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ε-Trimethyllysine dioxygenase (TMLD) is a non-heme Fe(II) and α-ketoglutarate dependent oxygenase that catalyzes the stereospecific hydroxylation of ε-trimethyl

Predicting the interaction modes and binding affinities of virtual compound libraries is of great interest in drug development. It reduces the cost and time of lead compound identification and selection. Here we apply path-based metadynamics simulations to characterise the binding of potential inhibitors to the Plasmodium falciparum aspartic protease plasmepsin V (plm V), a validated antimalarial drug target that has a highly mobile binding site. The potential plm V binders were identified in a high throughput virtual screening (HTVS) campaign and were experimentally verified in a fluorescence resonance energy transfer (FRET) assay. Our simulations allowed us to estimate compound binding energies and revealed putative transition states along binding/unbinding pathways in atomistic resolution. We believe that the method described allows the prioritisation of compounds for synthesis and enables rational structure-based drug design for targets that undergo considerable conformational changes upon inhibitor binding.

Heart-type fatty-acid binding protein (FABP3) is an essential cytosolic lipid transport protein found in cardiomyocytes. FABP3 binds fatty acids (FAs) reversibly and with high affinity. Acylcarnitines (ACs) are an esterified form of FAs that play an important role in cellular energy metabolism. However, an increased concentration of ACs can exert detrimental effects on cardiac mitochondria and lead to severe cardiac damage. In the present study, we evaluated the ability of FABP3 to bind long-chain ACs (LCACs) and protect cells from their harmful effects. We characterized the novel binding mechanism between FABP3 and LCACs by a cytotoxicity assay, nuclear magnetic resonance, and isothermal titration calorimetry. Our data demonstrate that FABP3 is capable of binding both FAs and LCACs as well as decreasing the cytotoxicity of LCACs. Our findings reveal that LCACs and FAs compete for the binding site of FABP3. Thus, the protective mechanism of FABP3 is found to be concentration dependent.

The microtubule associated protein tau forms disease-specific filamentous aggregates in several different neurodegenerative diseases. In order to understand how tau undergoes misfolding into a specific filament type and to be able to control this process for drug development purposes, it is crucial to studyin vitrotau aggregation methods and investigate the structures of the obtained filaments at atomic level. Here, we used the tau fragment dGAE(297-391), which aggregates spontaneously, to seed formation of fulllength tau filaments. The structures of dGAE and full-length tau filaments were investigated by solid-state MAS NMR showing that dGAE allows propagating a chronic traumatic encephalopathy (CTE)-like fold to full-length tau. This work demonstrates thatin vitropreparation of disease-specific types of full-length tau filaments is feasible.

While nuclear magnetic resonance (NMR) is regarded asa referencein fragment-based drug design, its implementation in a high-throughputmanner is limited by its lack of sensitivity resulting in long acquisitiontimes and high micromolar sample concentrations. Several hyperpolarizationapproaches could, in principle, improve the sensitivity of NMR alsoin drug research. However, photochemically induced dynamic nuclearpolarization (photo-CIDNP) is the only method that is directly applicablein aqueous solution and agile for scalable implementation using off-the-shelfhardware. With the use of photo-CIDNP, this work demonstrates thedetection of weak binders in the millimolar affinity range using lowmicromolar concentrations down to 5 mu M of ligand and 2 mu Mof target, thereby exploiting the photo-CIDNP-induced polarizationtwice: (i) increasing the signal-to-noise by one to two orders inmagnitude and (ii) polarization-only of the free non-bound moleculeallowing identification of binding by polarization quenching, yieldinganother factor of hundred in time when compared with standard techniques.The interaction detection was performed with single-scan NMR experimentsof a duration of 2 to 5 s. Taking advantage of the readiness of photo-CIDNPsetup implementation, an automated flow-through platform was designedto screen samples at a screening rate of 1500 samples per day. Furthermore,a 212 compounds photo-CIDNP fragment library is presented, openingan avenue toward a comprehensive fragment-based screening method., Journal of the American Chemical Society, 145 (22), ISSN:0002-7863, ISSN:1520-5126

Viral mRNA cap methyltransferases (MTases) are emerging targets for the development of broad-spectrum antiviral agents. In this work, we designed potential SARS-CoV-2 MTase Nsp14 and Nsp16 inhibitors by using bioisosteric substitution of the sulfonium and amino acid substructures of the cosubstrate S-adenosylmethionine (SAM), which serves as the methyl donor in the enzymatic reaction. The synthetically accessible target structures were prioritized using molecular docking. Testing of the inhibitory activity of the synthesized compounds showed nanomolar to submicromolar IC50 values for five compounds. To evaluate selectivity, enzymatic inhibition of the human glycine N-methyltransferase involved in cellular SAM/SAH ratio regulation was also determined, which indicated that the discovered compounds are nonselective inhibitors of the studied MTases with slight selectivity for Nsp16. No cytotoxic effects were observed; however, this is most likely a result of the poor cell permeability of all evaluated compounds.

Understanding molecular self-association in solution is vital for uncovering polymorph-selective crystal nucleation pathways. In this paper, we combine solution NMR spectroscopy and molecular dynamics simulations to shed light on the structural and dynamical features of p-aminobenzoic acid (pABA) in solution, and on their role in pABA crystals nucleation. pABA is known to yield different crystal forms (α, and β) depending on solvent choice and supersaturation conditions. NMR reveals that dominant interactions stabilizing pABA oligomers are markedly solvent-dependent: in organic solvents, hydrogen bonds dominate, while water promotes π-πstacking. Despite this clear preference, both types of interactions contribute to the variety of self-associated species in all solvents considered. MD simulations support this observation and show that pABA oligomers are short-lived and display a fluxional character, therefore indicating that the growth unit involved in pABA crystallization is likely to be a single molecule. Nevertheless, we note that the interactions dominating in pABA oligomers are indicative of the polymorph obtained from precipitation. In water, at low pABA concentrations - conditions that are known to yield crystals of the β form - carboxylic-carboxylic hydrogen bonds are exclusively asymmetric. At higher pABA concentration conditions in which the crystallization is known to yield the α form - a small but statistically significant fraction of symmetric carboxylic-carboxylic hydrogen-bonded dimers is present. We interpret the presence of these interactions in solvated pABA oligomers as indicative of the fact that a simultaneous and complete desolvation of two carboxylic groups, necessary to form the symmetric hydrogen-bonded dimer typical of the α crystal form, is accessible, therefore directing the nucleation pathway toward the nucleation of α-pABA.

Malarial aspartic protease, plasmepsin (Plm), is perspective antimalarial drug target. Following up our previosly identified 2-aminoquinazolin-4(3H)-one-based inhibitors, 2-aminopyrido[2,3-d]-, -[3,2-d]-, and -[4,3-d]pyrimidin-4(3H)-ones bearing subpocket-specific substituents at position 7 were prepared and tested for their Plm I, II, IV inhibition potency. The position of the nitrogen atom in 2-aminopyridopyrimidinones plays significant role in the inhibitory potency against Plms. Pyrido[2,3-d]pyrimidin-4(3H)-one derivatives showed poor inhibitory potency against Plms I, II, IV irrespectively of the substituent at position 7. However, pyrido[4,3-d]pyrimidin-4(3H)-ones and pyrido[3,2-d]pyrimidin-4(3H)-ones were more appropriate scaffolds for Plm inhibitor development. Particularly, 2-amino-7-[4-(3-phenylpropyl)phenyl]-3-[(tetrahydrofuran-2-yl)methyl]pyrido[3,2-d]pyrimidin-4(3H)-one showed very high potency against Plm IV subtype and high selectivity against human aspartic protease, cathepsin D.

The N-terminal (NT) domain of spider silk proteins (spidroins) is crucial for their storage at high concentrations and also regulates silk assembly. NTs from the major ampullate spidroin (MaSp) and the minor ampullate spidroin are monomeric at neutral pH and confer solubility to spidroins, whereas at lower pH, they dimerize to interconnect spidroins in a fiber. This dimerization is known to result from modulation of electrostatic interactions by protonation of well-conserved glutamates, although it is undetermined if this mechanism applies to other spidroin types as well. Here, we determine the solution and crystal structures of the flagelliform spidroin NT, which shares only 35% identity with MaSp NT, and investigate the mechanisms of its dimerization. We show that flagelliform spidroin NT is structurally similar to MaSp NT and that the electrostatic intermolecular interaction between Asp 40 and Lys 65 residues is conserved. However, the protonation events involve a different set of residues than in MaSp, indicating that an overall mechanism of pH-dependent dimerization is conserved but can be mediated by different pathways in different silk types.

S100A9 is a pro-inflammatory protein that co-aggregates with other proteins in amyloid fibril plaques. S100A9 can influence the aggregation kinetics and amyloid fibril structure of alpha-synuclein (α-syn), which is involved in Parkinson’s disease. Currently, there are limited data regarding their cross-interaction and how it influences the aggregation process. In this work, we analyzed this interaction using solution 19F and 2D 15N–1H HSQC NMR spectroscopy and studied the aggregation properties of these two proteins. Here, we show that α-syn interacts with S100A9 at specific regions, which are also essential in the first step of aggregation. We also demonstrate that the 4-fluorophenylalanine label in alpha-synuclein is a sensitive probe to study interaction and aggregation using 19F NMR spectroscopy.

Spider silk is the toughest fiber found in nature, and bulk production of artificial spider silk that matches its mechanical properties remains elusive. Development of miniature spider silk proteins (mini-spidroins) has made large-scale fiber production economically feasible, but the fibers' mechanical properties are inferior to native silk. The spider silk fiber's tensile strength is conferred by poly-alanine stretches that are zipped together by tight side chain packing in β-sheet crystals. Spidroins are secreted so they must be void of long stretches of hydrophobic residues, since such segments get inserted into the endoplasmic reticulum membrane. At the same time, hydrophobic residues have high β-strand propensity and can mediate tight inter-β-sheet interactions, features that are attractive for generation of strong artificial silks. Protein production in prokaryotes can circumvent biological laws that spiders, being eukaryotic organisms, must obey, and the authors thus design mini-spidroins that are predicted to more avidly form stronger β-sheets than the wildtype protein. Biomimetic spinning of the engineered mini-spidroins indeed results in fibers with increased tensile strength and two fiber types display toughness equal to native dragline silks. Bioreactor expression and purification result in a protein yield of ≈9 g L

Significance Amyloid-β (Aβ) is the subject of intense scrutiny because of its close association with Alzheimer’s disease (AD), which currently afflicts about 50 million people worldwide. The results reported in this manuscript focus on the new possibilities provided by ultrafast magic-angle spinning (MAS) 1H detection and fast-MAS dynamic nuclear polarization (DNP), which have ushered in a new era for NMR-based structural biology, but whose potential has not yet been fully exploited for the structural investigation of complex amyloid assemblies. This work demonstrates the expeditious structural analysis of amyloid fibrils, without requiring preparation of large sample amounts, and sets the stage for future studies of unlabeled AD peptides derived from tissue samples available in limited quantities., Several publications describing high-resolution structures of amyloid-β (Aβ) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments—namely, 1H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on 13C,15N-enriched, and fully protonated samples of M0Aβ1-42 fibrils by high-field 1H-detected NMR at 23.4 T and 18.8 T, and 13C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M0Aβ1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than 1H detection. These results suggest that 1H detection and DNP may be the spectroscopic approaches of choice for future studies of Aβ and other amyloid systems.

Malaria is a life-threatening infectious disease caused by Plasmodium parasites. Plasmepsins (proteolytic enzymes of the parasite) have been considered as promising targets for the development of antimalarial drugs. To date, much knowledge has been obtained regarding the interactions of inhibitors with plasmepsins, as well as the structure–activity relationships of the inhibitors. The discovery and characterization of the plasmepsin inhibitors that bind in open flap conformation have led to several inhibitor classes that show high selectivity over other human aspartic proteases. This Perspective addresses the flexibility of the plasmepsins that leads to inhibitor binding to the open flap conformation, summarizes known nonpeptidomimetic plasmepsin inhibitors, and discusses the role of the inhibitor flap pocket substituent.

Dynamic Nuclear Polarization (DNP) is an effective approach to alleviate the inherently low sensitivity of solid-state NMR (ssNMR) under magic angle spinning (MAS) towards large-sized multi-domain complexes and assemblies. DNP relies on a polarization transfer at cryogenic temperatures from unpaired electrons to adjacent nuclei upon continuous microwave irradiation. This is usually made possible via the addition in the sample of a polarizing agent. The first pioneering experiments on biomolecular assemblies were reported in the early 2000s on bacteriophages and membrane proteins. Since then, DNP has experienced tremendous advances, with the development of extremely efficient polarizing agents or with the introduction of new microwaves sources, suitable for NMR experiments at very high magnetic fields (currently up to 900 MHz). After a brief introduction, several experimental aspects of DNP enhanced NMR spectroscopy applied to biomolecular assemblies are discussed. Recent demonstration experiments of the method on viral capsids, the type III and IV bacterial secretion systems, ribosome and membrane proteins are then described.

Prion diseases are associated with conformational conversion of cellular prion protein into a misfolded pathogenic form, which resembles many properties of amyloid fibrils. The same prion protein sequence can misfold into different conformations, which are responsible for variations in prion disease phenotypes (prion strains). In this work, we use atomic force microscopy, FTIR spectroscopy and magic-angle spinning NMR to devise structural models of mouse prion protein fibrils prepared in three different denaturing conditions. We find that the fibril core region as well as the structure of its N- and C-terminal parts is almost identical between the three fibrils. In contrast, the central part differs in length of β-strands and the arrangement of charged residues. We propose that the denaturant ionic strength plays a major role in determining the structure of fibrils obtained in a particular condition by stabilizing fibril core interior-facing glutamic acid residues.

The stereoselective total synthesis of the proposed structure of a potent serotonin 5-HT1A receptor agonist uncarialin A (1) is described. By employing the readily available meroquinene tert-butyl ester as the chiral synthon, the target structure has been prepared in a six-step linear sequence with a 17% overall yield. In comparison to the sample isolated from natural sources, the synthetic product shows significant spectral differences, strongly suggesting that the structure of the natural product should be revised.

A nuclear magnetic resonance (NMR) spectrometer is a key instrument in the organic synthesis laboratory for structure determination, reaction control, and compound purity analysis. In addition to qualitative analysis, the application of NMR for quantitative analysis (qNMR) is gaining popularity. qNMR allows for simple quantification of crude product mixtures, determination of reaction yields, and purity of organic compounds. The determination of NMR yield requires the addition of an internal standard to each sample. Herein, we report a method where CDCl

The formation of amyloid fibril plaques in the brain creates inflammation and neuron death. This process is observed in neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases. Alpha-synuclein is the main protein found in neuronal inclusions of patients who have suffered from Parkinson’s disease. S100A9 is a calcium-binding, pro-inflammation protein, which is also found in such amyloid plaques. To understand the influence of S100A9 on the aggregation of α-synuclein, we analyzed their co-aggregation kinetics and the resulting amyloid fibril structure by Fourier-transform infrared spectroscopy and atomic force microscopy. We found that the presence of S100A9 alters the aggregation kinetics of α-synuclein and stabilizes the formation of a particular amyloid fibril structure. We also show that the solution’s ionic strength influences the interplay between S100A9 and α-synuclein, stabilizing a different structure of α-synuclein fibrils.

Pre-nucleation aggregates are important species on the crystallisation pathway. Here we combine heteronuclear Overhauser effect spectroscopy (HOESY) and molecular dynamics calculations to study the self-association of a model system – benzoic acid. Our findings indicate that heteronuclear Overhauser effects arise from diffusion-limited pre-nucleation aggregates and that self-association is solvent dependant.

Structure determination of adjuvant-coupled antigens is essential for rational vaccine development but has so far been hampered by the relatively low antigen content in vaccine formulations and by their heterogeneous composition. Here we show that magic-angle spinning (MAS) solid-state NMR can be used to assess the structure of the influenza virus hemagglutinin stalk long alpha helix antigen, both in its free, unformulated form and once chemically coupled to the surface of large virus-like particles (VLPs). The sensitivity boost provided by high-field dynamic nuclear polarization (DNP) and proton detection at fast MAS rates allows to overcome the penalty associated with the antigen dilution. Comparison of the MAS NMR fingerprints between the free and VLP-coupled forms of the antigen provides structural evidence of the conservation of its native fold upon bioconjugation. This work demonstrates that high-sensitivity MAS NMR is ripe to play a major role in vaccine design, formulation studies, and manufacturing process development.

Understanding molecular self-association in solution is vital for uncovering polymorph- selective crystal nucleation pathways. In this paper, we combine solution NMR spectroscopy and molecular dynamics simulations to shed light on the structural and dynamical features of para-aminobenzoic acid (pABA) in solution, and on their role in pABA crystals nucleation. pABA is known to yield different crystal forms (α, and β) depending on solvent choice and su- persaturation conditions. NMR reveals that dominant interactions stabilising pABA oligomers are markedly solvent-dependent: in organic solvents, hydrogen bonds dominate, while water promotes π - π stacking. Despite this clear preference, both types of interactions contribute to the variety of self-associated species in all solvents considered. MD simulations support this observation and show that pABA oligomers are short-lived and display a fluxional character, therefore indicating that the growth unit involved in pABA crystallisation is likely to be a single molecule. Nevertheless, we note that the interactions dominating in pABA oligomers are indicative of the polymorph obtained from precipitation. In water, at low pABA concen- trations - conditions that are known to yield crystals of the β form - carboxylic-carboxylic hydrogen bonds are exclusively asymmetric. At higher pABA concentration conditions in which the crystallisation is known to yield the α form - a small but statistically significant fraction of symmetric carboxylic-carboxylic hydrogen-bonded dimers is present. We interpret the presence of these interactions in solvated pABA oligomers as indicative of the fact that a simultaneous and complete desolvation of two carboxylic groups, necessary to form the sym- metric hydrogen-bonded dimer typical of the α crystal form, is accessible, therefore directing the nucleation pathway towards the nucleation of α-pABA.

Lyme disease is the most widespread vector‐transmitted disease in North America and Europe, caused by infection with Borrelia burgdorferi sensu lato complex spirochetes. We report the solution NMR structure of the B. burgdorferi outer surface lipoprotein BBP28, a member of the multicopy lipoprotein (mlp) family. The structure comprises a tether peptide, five α‐helices and an extended C‐terminal loop. The fold is similar to that of Borrelia tunicate outer surface protein BTA121, which is known to bind lipids. These results contribute to the understanding of Lyme disease pathogenesis by revealing the molecular structure of a protein from the widely found mlp family. This article is protected by copyright. All rights reserved.

Amyloidogenesis is associated with more than 30 diseases, but the molecular mechanisms involved in cell toxicity and fibril formation remain largely unknown. The inherent tendency of amyloid-forming proteins to aggregate renders expression, purification, and experimental studies challenging. NT* is a solubility tag derived from a spider silk protein that was recently introduced for the production of several aggregation-prone peptides and proteins at high yields. Herein, we investigate whether fusion to NT* can prevent amyloid fibril formation and enable controlled aggregation for experimental studies. As an example of an amyloidogenic protein, we chose the de novo-designed polypeptide β17. The fusion protein NT*-β17 was recombinantly expressed in Escherichia coli to produce high amounts of soluble and mostly monomeric protein. Structural analysis showed that β17 is kept in a largely unstructured conformation in fusion with NT*. After proteolytic release, β17 adopts a β-sheet conformation in a pH- and salt-dependent manner and assembles into amyloid-like fibrils. The ability of NT* to prevent premature aggregation and to enable structural studies of prefibrillar states may facilitate investigation of proteins involved in amyloid diseases.