- by Stasyuk, A., Smith, C. A.Over 1370 class D {beta}-lactamases are currently known, and they pose a serious threat to the effective treatment of many infectious diseases, particularly in some pathogenic bacteria where evolving carbapenemase activity has been reported. Detailed understanding of their molecular biology, enzymology and structural biology are critically important but the lack of a standardized residue numbering scheme and inconsistent secondary structure annotation has made comparative analyses sometimes difficult and cumbersome. Compounding this, in the post-AlphaFold world where we currently find ourselves, […]
- by Yang, J. G., Chen, H. Y., Guardado, J. H., Gardner, M., Foronda, M. S.The Escherichia coli MetNI-Q importer, an ATP-binding cassette (ABC) transporter, mediates the uptake of both L- and D-enantiomers of methionine. Original in vivo uptake studies show a strong preference for L-Met over D-Met, but the molecular basis of this selectivity is unclear. In this work, we systematically examine substrate discrimination by the MetNI transporter and MetQ substrate binding protein using an array of biophysical and biochemical techniques. Based on the kinetic and thermodynamic parameters of individual intermediates in the transport […]
- by Carpenter, E. J., Peng, C., Greiner, R., Derda, R.Cross-chiral recognition in glycobiology is the interactions between biologically conventional proteins and the enantiomers of biological glycans (e.g., L-proteins binding with L-hexoses) from organisms across all kingdoms of life. By symmetry, it also describes the interactions of chirally mirrored proteins with normal D-glycans. Knowledge of cross-chiral recognition is critical to understanding the potential interactions of existing life forms with artificial mirror-life forms, but currently known rules of protein-glycan interaction are insufficient. To build a methodology for learning such interactions, we […]
- by Burdina, N., Liebsch, F., Macha, A., Ortuno Gil, J. L., Frommelt, P., Rais, I., Basler, F., Poepsel, S., Schwarz, G.Gephyrin is the main scaffolding protein at inhibitory synapses clustering glycine and GABA type A receptors. At specific GABAergic synapses, the nucleotide exchange factor collybistin recruits gephyrin to the postsynaptic membrane via interaction with phosphoinositides. However, the molecular mechanisms underlying the formation, maintenance and regulation of collybistin-dependent gephyrin clusters remain poorly understood. This study sheds light on the molecular mechanism of gephyrin cluster formation based on gephyrin self-oligomerization induced by collybistin, leading to the formation of a high-molecular weight (>5 […]
- by Hsu, C.-I., Mei, S., Demmerle, J., Ruttenberg, S., Sahn, M., Yu, J.-R.NSD1 is a key lysine methyltransferase for di-methylation of lysine 36 of histone H3 (H3K36me2) essential for the establishment of active chromatin domains. While the loss of NSD1 catalytic activity halts embryonic development and a gain of that drives oncogenesis in leukemia and glioma, the regulatory mechanisms that control NSD1 activity in these processes remain poorly understood. Here, we uncover that NSD1 requires allosteric activation through the aromatic pocket of its PWWP2 domain. Surprisingly, we identify that NSD1-PWWP2 binds to […]
- by Salinero-Lanzarote, A., Lian, J., Namkoong, G., Suess, D. L. M., Rubio, L. M., Dean, D. R., Perez-Gonzalez, A.The free-living diazotroph Azotobacter vinelandii produces three genetically distinct but functionally and mechanistically similar nitrogenase isozymes, designated as Mo-dependent, V-dependent, and Fe-only. They respectively harbor nearly identical catalytic cofactors that are distinguished by a heterometal site occupied by Mo (FeMo-cofactor), V (FeV-cofactor), or Fe (FeFe-cofactor). Completion of FeMo-cofactor and FeV-cofactor formation occurs on molecular scaffolds prior to delivery to their catalytic partners. In contrast, completion of FeFe-cofactor assembly occurs directly within its cognate catalytic partner. Because hybrid nitrogenase species that […]
- by Hirt, H., Shekhawat, K., Fatima, A., Alhoraibi, H. M., Abulfaraj, A. A., Rayapuram, N., Manickam, P.Plants employ diverse strategies to cope with different types of heat stress. The response to short-term acute heat stress differs significantly from that to moderate heat stress followed by severe stress events. After experiencing moderate heat stress, plants exhibit a more robust response to subsequent severe stress, a phenomenon known as thermopriming or acquired thermotolerance. Thermopriming creates a memory by maintaining the heat stress (HS) memory-related genes in an alert state. In this work, we investigated the role of Arabidopsis […]
- by Yu, Z., Liu, Y., Lin, G., Jiang, W., Chen, M.Proteins often adopt multiple ensemble conformations to perform essential functions such as catalysis, transport, and signal transduction. Traditional physics-based methods for generating these conformations, including molecular dynamics and Monte Carlo simulations, are computationally expensive and time-consuming, limiting their practicality for high-throughput applications like screening. Recent advances in machine learning, particularly deep generative models, offer a promising alternative for protein conformation ensemble generation. However, these models are often task-specific or rely on strong assumptions to generalize. Here, we introduce ESMAdam, a […]
- by Miliordini, G., Zacco, E., Armaos, A., Di Palma, F., Masi, M., Giloid, M., Rupert, J., Broglia, L., Varignani, G., Oneto, M., Scotto, M., Marotta, R., GIROTTO, S., Cavalli, A., Tartaglia, G. G.The interaction between RAD51 and BRCA2 plays a key role in homologous recombination (HR), a critical DNA repair mechanism essential for the survival of cancer cells. Disrupting this interaction increases the sensitivity of cancer cells to chemotherapeutic agents. Here, we employed in silico methods to design a novel class of aptamers–customized single-stranded oligonucleotides–specifically engineered to bind RAD51. These aptamers were developed with the aim of selectively modulating RAD51s nuclear recruitment and its role in DNA repair processes. The leading candidate […]
- by Kong, X., Jiao, R., Lin, H., Guo, R., Huang, W., Ma, W.-Y., Wang, Z., Liu, Y., Ma, J.Peptides offer distinct advantages for targeted therapy, including oral bioavailability, cellular permeability, and high specificity, which set them apart from conventional small molecules and biologics. In this work, we developed an AI algorithm, named PepMimic, to transform a known protein receptor or an existing antibody of a target into a short peptide drug by mimicking the binding interfaces between targets and known binders. The structural root mean square deviation and interface DockQ with reference binders were 61% and 75% better […]
- by Xu, Y., Teng, Y., Liao, J., Leung, P. M., Dai, S., Sun, Y., Hu, W., Wang, H., Li, Y., Huang, Y., Guo, Z., Pan, X., Dong, X., Luo, Y., Greening, C.Carbon monoxide (CO) is both a potent poison for many aerobic organisms and a desirable energy source for diverse microorganisms. Atmospheric emissions of this gas have increased since industrialization and their levels are highly elevated in many urban and natural environments; however, it is unresolved whether elevated levels of CO at environmentally relevant concentrations are primarily stimulatory or inhibitory to soil microbial communities. Here, we showed that CO exposure minimally affects microbial abundance, richness, and composition in diverse ecosystem soils, […]
- by Filandr, F., Hepburn, M., Sarpe, V., Crowder, D. A., Hassannia, M., Coales, S., Shi, Y., Viner, R., Rossotti, M. A., Sheff, J. G., Tanha, J., Schriemer, D. C.Hydrogen/deuterium exchange mass spectrometry (HX-MS) is a method used to study solution-phase protein structure and dynamics. Despite its many applications, HX-MS is limited in throughput because manual data analysis is still the norm. We previously developed HX-MS2 technology to add a second dimension of deuteration data and promote automated data processing. Data-independent acquisition (DIA) techniques enable this approach, but we require optimized methods for best performance. Using an Orbitrap Eclipse for illustration, we show that ion optics and collision energy […]
- by Welland, J. W. J., Barrow, H. G., Stansfeld, P. J., Deane, J. E.Glycosphingolipids (GSLs) are crucial membrane components involved in essential cellular pathways. Complex GSLs, known as gangliosides, are synthesised in the ER/Golgi by a series of glycosyltransferase enzymes. Imbalances in GSL metabolism can lead to severe diseases, often affecting the nervous system. B4GALNT1 is a key enzyme in the ganglioside synthetic pathway, synthesising complex gangliosides including GM2 and GD2 from GM3 and GD3. These products are precursors to the major brain gangliosides. Loss of B4GALNT1 function causes hereditary spastic paraplegia 26 […]
- by Liu, Y., Shi, L., Brosius, J., Mo, D.Efficient RNA production is essential for therapeutic applications. Currently, mRNA is generated by in vitro transcription with RNA polymerases at a constant temperature. The success of these transcriptions depends on various factors including sequence composition and size. Sometimes, especially for long RNAs, achieving efficient transcript yields are challenging. We developed a novel approach by incorporating a high-temperature denaturation step. Our protocol includes the following two steps of thermal cycling RNA transcription: the initial enzyme-driven RNA transcription step is done at […]
- by Yin, B., Hu, H.-H., Zhong, J., Liu, J.-L.Cryo-electron microscopy (cryo-EM) single-particle analysis has become a widely used technique for high-resolution structural determination of biological macromolecules and complexes. However, the determination of the structure of small molecule proteins remains a limitation of this technology. To address this issue, here we develop a novel approach termed Visualizing interested target via integrated scaffold of cytoophidium (VitisC). We use a filamentous structure formed by Escherichia coli CTP synthase (CTPS) as the scaffold, termed the scaffold of cytoophidium. Through artificial design and […]
- by Dent, M. R., DeMartino, A. W., Xu, Q., Chen, X., Ghandi, A., Hwang, J., Bocian, K. A., Alipour, E., Ucer, B., Baker, S. R., Srimath Kandada, A. R., Bulbul, A., Kim-Shapiro, D. B., Rose, J. J., Tejero, J., Gladwin, M. T.Carbon monoxide (CO) poisoning causes 50,000-100,000 emergency department visits and [~]1,500 deaths in the United States annually. Current treatments are limited to supplemental and/or hyperbaric oxygen to accelerate CO elimination. Even with oxygen therapy, nearly half of CO poisoning survivors suffer long-term cardiac and neurocognitive deficits related to slow CO clearance, highlighting a need for point of care antidotal therapies. Given the natural interaction between CO and ferrous heme, we hypothesized that the hemoprotein RcoM, a transcriptional regulator of microbial […]
- by Moser, S., Hans, G., Saiardi, A., Bru, S., Taskin, A., Meisinger, C., Jessen, H. J.Inorganic polyphosphate (polyP) is a ubiquitous biopolymer composed of multiple orthophosphates connected by energy-rich phosphoanhydride bonds. In organisms, polyP is digested by two types of enzymes: exopolyphosphatases, which shorten the chain from the ends by cleaving off monophosphate units, and endopolyphosphatases, which cut the chain internally. While several continuous methods are available to monitor exopolyphosphatase activity, endopolyphosphatase activity assays are less common and typically involve multiple tedious steps. Here, we introduce FRET-polyP8, a novel probe for real-time detection of endopolyphosphatase […]
- by Gauger, S. J., Palmelund, L. B., Tian, Y., Marek, A., Namini, M. R., Griem-Krey, N., Kickinger, S., Mortensen, J. S., Frolund, B., Wellendorph, P.The Ca2+/CaM-dependent protein kinase II alpha (CaMKII) is a highly important synaptic protein, which comprises a unique holoenzyme structure organized via the central hub domain. Recently, a distinct binding pocket in the CaMKII hub domain was identified for the endogenous neuromodulator {gamma}-hydroxybutyric acid (GHB) and related synthetic analogues. Key interacting residues in CaMKII were revealed, but the pronounced selectivity towards the alpha variant of CaMKII has remained unresolved. Aimed at elucidating the molecular determinants for this selectivity, we here conducted […]
- by Wang, F., Zhang, T., Zhu, J., Zhang, X., Zhang, C., Lai, L.Cyclic peptides have become a new focus in drug discovery due to their ability to bind challenging targets, including "undruggable" protein-protein interactions, with low toxicity. Despite their potential, general methods for de novo design of cyclic peptide ligands based on target protein structures remain limited. Here, we developed CYC_BUILDER, a reinforcement learning based fragment growing method for efficient assembly of peptide fragments and cyclization to generate diverse cyclic peptide binders for target proteins. CYC_BUILDER employs a Monte Carlo Tree Search […]
- by Yang, X., Medina, P. C., Gopalswamy, M., Vaidya, A., Schavier, S., Oltzen, S., Mossner, S., Huang, A., Qi, J., Floss, D., Uhrberg, M., Gohlke, H., Scheu, S.Beauvericin (BEA), a cyclic depsipeptide, is a mycotoxin of the enniatin family and the secondary metabolite of various toxigenic fungi. Multiple biological functions of BEA have been well investigated, such as anti-cancer, anti-inflammatory, anti-microbial, and immune-activating functions. In a recent study, we showed that BEA can target Toll-like receptor 4 (TLR4) to induce dendritic cell (DC) activation. In an in silico screen, we identified Cathepsin B (CTSB) as a potential additional interaction partner for BEA, which has been verified recently […]
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