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 F nucleus from biomolecules results in background-free, high-resolution F NMR spectra. The introduction of F probes in a few selected locations in biomolecules reduces spectral crowding despite its increased line width in comparison to typical H NMR line widths and allows rapid site-specific measurements from simple 1D spectra alone. The design and synthesis of novel 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 F NMR spectroscopy. The increased interest and widespread use of F NMR spectroscopy of biomolecules is gradually establishing it as a sensitive and high-resolution probe of biomolecular structure and dynamics, supplementing traditional C/N-based methods. This Review focuses on the advances in F solution NMR spectroscopy of proteins in the past 5 years, with an emphasis on novel F tags and labeling techniques, NMR experiments to probe protein structure and conformational dynamics in vitro, and in-cell NMR applications.

Influenza B viruses have cocirculated during most seasonal flu epidemics and can cause significant human morbidity and mortality due to their rapid mutation, emerging drug resistance, and severe impact on vulnerable populations. The influenza B M2 proton channel (BM2) plays an essential role in viral replication, but the mechanisms behind its symmetric proton conductance and the involvement of a second histidine (His27) cluster remain unclear. Here we performed membrane-enabled continuous constant-pH molecular dynamics simulations on wildtype BM2 and a key H27A mutant channel to explore its pH-dependent conformational switch. Simulations captured the activation as the first histidine (His19) protonates and revealed the transition at lower pH values compared to AM2 is a result of electrostatic repulsions between His19 and preprotonated His27. Crucially, we provided an atomic-level understanding of the symmetric proton conduction by identifying preactivating channel hydration in the C-terminal portion. This research advances our understanding of the function of BM2 function and lays the groundwork for further chemically reactive modeling of the explicit proton transport process as well as possible antiflu drug design efforts.

Fungal ribosomally synthesized and post-translationally modified peptides (RiPPs) are a vital class of natural products known for their biological activities including anticancer, antitubulin, antinematode, and immunosuppressant properties. These bioactive fungal RiPPs play key roles in chemical ecology and have a significant therapeutic potential. Their structural diversity, which arises from intricate post-translational modifications of precursor peptides, is particularly remarkable. Despite their biological and ecological importance, the discovery of fungal RiPPs has been historically challenging and only a limited number have been identified. To date, known fungal RiPPs are primarily grouped into three groups: cycloamanides and borosins from basidiomycetes and dikaritins from ascomycetes. Recent advancements in bioinformatics have revealed the vast untapped potential of fungi to produce RiPPs, offering new opportunities for their discovery. This review highlights recent progress in fungal RiPP biosynthesis and genome-guided discovery strategies. We propose that combining the knowledge of fungal RiPP biosynthetic pathways with advanced gene-editing technologies and bioinformatic tools will significantly accelerate the discovery of novel bioactive fungal RiPPs.

Antibiotics are essential components of current medical practice, but their effectiveness is being eroded by the increasing emergence of antimicrobial-resistant infections. At the same time, the rate of antibiotic discovery has slowed, and our future ability to treat infections is threatened. Among Christopher T. Walsh's many contributions to science was his early recognition of this threat and the potential of biosynthesis─genes and mechanisms─to contribute solutions. Here, we revisit a 2006 review by Walsh and co-workers that highlighted a major challenge in antibiotic natural product discovery: the daunting odds for identifying new naturally occurring antibiotics. The review described strategies to mitigate the odds challenge. These strategies have been used extensively by the natural product discovery community in the years since and have resulted in some promising discoveries. Despite these advances, the rarity of novel antibiotic natural products remains a barrier to discovery. We compare the challenge of discovering natural product antibiotics to the process of identifying new prime numbers, which are also challenging to find and an essential, if underappreciated, element of modern life. We propose that inclusion of filters for functional compounds early in the discovery pipeline is key to natural product antibiotic discovery, review some recent advances that enable this, and discuss some remaining challenges that need to be addressed to make antibiotic discovery sustainable in the future.

Within the framework of liquid-liquid phase separation (LLPS), biomolecular condensation orchestrates vital cellular processes, and its dysregulation is implicated in severe pathological conditions. Recent studies highlight the role of intrinsically disordered proteins (IDPs) in LLPS, yet the influence of microenvironmental factors has remained a puzzling factor. Here, via computational simulation of the impact of solution conditions on LLPS behavior of neurologically pathogenic IDP Aβ40, we chanced upon a salt-driven reentrant condensation phenomenon, wherein Aβ40 aggregation increases with low salt concentrations (25-50 mM), followed by a decline with further salt increments. An exploration of the thermodynamic and kinetic signatures of reentrant condensation unveils a nuanced interplay between protein electrostatics and ionic strength as potential drivers. Notably, the charged residues of the N-terminus exhibit a nonmonotonic response to salt screening, intricately linked to the recurrence of reentrant behavior in hydrophobic core-induced condensation. Intriguingly, our findings also unveil the reappearance of similar reentrant condensation phenomena under varying temperature conditions. Collectively, our study illuminates the profoundly context-dependent nature of Aβ40s liquid-liquid phase separation behavior, extending beyond its intrinsic molecular framework, where microenvironmental cues wield significant influence over its aberrant functionality.

Nucleic acid mimics (NAMs) have demonstrated high potential as antibacterial drugs. However, very few studies have assessed their possible diffusion across the bacterial envelope. In this work, we studied NAMs' diffusion in lipid bilayer systems that mimic the bacterial outer membrane using molecular dynamics (MD) simulations. Additionally, we examined the interactions of a NAM sequence with lipid membranes and ascertained the partition constants () through MD and spectroscopic investigations. The NAM sequences were composed of locked nucleic acid (LNA) and 2'-O-methyl (2'-OMe) residues, whereas the membrane models were composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) or 1-palmitoyl-2-oleoyl--glycero-3-phospho-(1'-rac-glycerol) (POPG) phospholipids. The parametrization protocol followed was validated against literature data and demonstrated the reliability of our approach for simulating NAM sequences. Investigation into the interaction of the sequences with zwitterionic and anionic membranes revealed a preference for hydrogen bond formation with the anionic model over the zwitterionic one. Additionally, potential of mean force (PMF) calculations unveiled a lower free energy barrier for translocation across the zwitterionic bilayer model. Contrarily, the partition constants derived suggested a slightly higher partitioning within the anionic membrane, emphasizing a nuanced interplay of factors. Finally, spectroscopic partition measurements with liposomes presented challenges in quantifying the partition of NAMs due to minimal signal variations. However, a tendency for quenching in anionic vesicles suggested a potential, albeit small, partitioning effect that warrants further investigation. In summary, our study revealed that NAMs will not, in principle, be able to cross an intact bacterial outer membrane by passive diffusion.

In cells, TDP-43 is a crucial protein that can form harmful amyloid aggregates linked to fatal and incurable human neurodegenerative disorders. Normally, TDP-43 exists in a smaller soluble native state that prevents aggregation. However, aging and stress can destabilize this native state, leading to the formation of disease-causing amyloid aggregates via the formation of partially unfolded, high-energy intermediates with a greater tendency to aggregate. These intermediates are crucial in the early stages of amyloid formation and are challenging to study due to their low stability. Understanding the structure of these early aggregation-prone states of TDP-43 is essential for designing effective treatments for TDP-43 proteinopathies. Targeting these initial intermediates could be more effective than focusing on fully formed amyloid aggregates. By disrupting the aggregation process at this early stage, we may be able to prevent the progression of diseases related to TDP-43 aggregation. Hence, we decided to uncover the hidden, high-energy intermediates in equilibrium with the native states of TDP-43 by modulating the thermodynamic stability of the soluble native dimer (N form) and monomeric molten globular state (MG form) of full-length TDP-43. The thermodynamic modulation performed in the current study successfully revealed the highly aggregation-prone intermediate of full-length TDP-43, i.e., PUF. Moreover, we observed that along with high aggregation propensity, the aggregation kinetics and mechanisms of PUF differ from previously identified intermediates of full-length TDP-43 (the MG and I forms). The information regarding the initial aggregation-prone state of full-length TDP-43 could lead to therapies for amyloid diseases by halting early protein aggregation.

Chlamydia protein associating with death domains (CADD) is involved in the biosynthesis of -aminobenzoic acid (pABA) for integration into folate, a critical cofactor that is required for pathogenic survival. CADD activates dioxygen and utilizes its own tyrosine and lysine as synthons to furnish the carboxylate, carbon backbone, and amine group of pABA in a complex multistep mechanism. Unlike other members of the heme oxygenase-like dimetal oxidase (HDO) superfamily that typically house an Fe cofactor, previous activity studies have shown that CADD likely uses a heterobimetallic Fe/Mn center. The structure of the Fe/Mn cofactor and how the conserved HDO scaffold mediates metal selectivity have remained enigmatic. Adopting an metalation approach, CADD was solved in the apo, Fe, Mn, and catalytically active Fe/Mn forms to identify the probable site for Mn binding. The analysis of CADD active-site variants further reinforces the importance of the secondary coordination sphere on cofactor preference for competent pABA formation. Rapid kinetic optical and electron paramagnetic resonance (EPR) studies show that the heterobimetallic cofactor selectively reacts with dioxygen and likely initiates pABA assembly through the formation of a transient tyrosine radical intermediate and a resultant heterobimetallic Mn/Fe cluster.

Fungal polysaccharide monooxygenases (PMOs) oxidatively degrade cellulose and other carbohydrate polymers via a mononuclear copper active site using either O or HO as a cosubstrate. Cellulose-active fungal PMOs in the auxiliary activity 9 (AA9) family have a conserved second-sphere hydrogen-bonding network consisting of histidine, glutamine, and tyrosine residues. The second-sphere histidine has been hypothesized to play a role in proton transfer in the O-dependent PMO reaction. Here the role of the second-sphere histidine (H157) in an AA9 PMO, PMO9E, was investigated. This PMO is active on soluble cello-oligosaccharides such as cellohexaose (Glc6), thus enabling kinetic analysis with the point variants H157A and H157Q. The variants appeared to fold similarly to the wild-type (WT) enzyme and yet exhibited weaker affinity toward Glc6 than WT (WT = 20 ± 3 μM). The variants had comparable oxidase (O reduction to HO) activity to WT at all pH values tested. Using O as a cosubstrate, the variants were less active for Glc6 hydroxylation than WT, with H157A being the least active. Similarly, H157Q was competent for Glc6 hydroxylation with HO, but H157A was less active. Comparison of the crystal structures of H157Q and WT PMO9E reveals that a terminal heteroatom of Q157 overlays with N of H157. Altogether, the data suggest that H157 is not important for proton transfer, but support a role for H157 as a hydrogen-bond donor to diatomic-oxygen intermediates, thus facilitating catalysis with either O or HO.

The conventional idea that a well-defined protein structure governs its functions is being challenged by the evolving significance of conformational flexibility and disorder in influencing protein activity. Here, we focus on the Small Ubiquitin-like MOdifier 1 (SUMO1) protein, a post-translational modifier, which binds various target proteins during the process of SUMOylation. We present evidence supporting the presence of both folded and "ordered" molten globule (MG) states in SUMO1 under physiological conditions. We investigate the MG state using a combination of near-UV and far-UV circular dichroism (CD) experiments. Moreover, we dissect the information from the near-UV CD data to gain specific insights about the MG intermediate. This is achieved by mutating specific aromatic amino acids, particularly creating a single-tyrosine mutant S1Y51 (by introducing Y21F and Y91F mutations) and a tryptophan mutant S1F66W. Spectroscopic studies of the mutants as a function of temperature revealed multiple insights. The transition from the folded to the MG state involves a site-specific loss of tertiary packing near Y51 but the region surrounding F66 retained most of its tertiary contacts, suggesting an ordered MG structure. We further demonstrate the increased solvent exposure of Y51 in the MG state by using time-resolved fluorescence and steady-state quenching experiments. The observed conformational flexibility and solvent accessibility, particularly around Y51 that is known to be involved in binding the cognate ligands such as PIASX and its peptide analogues, have biological and functional implications in mediating protein-protein interactions during the SUMOylation process.

Transmissible Spongiform Encephalopathies are fatal neurodegenerative diseases caused by the misfolding of the cellular prion protein (PrP) into its pathological isoform (PrP). Efficient transmission of PrP occurs within the same species, but a species barrier limits interspecies transmission. While PrP structure is largely conserved among mammals, variations at the β2-α2 loop are observed, and even minor changes in the amino acid sequence of the β2-α2 loop can significantly affect transmission efficiency. The present study shows that the introduction of the elk/deer-specific amino acid substitutions at positions 169 (Ser to Asn) and 173 (Asn to Thr) into the mouse prion protein, which are associated with the structural rigidity of the β2-α2 loop, has a substantial impact on protein dynamics as well as on the misfolding pathways of the protein. Native state hydrogen-deuterium exchange studies coupled with mass spectrometry, show that the rigid loop substitutions stabilize not only the β2-α2 loop but also the C-terminal end of α3, suggesting that molecular interactions between these two segments are strengthened. Moreover, the energy difference between the native state and multiple misfolding-prone partially unfolded forms (PUFs) present at equilibrium, is increased. The decreased accessibility of the PUFs from the native state leads to a slowing down of the misfolding of the protein. The results of this study provide important insights into the early events of conformational conversion of prion protein into β-rich oligomers, and add to the evidence that the β2-α2 loop is a key determinant in prion protein aggregation.

Berberrubine (BRB), belonging to the benzylisoquinoline alkaloid, is a main metabolite of berberine . BRB was previously proven to undergo metabolic activation mediated by P450s. In this study, the chemical interactions between BRB and CYP2D6 enzyme were investigated. First, a variety of P450s participated in the metabolism of berberine transformed to BRB, but CYP2D6 was the most involved enzyme. A time-, concentration-, and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent inhibition of CYP2D6 was caused by BRB. The inhibitory effect of BRB on CYP2D6 was irreversible. The maximum reaction rate constants of inactivation () and half-maximal inactivation () of BRB on CYP2D6 were 0.0410 min and 3.798 μM, respectively. Metoprolol, a classic substrate of CYP2D6, attenuated CYP2D6 from inactivation by BRB. Glutathione (GSH) and catalase/superoxide dismutase failed to protect against the inactivation of CYP2D6 caused by BRB. Three cys-based adducts derived from the reaction of electrophilic metabolites of BRB with CYP2D6 were detected by ultra performance liquid chromatography-mass spectrometry (UPLC-MS)/MS. The reactive metabolites derived from BRB might be responsible for the inactivation of CYP2D6. In summary, BRB was characterized as a mechanism-based inactivator of CYP2D6.

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 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 . We characterized the enzyme variants 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 .

Glycosylation is biochemically complex and functionally critical to a wide range of processes and disease states, making it a vibrant area of contemporary research. Here, we highlight a selection of notable recent advances in the glycobiology of SARS-CoV-2 infection and immunity, cancer biology and immunotherapy, and newly discovered glycosylated RNAs. Together, these studies illustrate the significance of glycosylation in normal biology and the great promise of manipulating glycosylation for therapeutic benefit in disease.

Sabinene is a plant natural product with a distinctive strained [3.1.0] bicyclic ring system that is used commercially as a spicy and pine-like fragrance with citrus undertones. This unusual monoterpene has also been studied as an antifungal and anti-inflammatory agent as well as a next-generation biofuel. In order to understand the molecular determinants of [3.1.0] bicyclic ring formation in sabinene biosynthesis, we now report three X-ray crystal structures of sabinene synthase from Western red cedar, (TpSS), with open and partially closed active site conformations at 2.21-2.72 Å resolution. We additionally report the complete biochemical characterization of sabinene synthase, including steady-state kinetics, active site mutagenesis, and product array profiling. The catalytic metal ion requirement is unexpectedly broad for a class I terpene cyclase: optimal catalytic activity was measured using Mn or Co, with more modest activity observed using Mg or Ni. Kinetic parameters were determined for both full-length TpSS and a deletion variant lacking the putative N-terminal plastidial targeting sequence, designated ΔTpSS. Monoterpene product profiles for both indicated similar product arrays independent of the catalytic metal ion used, with sabinene comprising nearly 90% of the total products generated. Site-directed mutagenesis was utilized to probe the function of active site residues, and several mutants yielded altered product arrays. Most notably, the G458A substitution converted ΔTpSS into a high-activity α-pinene synthase. α-Pinene contains a bicyclic [3.1.1] ring system; structural and mechanistic analyses suggest a molecular rationale for the reprogrammed transannulation reaction, leading to the alternative bicyclic product.

Cyclodipeptide synthases (CDPSs) catalyze the synthesis of diverse cyclodipeptides (CDPs) by utilizing two aminoacyl-tRNA (aa-tRNA) substrates in a sequential ping-pong reaction mechanism. Numerous CDPSs have been characterized to provide precursors for diketopiperazines (DKPs) with diverse structural characteristics and biological activities. BcmA, belonging to the XYP subfamily, is a cyclo(l-Ile-l-Leu)-synthesizing CDPS involved in the biosynthesis of the antibiotic bicyclomycin. The structural basis and determinants influencing BcmA enzyme activity and substrate selectivity are not well understood. Here, we report the crystal structure of BcmA from . Through structural comparison and systematic site-directed mutagenesis, we highlight the significance of key residues located in the aminoacyl-binding pocket for enzyme activity and substrate specificity. In particular, the nonconserved residues D161 and K165 in pocket P2 are essential for the activity of BcmA without significant alteration of the substrate specificity, while the conserved residues F158 as well as F210 and S211 in P2 are responsible for determining substrate selectivity. These findings facilitate the understanding of how CDPSs selectively accept hydrophobic substrates and provide additional clues for the engineering of these enzymes for synthetic biology applications.

The main protease (M) of SARS-CoV-2 is essential for viral replication and is, therefore, an important drug target. Here, we investigate two flexible loops in M that play a role in catalysis. Using all-atom molecular dynamics simulations, we analyze the structural ensemble of M in an apo state and substrate-bound state. We find that the flexible loops can adopt open, intermediate (partly open), and closed conformations in solution, which differs from the partially closed state observed in crystal structures of M. When the loops are in closed or intermediate states, the catalytic residues are more likely to be in close proximity, which is crucial for catalysis. Additionally, we find that substrate binding to one protomer of the homodimer increases the frequency of intermediate states in the bound protomer while also affecting the structural propensity of the apo protomer's flexible loops. Using dynamic network analysis, we identify multiple allosteric pathways connecting the two active sites of the homodimer. Common to these pathways is an allosteric hotspot involving the N-terminus, a critical region that comprises part of the binding pocket. Taken together, the results of our simulation study provide detailed insight into the relationships between the flexible loops and substrate binding in a prime drug target for COVID-19.

Extracellular signals perceived by 7-transmembrane (7TM)-spanning receptors initiate desensitization that involves the removal of these receptors from the plasma membrane. Agonist binding often evokes phosphorylation in the flexible C-terminal region and/or intracellular loop 3 of many 7TM G-protein-coupled receptors in animal cells, which consequently recruits a cytoplasmic intermediate adaptor, β-arrestin, resulting in clathrin-mediated endocytosis (CME) and downstream signaling such as transcriptional changes. Some 7TM receptors undergo CME without recruiting β-arrestin, but it is not clear how. Arrestins are not encoded in the genome, yet cells have a well-characterized signal-induced CME of a 7TM protein, designated Regulator of G Signaling 1 (AtRGS1). Here we show that a component of the retromer complex, Vacuolar Protein Sorting-Associated 26 (VPS26), binds the phosphorylated C-terminal region of AtRGS1 as a VPS26A/B heterodimer to form a complex that is required for downstream signaling. We propose that VPS26 moonlights as an arrestin-like adaptor in the CME of AtRGS1.

Markov State Models (MSMs) have been widely applied to understand protein folding mechanisms by predicting long time scale dynamics from ensembles of short molecular simulations. Most MSM estimators enforce detailed balance, assuming that trajectory data are sampled at an equilibrium. This is rarely the case for ab initio folding studies, however, and as a result, MSMs can severely underestimate protein folding stabilities from such data. To remedy this problem, we have developed an enhanced-sampling protocol in which (1) unbiased folding simulations are performed and sparse tICA is used to obtain features that best capture the slowest events in folding, (2) umbrella sampling along this reaction coordinate is performed to observe folding and unfolding transitions, and (3) the thermodynamics and kinetics of folding are estimated using multiensemble Markov models (MEMMs). Using this protocol, folding pathways, rates, and stabilities of a designed α-helical hairpin, Z34C, can be predicted in good agreement with experimental measurements. These results indicate that accurate simulation-based estimates of absolute folding stabilities are within reach, with implications for the computational design of folded miniproteins and peptidomimetics.

The chemoproteomics technique, activity-based protein profiling (ABPP), has proven to be an invaluable tool in assigning functions to enzymes. The serine hydrolase (SH) enzyme superfamily, in particular, has served as an excellent example in displaying the versatility of various ABPP platforms and has resulted in a comprehensive cataloging of the biochemical activities associated within this superfamily. Besides SHs, in mammals, several other enzyme classes have been thoroughly investigated using ABPP platforms. However, the utility of ABPP platforms in fly models remains underexplored. Realizing this knowledge gap, leveraging complementary ABPP platforms, we reported the full array of SH activities during various developmental stages and adult tissues in the fruit fly (). Following up on this study, using ABPP, we mapped SH activities in adult fruit flies in an infection model and found that a gut-resident lipase CG17192 showed increased activity during infection. To assign a biological function to this uncharacterized lipase, we performed an untargeted lipidomics analysis and found that phosphatidylinositols were significantly elevated when was depleted in the adult fruit fly gut. Next, we overexpressed this lipase in insect cells, and using biochemical assays, we show that CG17192 is a secreted enzyme that has phospholipase C (PLC) type activity, with phosphatidylinositol being a preferred substrate. Finally, we show during infection that heightened CG17192 regulates phosphatidylinositol levels and, by doing so, likely modulates signaling pathways in the adult fruit fly gut that might be involved in the resolution of this pathophysiological condition.

An attractive strategy for combating antibacterial resistance involves the development of new antibiotics whose mechanisms differ from those of existing ones in the clinic. Elfamycin antibiotics, whose prototypes include kirromycin and aurodox, are illustrative examples based on their ability to target EF-Tu, an essential component for protein translation in bacteria. Our efforts to revisit this antibiotic class were enabled by two developments. First, we produced L-681,217, an understudied member of this polyketide family harboring a terminal carboxylic acid in place of a hydroxypyridone ring, and synthesized a biotinylated derivative with comparable activity to the natural product. Second, we established a sensitive cell-free protein synthesis (CFPS) assay in which superfolder green fluorescent protein (sfGFP) production was inhibited by L-681,217. Biotinyl-L-681,217 was used to drain the CFPS system of endogenous EF-Tu, allowing replenishment with orthologs to interrogate pathogen selectivity and propensity toward resistance. Comparative analysis of kirromycin and L-681,217 showed that, while both antibiotics are equipotent in CFPS assays, they interact distinctly with purified EF-Tu, a feature that presumably correlates with prior observations that kirromycin enhances GTP hydrolysis by EF-Tu whereas L-681,217 does not. Analysis of L-681,217 and kirromycin accumulation in selected mutant strains also revealed that antibiotic import and efflux contributed to resistance. The promise of L-681,217 as a medicinal lead was underscored by the observation that, unlike aurodox, this polyketide does not inhibit adenylosuccinate synthase.