The 50 most-cited articles in Biochemistry, 2024

High Resolution Image Download MS PowerPoint Slide Polyethylene glycol (PEG) is a flexible, nontoxic polymer commonly used in biological and medical research, and it is generally regarded as biologically inert. PEG molecules of variable sizes are also used as crowding agents to mimic intracellular environments. A recent study with PEG crowders revealed decreased catalytic activity of Escherichia coli prolyl-tRNA synthetase (Ec ProRS), where the smaller molecular weight PEGs had the maximum impact. The molecular mechanism of the crowding effects of PEGs is not clearly understood. PEG may impact protein conformation and dynamics, thus its function. In the present study, the effects of PEG molecules of various molecular weights and concentrations on the conformation and dynamics of Ec ProRS were investigated using a combined experimental and computational approach including intrinsic tryptophan fluorescence spectroscopy, atomic force microscopy, and atomistic molecular dynamic simulations. Results of the present study suggest that lower molecular weight PEGs in the dilute regime have…

Pollution by plastics such as polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyurethane (PUR), polyamide (PA), polystyrene (PS), and poly(ethylene terephthalate) (PET) is now gaining worldwide attention as a critical environmental issue, closely linked to climate change. Among them, PET is particularly prone to hydrolysis, breaking down into its constituents, ethylene glycol (EG) and terephthalate (TPA). Biorecycling or bioupcycling stands out as one of the most promising methods for addressing PET pollution. For dealing with pollution by the macrosize PET, a French company Carbios has developed a pilot-scale plant for biorecycling waste PET beverage bottles into new bottles using derivatives of thermophilic leaf compost cutinase (LCC). However, this system still provides significant challenges in its practical implementation. For the micro- or nanosize PET pollution that poses significant human health risks, including cancer, no industrial-scale approach has been established so far, despite the need to develop such technologies. In this Perspective, we explore…

High Resolution Image Download MS PowerPoint Slide A central goal of photoprotective energy dissipation processes is the regulation of singlet oxygen ( 1 O 2 *) and reactive oxygen species in the photosynthetic apparatus. Despite the involvement of 1 O 2 * in photodamage and cell signaling, few studies directly correlate 1 O 2 * formation to nonphotochemical quenching (NPQ) or lack thereof. Here, we combine spin-trapping electron paramagnetic resonance (EPR) and time-resolved fluorescence spectroscopies to track in real time the involvement of 1 O 2 * during photoprotection in plant thylakoid membranes. The EPR spin-trapping method for detection of 1 O 2 * was first optimized for photosensitization in dye-based chemical systems and then used to establish methods for monitoring the temporal dynamics of 1 O 2 * in chlorophyll-containing photosynthetic membranes. We find that the apparent 1 O 2 * concentration in membranes changes throughout a 1 h period of continuous illumination. During an initial response to high light intensity, the concentration of 1 O 2 * decreased in parallel…

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by a macrolactam linkage between the N-terminus and the side chain of an internal aspartic acid or glutamic acid residue. Instead of adopting a branched-cyclic conformation, lasso peptides are "threaded", with the C-terminal tail passing through the macrocycle to present a kinetically trapped rotaxane conformation. The availability of enhanced bioinformatics methods has led to a significant increase in the number of secondary modifications found on lasso peptides. To uncover new ancillary modifications in a targeted manner, a bioinformatic strategy was developed to discover lasso peptides with modifications to tryptophan. This effort identified numerous putative lasso peptide biosynthetic gene clusters with core regions of the precursor peptides enriched in tryptophan. Parsing of these tryptophan (Trp)-rich biosynthetic gene clusters uncovered several putative ancillary modifying enzymes, including halogenases and dimethylallyltransferases expected to act upon Trp.…

Berberine bridge enzyme-like oxidases are often involved in natural product biosynthesis and are seen as essential enzymes for the generation of intricate pharmacophores. These oxidases have the ability to transfer a hydride atom to the FAD cofactor, which enables complex substrate modifications and rearrangements including (intramolecular) cyclizations, carbon-carbon bond formations, and nucleophilic additions. Despite the diverse range of activities, the mechanistic details of these reactions often remain incompletely understood. In this Review, we delve into the complexity that BBE-like oxidases from bacteria, fungal, and plant origins exhibit by providing an overview of the shared catalytic features and emphasizing the different reactivities. We propose four generalized modes of action by which BBE-like oxidases enable the synthesis of natural products, ranging from the classic alcohol oxidation reactions to less common amine and amide oxidation reactions. Exploring the mechanisms utilized by nature to produce its vast array of natural products is a subject of considerable…

High Resolution Image Download MS PowerPoint Slide Ferroptosis is a recently identified form of regulated cell death, characterized by excessive iron-dependent lipid peroxidation. Recent studies have demonstrated that protein disulfide isomerase (PDI) is an important mediator of chemically induced ferroptosis and also a new target for protection against ferroptosis-associated cell death. In the present study, we identified that 4-hydroxyestrone (4-OH-E 1 ), a metabolic derivative of endogenous estrogen, is a potent small-molecule inhibitor of PDI, and can strongly protect against chemically induced ferroptotic cell death in the estrogen receptor-negative MDA-MB-231 human breast cancer cells. Pull-down and CETSA assays demonstrated that 4-OH-E 1 can directly bind to PDI both in vitro and in intact cells. Computational modeling analysis revealed that 4-OH-E 1 forms two hydrogen bonds with PDI His256, which is essential for its binding interaction and thus inhibition of PDI’s catalytic activity. Additionally, PDI knockdown attenuates the protective effect of 4-OH-E 1 as well as…

Siderophores are metal-binding secondary metabolites that assist in iron homeostasis and have been of interest to the scientific community for the last half century. Foundational siderophore research has enabled several translational applications including siderophore-antibiotic and siderophore-peptide conjugates, identification of new antimicrobial targets, advances in disease imaging, and novel therapeutics. This review aims to connect the basic science research (biosynthesis, cellular uptake, gene regulation, and effects on homeostasis) of well-known siderophores with the successive translational application that results. Intertwined throughout are connections to the career of Christopher T. Walsh, his impact on the field of chemical biology, and the legacy of his trainees who continue to innovate.

Glycogen synthase kinase 3 (GSK3) plays a pivotal role in signaling pathways involved in insulin metabolism and the pathogenesis of neurodegenerative disorders. In particular, the GSK3β isoform is implicated in Alzheimer's disease (AD) as one of the key kinases involved in the hyperphosphorylation of tau protein, one of the neuropathological hallmarks of AD. As a constitutively active serine/threonine kinase, GSK3 is inactivated by Akt/PKB-mediated phosphorylation of Ser9 in the N-terminal disordered domain, and for most of its substrates, requires priming (prephosphorylation) by another kinase that targets the substrate to a phosphate-specific pocket near the active site. GSK3 has also been shown to be post-translationally modified by O-linked β-N-acetylglucosaminylation (O-GlcNAcylation), with still unknown functions. Here, we have found that binding of Akt inhibits GSK3β kinase activity on both primed and unprimed tau substrates. Akt-mediated Ser9 phosphorylation restores the GSK3β kinase activity only on primed tau, thereby selectively inactivating GSK3β toward unprimed tau…

The class A orphan G protein-coupled receptor (GPCR), GPR3, has been implicated in a variety of conditions, including Alzheimer’s and premature ovarian failure. GPR3 constitutively couples with Gαs, resulting in the production of cAMP in cells. While tool compounds and several putative endogenous ligands have emerged for the receptor, its endogenous ligand, if it exists, remains a mystery. As novel potential drug targets, the structures of orphan GPCRs have been of increasing interest, revealing distinct modes of activation, including autoactivation, presence of constitutively activating mutations, or via cryptic ligands. Here, we present a cryo-electron microscopy (cryo-EM) structure of the orphan GPCR, GPR3 in complex with DNGαs and Gβ 1 γ 2 . The structure revealed clear density for a lipid-like ligand that bound within an extended hydrophobic groove, suggesting that the observed “constitutive activity” was likely due to activation via a lipid that may be ubiquitously present. Analysis of conformational variance within the cryo-EM data set revealed twisting motions of the GPR3…

ABSTRACT Production of soluble proteins is essential for structure/function studies, however, this usually requires milligram amounts of protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacterium Vibrio natriegens appeared as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived from V. natriegens (Vmax TM X2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve in Escherichia coli . These proteins include the cholera toxin (CT) and N -acetyl glucosamine binding protein A (GbpA) from Vibrio cholerae , the heat-labile enterotoxin (LT) from E. coli and the fungal nematotoxin CCTX2 from Coprinopsis cinerea . CT, GbpA and LT are secreted by the Type II secretion system in their natural hosts. When these three proteins were produced in Vmax, they were also secreted, and could be recovered from the growth media. This simplified the downstream purification procedure and resulted in considerably higher protein yields…

Global substitution of leucine for analogues containing CH 2 F instead of methyl groups delivers proteins with multiple sites for monitoring by 19 F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa Escherichia coli peptidyl–prolyl cis–trans isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2 S,4 S )-5-fluoroleucine, (2 S,4 R )-5-fluoroleucine, or 5,5′-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. 19 F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The 19 F chemical shifts correlate with the three-bond 1 H– 19 F couplings ( 3 J HF ), providing the first experimental verification of the γ-gauche effect predicted by [ Feeney, J. J. Am. Chem. Soc. 1996, 118, 8700–8706] and establishing the effect as the predominant determinant of the 19 F chemical shifts of CH 2 F groups. Individual CH 2 F groups can be confined to single rotameric states by the protein environment, but most CH 2 F groups exchange between…

-adenosyl-l-methionine (SAM) enzymes catalyze radical mediated chemical transformations notable for their diversity. The radical mediated reactions that take place in their catalytic cycles can be characterized with respect to one or more phases of initiation, propagation, and termination. Mechanistic models abound regarding these three phases of catalysis being regularly informed and updated by new discoveries that offer insights into their detailed workings. However, questions continue to be raised that touch on fundamental aspects of their mechanistic enzymology. Radical SAM enzymes are consequently far from fully understood, and this Perspective aims to outline some of the current models of radical SAM chemistry with an emphasis on lines of investigation that remain to be explored.

Telomeres are specialized structures, found at the ends of linear chromosomes in eukaryotic cells, that play a crucial role in maintaining the stability and integrity of genomes. They are composed of repetitive DNA sequences, ssDNA overhangs, and several associated proteins. The length of telomeres is linked to cellular aging in humans, and deficiencies in their maintenance are associated with various diseases. Key structural motifs at the telomeres serve to protect vulnerable chromosomal ends. Telomeric DNA also has the ability to form diverse complex DNA higher-order structures, including T-loops, D-loops, R-loops, G-loops, G-quadruplexes, and i-motifs, in the complementary C-rich strand. While many essential proteins at telomeres have been identified, the intricacies of their interactions and structural details are still not fully understood. This Perspective highlights recent advancements in comprehending the structures associated with human telomeres. It emphasizes the significance of telomeres, explores various telomeric structural motifs, and delves into the structural…

Inositol pyrophosphates (PP-InsPs) are eukaryote-specific second messengers that regulate diverse cellular processes, including immunity, nutrient sensing, and hormone signaling pathways in plants. These energy-rich messengers exhibit high sensitivity to the cellular phosphate status, suggesting that the synthesis and degradation of PP-InsPs are tightly controlled within the cells. Notably, the molecular basis of PP-InsP hydrolysis in plants remains largely unexplored. In this study, we report the functional characterization of MpDDP1, a diadenosine and diphosphoinositol polyphosphate phosphohydrolase encoded by the genome of the liverwort, Marchantia polymorpha . We show that MpDDP1 functions as a PP-InsP phosphohydrolase in different heterologous organisms. Consistent with this finding, M. polymorpha plants defective in MpDDP1 exhibit elevated levels of 1/3-InsP 7 and 1/3,5-InsP 8, highlighting the contribution of MpDDP1 in regulating PP-InsP homeostasis in planta . Furthermore, our study reveals that MpDDP1 controls thallus development and vegetative reproduction in M. polymorpha…

carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.

Proteins produced with leucine analogues, where CH 2 F groups substitute specific methyl groups, can readily be probed by 19 F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH 2 F groups is unclear. We present high-resolution crystal structures of Escherichia coli peptidyl-prolyl cis – trans isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2 S,4 S )-5-fluoroleucine, (2 S,4 R )-5-fluoroleucine, or 5,5′-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH 2 F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of…

High Resolution Image Download MS PowerPoint Slide Vital to the treatment of influenza is the use of antivirals such as Oseltamivir (Tamiflu) and Zanamivir (Relenza); however, antiviral resistance is becoming an increasing problem for these therapeutics. The RNA-dependent RNA polymerase acidic N-terminal (PA N ) endonuclease, a critical component of influenza viral replication machinery, is an antiviral target that was recently validated with the approval of Baloxavir Marboxil (BXM). Despite its clinical success, BXM has demonstrated susceptibility to resistance mutations, specifically the I38T, E23K, and A36 V mutants of PA N . To better understand the effects of these mutations on BXM resistance and improve the design of more robust therapeutics, this study examines key differences in protein–inhibitor interactions with two inhibitors and the I38T, E23K, and A36 V mutants. Differences in inhibitor binding were evaluated by measuring changes in binding to PA N using two biophysical methods. The binding mode of two distinct inhibitors was determined crystallographically with both…

Per and polyfluoroalkyl substances (PFAS) are a large family of anthropogenic fluorinated chemicals of increasing environmental concern. Over recent years, numerous microbial communities have been found to be capable of metabolizing some polyfluoroalkyl substances, generating a range of low-molecular-weight PFAS metabolites. One proposed pathway for the microbial breakdown of fluorinated carboxylates includes β-oxidation, this pathway is initiated by the formation of a CoA adduct. However, until recently no PFAS-CoA adducts had been reported. In a previous study, we were able to use a bacterial medium-chain acyl-CoA synthetase (mACS) to form CoA adducts of fluorinated adducts of propanoic acid and pentanoic acid but were not able to detect any products of fluorinated hexanoic acid analogues. Herein, we expressed and purified a long-chain acyl-CoA synthetase (lACS) and a A461K variant of mACS from the soil bacterium Gordonia sp. strain NB4–1Y and performed an analysis of substrate scope and enzyme kinetics using fluorinated and nonfluorinated carboxylates. We determined that lACS can…

Despite the importance of proline conformational equilibria (trans versus cis amide, exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and of cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0–3 ppm) when a trans X–Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5–12 ppm) difference in chemical shift in a cis X–Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that the ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e. the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e. pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a…

The identification of chemical starting points for the development of molecular glues is challenging. Here, we employed fragment screening and identified an allosteric stabilizer of the complex between 14-3-3 and a TAZ-derived peptide. The fragment binds preferentially to the 14-3-3/TAZ peptide complex and shows moderate stabilization in differential scanning fluorimetry and microscale thermophoresis. The binding site of the fragment was predicted by molecular dynamics calculations to be distant from the 14-3-3/TAZ peptide interface, located between helices 8 and 9 of the 14-3-3 protein. This site was confirmed by nuclear magnetic resonance and X-ray protein crystallography, revealing the first example of an allosteric stabilizer for 14-3-3 protein-protein interactions.

/bicarbonate from 2OG or couples the decarboxylation of 2OG to C5 hydroxylation of l-Arg. l-Arg binds with C5 toward the metal center, causing 2OG to change from monodentate to chelate metal interaction and OD1 to OD2 switch of D191 metal coordination. We applied anaerobic UV-visible spectroscopy, X-ray crystallography, and computational approaches to three EFE systems with high-resolution structures. The ineffective l-Arg analogue l-canavanine binds to the EFE with O5 pointing away from the metal center while promoting chelate formation by 2OG but fails to switch the D191 metal coordination from OD1 to OD2. Substituting alanine for R171 that interacts with 2OG and l-Arg inactivates the protein, prevents metal chelation by 2OG, and weakens l-Arg binding. The R171A EFE had electron density at the 2OG binding site that was identified by mass spectrometry as benzoic acid. The substitution by alanine of Y306 in the EFE, a residue 12 Å away from the catalytic metal center, generates an interior cavity that leads to multiple local and distal structural changes that reduce l-Arg binding…

Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for the mechanisms of their biological functions as well as their potential as therapeutic targets.

High Resolution Image Download MS PowerPoint Slide Class Ia ribonucleotide reductases (RNRs) are allosterically regulated by ATP and dATP to maintain the appropriate deoxyribonucleotide levels inside the cell for DNA biosynthesis and repair. RNR activity requires precise positioning of the β 2 and α 2 subunits for the transfer of a catalytically essential radical species. Excess dATP inhibits RNR through the creation of an α–β interface that restricts the ability of β 2 to obtain a position that is capable of radical transfer. ATP breaks the α–β interface, freeing β 2 and restoring enzyme activity. Here, we investigate the molecular basis for allosteric activity regulation in the well-studied Escherichia coli class Ia RNR through the determination of six crystal structures and accompanying biochemical and mutagenesis studies. We find that when dATP is bound to the N-terminal regulatory cone domain in α, a helix unwinds, creating a binding surface for β. When ATP displaces dATP, the helix rewinds, dismantling the α–β interface. This reversal of enzyme inhibition requires that two ATP…

Optimization of aptamers in length and chemistry is crucial for industrial applications. Here, we developed aptamers against the SARS-CoV-2 spike protein and achieved optimization with a deep-learning-based algorithm, RaptGen. We conducted a primer-less SELEX against the receptor binding domain (RBD) of the spike with an RNA/DNA hybrid library, and the resulting sequences were subjected to RaptGen analysis. Based on the sequence profiling by RaptGen, a short truncation aptamer of 26 nucleotides was obtained and further optimized by a chemical modification of relevant nucleotides. The resulting aptamer is bound to RBD not only of SARS-CoV-2 wildtype but also of its variants, SARS-CoV-1, and Middle East respiratory syndrome coronavirus (MERS-CoV). We concluded that the RaptGen-assisted discovery is efficient for developing optimized aptamers.

The 13 Hsp70 proteins in humans act on unique sets of substrates with diversity often being attributed to J-domain-containing protein (Hsp40 or JDP) cofactors. We were therefore surprised to find drastically different binding affinities for Hsp70-peptide substrates, leading us to probe substrate specificity among the 8 canonical Hsp70s from humans. We used peptide arrays to characterize Hsp70 binding and then mined these data using machine learning to develop an algorithm for isoform-specific prediction of Hsp70 binding sequences. The results of this algorithm revealed recognition patterns not predicted based on local sequence alignments. We then showed that none of the human isoforms can complement heat-shocked DnaK knockout Escherichia coli cells. However, chimeric Hsp70s consisting of the human nucleotide-binding domain and the substrate-binding domain of DnaK complement during heat shock, providing further evidence in vivo of the divergent function of the Hsp70 substrate-binding domains. We also demonstrated that the differences in heat shock complementation among the chimeras…

It is now well-established that microbial genomes carry sparingly expressed biosynthetic gene clusters (BGCs) that need to be induced in order to characterize their products. To do so, we herein subjected two well-known producers, Burkholderia plantarii and Burkholderia gladioli, to high-throughput elicitor screening (HiTES), an emerging approach for accessing the products of these “cryptic” BGCs. Both organisms have previously been examined extensively in liquid cultures. We therefore applied HiTES on agar and found several novel natural products that are only produced in this format and not in liquid cultures. Most notably we found two metabolites, termed burkethyl A and B, that contain an unusual m -ethylbenzoyl group and for which we identified the cognate BGC using bioinformatic and genetic studies. Our results indicate that agar-based HiTES is a promising approach for natural product discovery and are in line with the notion that even “drained” strains remain sources of new metabolites as long as alternative approaches are employed.

As a traceless, bioreversible modification, the esterification of carboxyl groups in peptides and proteins has the potential to increase their clinical utility. An impediment is the lack of strategies to quantify esterase-catalyzed hydrolysis rates for esters in esterified biologics. We have developed a continuous Förster resonance energy transfer (FRET) assay for esterase activity based on a peptidic substrate and a protease, Glu-C, that cleaves a glutamyl peptide bond only if the glutamyl side chain is a free acid. Using pig liver esterase (PLE) and human carboxylesterases, we validated the assay with substrates containing simple esters ( e.g., ethyl) and esters designed to be released by self-immolation upon quinone methide elimination. We found that simple esters were not cleaved by esterases, likely for steric reasons. To account for the relatively low rate of quinone methide elimination, we extended the mathematics of the traditional Michaelis–Menten model to conclude with a first-order intermediate decay step. By exploring two regimes of our substrate → intermediate → product…

Active site lids are common features of enzymes and typically undergo conformational changes upon substrate binding to promote catalysis. Iodotyrosine deiodinase is no exception and contains a lid segment in all of its homologues from human to bacteria. The solution-state dynamics of the lid have now been characterized using 19 F NMR spectroscopy with a CF 3 -labeled enzyme and CF 3 O-labeled ligands. From two-dimensional 19 F– 19 F NMR exchange spectroscopy, interconversion rates between the free and bound states of a CF 3 O-substituted tyrosine (45 ± 10 s –1 ) and the protein label (40 ± 3 s –1 ) are very similar and suggest a correlation between ligand binding and conformational reorganization of the lid. Both occur at rates that are ∼100-fold faster than turnover, and therefore these steps do not limit catalysis. A simple CF 3 O-labeled phenol also binds to the active site and induces a conformational change in the lid segment that was not previously detectable by crystallography. Exchange rates of the ligand (130 ± 20 s –1 ) and protein (98 ± 8 s –1 ) in this example are faster…

High Resolution Image Download MS PowerPoint Slide DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including Mycobacterium tuberculosis, which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (A 2 B 2 ) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of M. tuberculosis GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other…

ABSTRACT The Ras GTPase-activating protein SH3-domain-binding protein 1 (G3BP1) serves as a formidable barrier to viral replication by generating stress granules (SGs) in response to viral infections. Interestingly, viruses, including SARS-CoV-2, have evolved defensive mechanisms to hijack SG proteins like G3BP1 for the dissipation of SGs that lead to the evasion of host’s immune responses. Previous research has demonstrated that the interaction between the NTF2-like domain of G3BP1 (G3BP1 NTF-2 ) and the intrinsically disordered N-terminal domain (NTD-N 1-25 ) of the N protein plays a crucial role in regulating viral replication and pathogenicity. Interestingly, the current study identified an additional upstream stretch of residues (128KDGIIWVATEG138) (N 128-138 ) within the N-terminal domain of the N protein (NTD-N 41-174 ) that also forms molecular contacts with the G3BP1 protein, as revealed through in silico analysis, site-directed mutagenesis and biochemical analysis. Remarkably, WIN-62577, and fluspirilene, the small molecules targeting the conserved peptide binding pocket…

19 F NMR spectroscopy has recently witnessed a resurgence as an attractive analytical tool for the study of the structure and dynamics of biomolecules in vitro and in cells, despite reports of its applications in biomolecular NMR since the 1970s. The high gyromagnetic ratio, large chemical shift dispersion, and complete absence of the spin 1/2 19 F nucleus from biomolecules results in background-free, high-resolution 19 F NMR spectra. The introduction of 19 F probes in a few selected locations in biomolecules reduces spectral crowding despite its increased line width in comparison to typical 1 H NMR line widths and allows rapid site-specific measurements from simple 1D spectra alone. The design and synthesis of novel 19 F probes with reduced line widths and increased chemical shift sensitivity to the surrounding environment, together with advances in labeling techniques, NMR methodology, and hardware, have overcome several drawbacks of 19 F NMR spectroscopy. The increased interest and widespread use of 19 F NMR spectroscopy of biomolecules is gradually establishing it as a sensitive…

(2 S,3 S )-4-Fluorovaline (FVal) is an analogue of valine, where a single CH 3 group is substituted by a CH 2 F group. In the absence of valine, E. coli valyl-tRNA synthetase uses FVal as a substitute, enabling the production of proteins uniformly labeled with FVal. Here, we describe the production and analysis of E. coli peptidyl–prolyl isomerase B where all 16 valine residues have been replaced by FVal synthesized with a 13 C-labeled CH 2 F group. Although the melting temperature is lower by about 11 °C relative to the wild-type protein, the three-dimensional protein structure is almost completely conserved, as shown by X-ray crystallography. The CH 2 F groups invariably populate staggered rotamers. Most CH 2 F groups populate two different rotamers. The increased space requirement of fluorine versus hydrogen does not prohibit rotamers that position fluorine next to a backbone carbonyl carbon. 19 F NMR spectra show a signal dispersion over 25 ppm. The most high-field shifted 19 F resonances correlate with large 3 J HF coupling constants, confirming the impact of the γ- gauche…

Cholesterol is central to mammalian lipid metabolism and serves many critical functions in the regulation of diverse physiological processes. Dysregulation in cholesterol metabolism is causally linked to numerous human diseases, and therefore, in vivo, the concentrations and flux of cholesterol and cholesteryl esters (fatty acid esters of cholesterol) are tightly regulated. While mass spectrometry has been an analytical method of choice for detecting cholesterol and cholesteryl esters in biological samples, the hydrophobicity, chemically inert nature, and poor ionization of these neutral lipids have often proved a challenge in developing lipidomics compatible liquid chromatography-mass spectrometry (LC-MS) methods to study them. To overcome this problem, here, we report a reverse-phase LC-MS method that is compatible with existing high-throughput lipidomics strategies and capable of identifying and quantifying cholesterol and cholesteryl esters from mammalian cells and tissues. Using this sensitive yet robust LC-MS method, we profiled different mammalian cell lines and tissues and…

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) frameshift stimulatory element (FSE) is necessary for programmed -1 ribosomal frameshifting (-1 PRF) and optimized viral efficacy. The FSE has an abundance of context-dependent alternate conformations, but two of the structures most crucial to -1 PRF are an attenuator hairpin and a three-stem H-type pseudoknot structure. A crystal structure of the pseudoknot alone features three RNA stems in a helically stacked linear structure, whereas a 6.9 Å cryo-EM structure including the upstream heptameric slippery site resulted in a bend between two stems. Our previous research alluded to an extended upstream multibranch loop that includes both the attenuator hairpin and the slippery site-a conformation not previously modeled. We aim to provide further context to the SARS-CoV-2 FSE via computational and medium resolution cryo-EM approaches, by presenting a 6.1 Å cryo-EM structure featuring a linear pseudoknot structure and a dynamic upstream multibranch loop.

Parkinson's disease (PD) is characterized by the toxic oligomeric and fibrillar phases formed by monomeric alpha-synuclein (α-syn). Certain nanoparticles have been demonstrated to promote protein aggregation, while other nanomaterials have been found to prevent the process. In the current work, we use nuclear magnetic resonance spectroscopy in conjunction with isothermal titration calorimetry to investigate the cause and mechanism of these opposing effects at the amino acid protein level. The interaction of α-syn with two types of nanomaterials was considered: citrate-capped gold nanoparticles (AuNPs) and graphene oxide (GO). In the presence of AuNPs, α-syn aggregation is accelerated, whereas in the presence of GO, aggregation is prevented. The study indicates that GO sequesters the NAC region of α-syn monomers through electrostatic and hydrophobic interactions, leading to a reduced elongation rate, and AuNPs leave the NAC region exposed while binding the N-terminus, leading to higher aggregation. The protein's inclination toward quicker aggregation is explained by the binding of…

Herein, we present a novel esterase enzyme, Ade1, isolated from a metagenomic library of Amazonian dark earths soils, demonstrating its broad substrate promiscuity by hydrolyzing ester bonds linked to aliphatic groups. The three-dimensional structure of the enzyme was solved in the presence and absence of substrate (tributyrin), revealing its classification within the α/β-hydrolase superfamily. Despite being a monomeric enzyme, enzymatic assays reveal a cooperative behavior with a sigmoidal profile (initial velocities vs substrate concentrations). Our investigation brings to light the allokairy/hysteresis behavior of Ade1, as evidenced by a transient burst profile during the hydrolysis of substrates such as p -nitrophenyl butyrate and p -nitrophenyl octanoate. Crystal structures of Ade1, coupled with molecular dynamics simulations, unveil the existence of multiple conformational structures within a single molecular state (E̅ 1 ). Notably, substrate binding induces a loop closure that traps the substrate in the catalytic site. Upon product release, the cap domain opens simultaneously…

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15 N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent…

The bacterial metabolic enzyme 1-deoxy- d -xylulose-5-phosphate synthase (DXPS) catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and d -glyceraldehyde-3-phosphate ( d -GAP). DXP is an essential bacteria-specific metabolite that feeds into the biosynthesis of isoprenoids, pyridoxal phosphate (PLP), and ThDP. DXPS catalyzes the activation of pyruvate to give the C2α-lactylThDP (LThDP) adduct that is long-lived on DXPS in a closed state in the absence of the cosubstrate. Binding of d -GAP shifts the DXPS-LThDP complex to an open state which coincides with LThDP decarboxylation. This gated mechanism distinguishes DXPS in ThDP enzymology. How LThDP persists on DXPS in the absence of cosubstrate, while other pyruvate decarboxylases readily activate LThDP for decarboxylation, is a long-standing question in the field. We propose that an active site network functions to prevent LThDP activation on DXPS until the cosubstrate binds. Binding of d -GAP coincides with a conformational shift and disrupts the network causing changes in the active site that promote…

The innate immune system relies on molecular sensors to detect distinctive molecular patterns, including viral double-stranded RNA (dsRNA), which triggers responses resulting in apoptosis and immune infiltration. Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I), serving as a mechanism to distinguish self from non-self RNA and prevent aberrant immune activation. Loss-of-function mutations in the ADAR1 gene are one cause of Aicardi Goutières Syndrome (AGS), a severe autoimmune disorder in children. Although seven out of the eight AGS-associated mutations in ADAR1 occur within the catalytic domain of the ADAR1 protein, their specific effects on the catalysis of adenosine deamination remain poorly understood. In this study, we carried out a biochemical investigation of four AGS-causing mutations (G1007R, R892H, K999N, and Y1112F) in ADAR1 p110 and truncated variants. These studies included adenosine deamination rate measurements with two different RNA substrates derived from human transcripts known to be edited by ADAR1 p110…

ADVERTISEMENT RETURN TO ARTICLES ASAPViewpointNEXTBeyond the Crystal Structure of Human Macrophage C-Type LectinAdele Gabba*Adele GabbaDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United StatesSchool of Biological and Chemical Sciences, University of Galway, University Rd., Galway H91 TK33, Ireland*Email: [email protected]More by Adele Gabbahttps://orcid.org/0000-0001-8240-6482, Paul V. MurphyPaul V. MurphySchool of Biological and Chemical Sciences, University of Galway, University Rd., Galway H91 TK33, IrelandSSPC, The Science Foundation Ireland Research Centre for Pharmaceuticals, University of Galway, University Rd., Galway H91 TK33, IrelandMore by Paul V. Murphyhttps://orcid.org/0000-0002-1529-6540, Laura L. KiesslingLaura L. KiesslingDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United StatesMore by Laura L. Kiesslinghttps://orcid.org/0000-0001-6829-1500, and Gabriel BirraneGabriel BirraneDivision of Experimental Medicine, Department of Medicine, Beth Israel Deaconess Medical…

G protein-coupled receptors (GPCRs) play a pivotal role in regulating numerous physiological processes through their interactions with two key effectors: G proteins and β-arrestins (βarrs). This makes them crucial targets for therapeutic drug development. Interestingly, the evolving concept of biased signaling where ligands selectively activate either the G proteins or the βarrs has not only refined our understanding of segregation of physiological responses downstream of GPCRs but has also revolutionized drug discovery, offering the potential for treatments with enhanced efficacy and minimal side effects. This Review explores the mechanisms behind biased agonism, exploring it through various lenses, including ligand, receptor, cellular systems, location, and tissue-specific biases. It also offers structural insights into both orthosteric and allosteric ligand-binding pockets, structural rearrangements associated with the loops, and how ligand-engineering can contribute to biased signaling. Moreover, we also discuss the unique conformational signature in an intrinsically biased…

High Resolution Image Download MS PowerPoint Slide Glycerol 3-phosphate dehydrogenase catalyzes reversible hydride transfer from glycerol 3-phosphate (G3P) to NAD + to form dihydroxyacetone phosphate; from the truncated substrate ethylene glycol to NAD + in a reaction activated by the phosphite dianion substrate fragment; and from G3P to the truncated nicotinamide riboside cofactor in a reaction activated by adenosine 5′-diphosphate, adenosine 5′-monophosphate, and ribose 5-phosphate cofactor fragments. The sum of the stabilization of the transition state for GPDH-catalyzed hydride transfer reactions of the whole substrates by the phosphodianion fragment of G3P and the ADP fragment of NAD + is 25 kcal/mol. Fourteen kcal/mol of this transition state stabilization is recovered as phosphite dianion and AMP activation of the reactions of the substrate and cofactor fragments. X-ray crystal structures for unliganded GPDH, for a binary GPDH·NAD + complex, and for a nonproductive ternary GPDH·NAD + ·DHAP complex show that the ligand binding energy is utilized to drive an extensive protein…

Engineered type I polyketide synthases (type I PKSs) can enable access to diverse polyketide pharmacophores and generate non-natural natural products. However, the promise of type I PKS engineering remains modestly realized at best. Here, we report that ketosynthase (KS) domains, the key carbon-carbon bond-forming catalysts, control which intermediates are allowed to progress along the PKS assembly lines and which intermediates are excluded. Using bimodular PKSs, we demonstrate that KSs can be exquisitely selective for the upstream polyketide substrate while retaining promiscuity for the extender unit that they incorporate. It is then the downstream KS that acts as a gatekeeper to ensure the fidelity of the extender unit incorporation by the upstream KS. We also demonstrate that these findings are not universally applicable; substrate-tolerant KSs do allow engineered polyketide intermediates to be extended. Our results demonstrate the utility for evaluating the KS-induced bottlenecks to gauge the feasibility of engineering PKS assembly lines.

Over the past two decades, the utilization of protein cages has witnessed exponential growth driven by their extensive applications in biotechnology and therapeutics. In the context of the recent Covid-19 pandemic, protein-cage-based scaffolds played a pivotal role in vaccine development. Beyond vaccines, these protein cages have proven valuable in diverse drug delivery applications thanks to their distinctive architecture and structural stability. Among the various types of protein cages, ferritin-based cages have taken the lead in drug delivery applications. This is primarily attributed to their ease of production, exceptional thermal stability, and nontoxic nature. While ferritin-based cages are commonly employed in anticancer drug delivery and contrast agent delivery, their efficacy in malarial drug delivery had not been explored until this study. In this investigation, several antimalarial drugs were encapsulated within horse spleen ferritin, and the binding and loading processes were validated through both experimental and computational techniques. The data unequivocally…

Abstract Methyl-coenzyme M reductase (MCR) is a central player in methane biogeochemistry, governing methanogenesis and the anaerobic oxidation of methane (AOM) in methanogens and anaerobic methanotrophs (ANME), respectively. The prosthetic group of MCR is coenzyme F 430 , a nickel-containing tetrapyrrole derivative. Additionally, a few modified versions of F 430 have been discovered, including the 17 2 -methylthio-F 430 (mt-F 430 ) that functions with ANME-1 MCR. This study employs molecular dynamics (MD) simulations to unravel the intricacies of the active-site dynamics of MCR from Methanosarcina acetivorans and ANME-1 when bound to the canonical F 430 compared to 17 2 -thioether coenzyme F 430 variants and substrates for methane formation. Overall, our simulations indicate that each MCR active site is optimized for a given version of F 430 and support the importance of the Gln to Val substitution in accommodating the 17 2 methylthio modification. Notably, modifications in the 17 2 position disrupt the canonical coordination among cofactors in M. acetivorans MCR, implicating…

N -Acetylnorloline synthase (LolO) is one of several iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases that catalyze sequential reactions of different types in the biosynthesis of valuable natural products. LolO hydroxylates C2 of 1- exo -acetamidopyrrolizidine before coupling the C2-bonded oxygen to C7 to form the tricyclic loline core. Each reaction requires cleavage of a C–H bond by an oxoiron(IV) (ferryl) intermediate; however, different carbons are targeted, and the carbon radicals have different fates. Prior studies indicated that the substrate-cofactor disposition (SCD) controls the site of H · abstraction and can affect the reaction outcome. These indications led us to determine whether a change in SCD from the first to the second LolO reaction might contribute to the observed reactivity switch. Whereas the single ferryl complex in the C2 hydroxylation reaction was previously shown to have typical Mössbauer parameters, one of two ferryl complexes to accumulate during the oxacyclization reaction has the highest isomer shift seen to date for such a complex and…

Src-homology-2-domain-containing PTP-2 (SHP2) is a widely expressed signaling enzyme whose misregulation is associated with multiple human pathologies. SHP2's enzymatic activity is controlled by a conformational equilibrium between its autoinhibited ("closed") state and its activated ("open") state. Although SHP2's closed state has been extensively characterized, the putative structure of its open form has only been revealed in the context of a highly activated mutant (E76K), and no systematic studies of the biochemical determinants of SHP2's open-state stabilization have been reported. To identify amino-acid interactions that are critical for stabilizing SHP2's active state, we carried out a mutagenic study of residues that lie at potentially important interdomain interfaces of the open conformation. The open/closed equilibria of the mutants were evaluated, and we identified several interactions that contribute to the stabilization of SHP2's open state. In particular, our findings establish that an ion pair between glutamate 249 on SHP2's PTP domain and arginine 111 on an…

The effects of guanidinium hydrochloride (GdmCl) on two intrinsically disordered proteins (IDPs) are investigated using simulations of the self-organized polymer-IDP (SOP-IDP) model. The impact of GdmCl is taken into account using the molecular transfer model (MTM). We show that due to the dramatic reduction in the stiffness of the highly charged Prothymosin-α (ProTα) with increasing concentration of GdmCl ([GdmCl]), the radius of gyration ( R g ) decreases sharply until about 1.0 M. Above 1.0 M, ProTα expands, caused by the swelling effect of GdmCl. In contrast, R g of α-Synuclein (αSyn) swells as continuously as [GdmCl] increases, with most of the expansion occurring at concentrations less than 0.2 M. Strikingly, the amplitude of the small-angle X-ray scattering (SAXS) profiles for ProTα increases until [GdmCl] ≈ 1.0 M and decreases beyond 1.0 M. The [GdmCl]-dependent SAXS profiles for αSyn, which has a pronounced bump at small wave vector ( q ∼ 0.5 nm –1 ) at low [GdmCl] (≤0.2 M), monotonically decrease at all values of [GdmCl]. The contrasting behavior predicted by the…

Prenylation consists of the modification of proteins with either farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP) at a cysteine near the C-terminus of target proteins to generate thioether-linked lipidated proteins. In recent work, metabolic labeling with alkyne-containing isoprenoid analogues including C15AlkOPP has been used to identify prenylated proteins and track their levels in different diseases. Here, a systematic study of the impact of isoprenoid length on proteins labeled with these probes was performed. Chemical synthesis was used to generate two new analogues, C15hAlkOPP and C20AlkOPP, bringing the total number of compounds to eight used in this study. Enzyme kinetics performed in vitro combined with metabolic labeling in cellulo, resulted in the identification of 8 proteins for C10AlkOPP, 70 proteins for C15AlkOPP, 41 proteins for C15hAlkOPP, and 7 proteins for C20AlkOPP. While C10AlkOPP was the most selective for farnesylated proteins and C20AlkOPP was most selective for geranylgeranylated proteins, the number of proteins identified using those probes…

Nonribosomal peptide synthetases (NRPS) biosynthesize numerous natural products with therapeutic, agricultural, and industrial significance. Reliably altering substrate selection in these enzymes has been a longstanding goal, as this would enable the production of tailor-made peptides with desired activities. In this study, the NRPS EntF and the associated biosynthesis of the siderophore enterobactin (ENT) were used as a model system to interrogate substrate selection by an adenylation (A) domain. We employed a directed evolution pipeline that harnesses an in vivo genetic selection for siderophore production to alter A domain substrate selection. Surprisingly, this led to the formation of a new, physiologically active catechol siderophore in Escherichia coli . We characterized the enzyme variants in vitro and demonstrated transferability of our findings to the well-studied TycC and GrsB NRPSs. This work identifies critical binding pocket residues that allow for altered substrate selection in our model system and expands upon our understanding of iron acquisition in E. coli .