W99, and Y115.

Fag s 1 is a member of the Pathogen Related protein family 10 (PR-10) and can elicit cross-reaction with IgE antibodies produced against the birch pollen allergen Bet v 1. The Nuclear Magnetic Resonance (NMR) structure of Fag s 1 is presented along with its dynamic properties. It shares 66% identity with Bet v 1 and exhibits the expected three α-helices and seven β-sheets arranged as a semi-beta barrel and exposing the residues mapped as the Bet v 1 IgE epitope. The structural dynamics of Fag s 1 were monitored on the fast and intermediate timescales, using relaxation rates. The complex dynamics of Fag s 1 are closely related to the internal cavity, and they modulate IgE and ligand binding.

The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca 2+ -binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30-40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca 2+ than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca 2+ -saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other.

Hypertrophic cardiomyopathy (HCM) is one of the most common cardiomyopathies and a major cause of sudden death in young athletes. The Ca 2+ sensor of the sarcomere, cardiac troponin C (cTnC), plays an important role in regulating muscle contraction. Although several cardiomyopathy-causing mutations have been identified in cTnC, the limited information about their structural defects has been mapped to the HCM phenotype. Here, we used high-resolution electron-spray ionization mass spectrometry (ESI-MS), Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD), and affinity measurements of cTnC for the thin filament in reconstituted papillary muscles to provide evidence of an allosteric mechanism in mutant cTnC that may play a role to the HCM phenotype. We showed that the D145E mutation leads to altered dynamics on a μs-ms time scale and deactivates both of the divalent cation-binding sites of the cTnC C-domain. CPMG-RD captured a low populated protein-folding conformation triggered by the Glu-145 replacement of Asp. Paradoxically, although D145E C-domain was unable to bind Ca 2+ , these changes along its backbone allowed it to attach more firmly to thin filaments than the wild-type isoform, providing evidence for an allosteric response of the Ca 2+ -binding site II in the N-domain. Our findings explain how the effects of an HCM mutation in the C-domain reflect up into the N-domain to cause an increase of Ca 2+ affinity in site II, thus opening up new insights into the HCM phenotype., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Parkinson's disease is a neurological disease in which aggregated forms of the α-synuclein (α-syn) protein are found. We used high hydrostatic pressure (HHP) coupled with NMR spectroscopy to study the dissociation of α-syn fibril into monomers and evaluate their structural and dynamic properties. Different dynamic properties in the non-amyloid-β component (NAC), which constitutes the Greek-key hydrophobic core, and in the acidic C-terminal region of the protein were identified by HHP NMR spectroscopy. In addition, solid-state NMR revealed subtle differences in the HHP-disturbed fibril core, providing clues to how these species contribute to seeding α-syn aggregation. These findings show how pressure can populate so far undetected α-syn species, and they lay out a roadmap for fibril dissociation via pathways not previously observed using other approaches. Pressure perturbs the cavity-prone hydrophobic core of the fibrils by pushing water inward, thereby inducing the dissociation into monomers. Our study offers the molecular details of how hydrophobic interaction and the formation of water-excluded cavities jointly contribute to the assembly and stabilization of the fibrils. Understanding the molecular forces behind the formation of pathogenic fibrils uncovered by pressure perturbation will aid in the development of new therapeutics against Parkinson's disease.

Unlabelled: Domain III of dengue virus E protein (DIII) participates in the recognition of cell receptors and in structural rearrangements required for membrane fusion and ultimately viral infection; furthermore, it contains epitopes for neutralizing antibodies and has been considered a potential vaccination agent. In this work, we addressed various structural aspects of DIII and their relevance for both the dengue virus infection mechanism and antibody recognition. We provided a dynamic description of DIII at physiological and endosomal pHs and in complex with the neutralizing human antibody DV32.6. We observed conformational exchange in the isolated DIII, in regions important for the packing of E protein dimers on the virus surface. This conformational diversity is likely to facilitate the partial detachment of DIII from the other E protein domains, which is required to achieve fusion to the host cellular membranes and to expose the epitopes of many anti-DIII antibodies. A comparison of DIII of two dengue virus serotypes revealed many common features but also some possibly unexpected differences. Antibody binding to DIII of dengue virus serotype 4 attenuated the conformational exchange in the epitope region but, surprisingly, generated exchange in other parts of DIII through allosteric effects., Importance: Many studies have provided extensive structural information on the E protein and particularly on DIII, also in complex with antibodies. However, there is very scarce information regarding the molecular dynamics of DIII, and almost nothing is available on the dynamic effect of antibody binding, especially at the quantitative level. This work provides one of the very rare descriptions of the effect of antibody binding on antigen dynamics., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.

Beta-parvalbumins from different fish species have been identified as the main elicitors of IgE-mediated reactions in fish-allergic individuals. Here, we report for the first time the NMR determination of the structure and dynamics of the major Atlantic cod (Gadus morhua) allergen Gad m 1 and compare them with other known parvalbumins. Although the Gad m 1 structure and accessibility of putative IgE epitopes are similar to parvalbumins in mackerel and carp, the charge distribution at the putative epitopes is different. The determination of the Gad m 1 structure contributes to a better understanding of cross-reactivity among fish parvalbumins. In addition, the high-pressure NMR and temperature variation experiments revealed the important contribution of the AB motif and other regions to the protein folding. This structural information could assist the future identification of hot spots for targeted mutations to develop hypoallergenic Ca(2+) -free forms for potential use in immunotherapy., (© 2014 Wiley Periodicals, Inc.)