- by Li, C., Efremov, R. G.Ryanodine receptors (RyRs) are intracellular tetrameric ion channels responsible for calcium; release from the sarcoplasmic and endoplasmic reticulum. Among the three known mammalian RyR isoforms, RyR1 is critical for muscle contraction and has been studied most extensively. The cytoplasm-exposed multidomain fragment of RyRs integrates multiple cellular signals that regulate channel gating and small deviations from the physiological open probability of RyRs leads to life-threatening diseases. While cryo-EM has been instrumental in revealing near-atomic details of RyR gating mechanisms, the open […]
- by Chauvire, T., Chandrasekaran, S., Dunleavy, R., Freed, J. H., CRANE, B. R.Flavin cofactors are attractive Electron Spin Resonance (ESR) probes for proteins because cellular reductants and light can generate their semiquinone states. We have used ESR spectroscopy to study the bacterial transmembrane aerotaxis receptor (Aer) in its native Escherichia coli membrane environment. Optimization of the spectroscopic (electronic relaxation times) and cell growth (isotopic labeling) conditions allowed for measurements of Aer with its partners – the histidine kinase (CheA) and the coupling protein (CheW) – in native signaling arrays. Continuous-wave ESR measurements […]
- by Guo, J., Zhang, Q., Cheng, F., Sang, M., Wang, X., Yu, H.-B., Hu, B., Wang, S., Zheng, L., Geng, C., Yang, C., Luo, L., Zhang, G., Du, L., Zhang, W., Wang, B., Li, S., Zhang, X.Thiolactomycin (1), which features a unique gama-thiolactone ring, is a promising antibiotic candidate that specifically targets bacterial type II fatty acid synthase. Despite extensive studies on its pharmacological activities, modes of action, and chemical synthesis, the enzymatic processes responsible for forming the activity-determining gama-thiolactone ring have remained largely unknown. Here, we resolve this problem by revealing that the condensation and heterocyclization (Cy) domain of the nonribosomal peptide synthetase (NRPS) TlnC (TlnCCy), along with the cytochrome P450 enzyme TlnA, cooperatively enable […]
- by Soubias, O., Foley, S. L., Jian, X., Jackson, R. A., Zhang, Y., Rosenberg, E. M., Li, J., Heinrich, F., Johnson, M. E., Sodt, A. J., Randazzo, P. A., Byrd, R. A.ASAP1 is a multidomain Arf GTPase-activating protein (ArfGAP) that catalyzes GTP hydrolysis on the small GTPase Arf1 and is implicated in cancer progression. The PH domain of ASAP1 enhances its activity greater than 7 orders of magnitude but the underlying mechanisms remain poorly understood. Here, we combined Nuclear Magnetic Resonance (NMR), Molecular Dynamic (MD) simulations and mathematical modeling of functional data to build a comprehensive structural-mechanistic model of the complex of Arf1 and the ASAP1 PH domain on a membrane […]
- by Calvario, J., Antunes, D., Cipriano, R., Kalafatovic, D., Mausa, G., Pina, A. S.Liquid-Liquid Phase Separation (LLPS) forms membraneless organelles, enhancing biochemical processes. The stickers-and-spacers model explains LLPS but is mainly validated in Prion-like RNA Binding Proteins. We explore peptide motifs in LLPS in broader protein contexts. We developed a computational approach for motif discovery, implemented in 178 Phase Separating Proteins (PhSePs), complemented by the FuzDrop and CIDER servers, which identified droplet-promoting regions (DPRs) and examined disorder-related characteristics. Our database of PhSePs was analyzed against proteins with low propensity for LLPS. This comparative […]
- by Shishikura, K., Li, J., Chen, Y., McKnight, N. R., Bustin, K. A., Barr, E. W., Chilkamari, S. R., Ayub, M., Kim, S. W., Lin, Z., Hu, R.-M., Hicks, K., Wang, X., O'Rourke, D. M., Bollinger, J. M., Binder, Z. A., Parsons, W. H., Martemyanov, K. A., Liu, A., Matthews, M. L.The vasodilator hydralazine (HYZ) has been used clinically for ~ 70 years and remains on the World Health Organization's List of Essential Medicines as a therapy for preeclampsia. Despite its longstanding use and the concomitant progress toward a general understanding of vasodilation, the target and mechanism of HYZ have remained unknown. We show that HYZ selectively targets 2-aminoethanethiol dioxygenase (ADO) by chelating its metal cofactor and alkylating one of its ligands. This covalent inactivation slows entry of proteins into the […]
- by Mann, S. G. A., Paz-Galeano, M., Shahsavarani, M., Perley, J. O., Guo, J., Garza-Garcia, J. J. O., Qu, Y.The Apocynaceae family produces a remarkable diversity of monoterpenoid indole alkaloids (MIAs), many of which possess significant pharmaceutical value. Among these, sarpagan and akuammiline alkaloids represent distinct subclasses characterized by their intricate stereochemical frameworks, derived through enzymatically controlled cyclization and rearrangement of the key precursor geissoschizine. In this study, we systematically investigated the products and stereochemical outcomes of sarpagan bridge enzymes (SBEs) and rhazimal synthases (RHS), key enzymes involved in geissoschizine cyclization and MIA diversification. Using two previously characterized enzymes […]
- by Campbell, L., Lowran, K., Cismas, E., Wu, C.Nucleic acid sequences that are rich in guanines can form G-quadruplex (G4) structures, which can impede DNA replication or repair. The FANCJ helicase plays a role in maintaining genomic stability by facilitating DNA replication through regions of DNA that form G4 structures. This activity has been associated with an AKKQ motif in FANCJ and it is thought that this motif retains the ability to also target 8-oxoguanine-modified G4 (8oxoG4) structures. We hypothesize that the molecular recognition of FANCJ AKKQ to […]
- by Jiang, Y., Gong, P., Li, Z., Li, Y., Wang, B., Peng, W., Gao, X., Li, S.Exploring and exploiting the catalytic promiscuity of enzymes is a central topic and captivating challenge in enzymology. CYP152 peroxygenases are attractive biocatalysts for diverse reactions under mild conditions using H2O2 as cofactor. However, their substrate scope is limited by a carboxyl group required for acid-base catalysis, following the well-accepted principle that heme-dependent H2O2-utilizing enzymes employ a carboxyl group within their active sites to facilitate H2O2 activation. Herein, we reveal for the first time that several CYP152 family members can directly […]
- by Eldeeb, M. H., Cosner, Z., Carlstrom, A., Mays, J.-N., Rodriguez, G. F., Berndtsson, J., Ott, M., Fontanesi, F.The mitochondrial respiratory chain (MRC) enzymatic complexes, essential for aerobic energy transduction in eukaryotic cells, are organized into evolutionarily conserved higher-order structures known as supercomplexes (SCs). The elucidation of the physiological relevance of respiratory SCs is essential for our understanding of mitochondrial function and cellular bioenergetics, yet it has been severely hampered by the limited availability of experimental models isolating SC formation as the sole variable. In the yeast Saccharomyces cerevisiae, where SCs are formed by the association of complexes […]
- by Dhahri, H., Lau, K. H., Saintilnord, W. N., Lopes, E., Damico, H. N., Palma, F. R., Melters, D. P., Chandler, D. P., Dalal, Y., Licht, J. D., Bonini, M. G., Fondufe-Mittendorf, Y.Histones scaffold genomic DNA and regulate access to the transcriptional machinery. However, naturally occurring histone variants can alter histone-DNA interactions, DNA and histone modifications, and the chromatin interactome. Hence, alterations in histone variant deposition can disrupt chromatin, and are increasingly recognized as a way to trigger various disease, including cancer. While significant attention has been placed on the biochemical and functional roles of H2A, H3, and H4 histone variants, the variants of H2B remain largely understudied. Here, we show that […]
- by Merrild, A., Svenningsen, T., Chevrette, M., Torring, T.Triculamin is a ribosomally synthesized and post-translationally modified peptide (RiPP) lasso peptide with potent antimycobacterial activity, produced by an unusual, non-canonical biosynthetic gene cluster (BGC). In this study, we elucidate the biosynthetic pathway of triculamin through heterologous expression and show that the biosynthesis proceeds in the presence of a precursor (triA), macrocyclase (triC), and acetyltransferase (triT). Through in vitro triT acetylation and bioactivity assays, we show that acetylation functions as a resistance mechanism. Genomic searches of triculamin BGC genes across […]
- by Mueller, F., Stejskal, K., Mechtler, K.The advancement of crosslinking mass spectrometry (CLMS) has significantly enhanced the ability to study protein-protein interactions and complex biological systems. This study evaluates the performance of the Orbitrap Astral and Eclipse mass spectrometers in CLMS workflows, focusing on the identification of low-abundance crosslinked peptides. The comparison employed consistent liquid chromatography setups and experimental conditions, using Cas9 crosslinked with PhoX and DSSO as quality control samples. Results demonstrated that the Astral analyzer outperformed the Eclipse, achieving over 40% more unique residue […]
- by Pfalzgraf, H. E., Rao, A. G., Sen, K., Adams, H. R., Edwards, M., Lu, Y., Yong, C., Jaho, S., Tosha, T., Sugimoto, H., Horrell, S., Beilsten-Edmands, J., Owen, R. L., Andrew, C. R., Worrall, J. A. R., Tews, I., Mulholland, A. J., Hough, M. A., Keal, T. W.Cytochromes P460 oxidise hydroxylamine within the nitrogen cycle and contain as their active site an unusual catalytic c-type heme where the porphyrin is cross-linked to the protein via a lysine residue in addition to the canonical cross links from cysteine residues. Understanding how enzymes containing P460 heme oxidise hydroxylamine into either nitrous oxide or nitric oxide has implications for climate change. Interestingly the P460 containing hydroxylamine oxidoreductase utilises a tyrosine cross link to heme and performs similar chemistry. Previous crystal […]
- by Archana, K., Bohra, B., Tripathi, R. K., Haldar, S.The Japanese encephalitis virus (JEV) is a mosquito-borne, enveloped flavivirus that causes acute encephalitis. Although JEV is increasingly recognized as a global threat, there is currently no FDA-approved treatment available for JEV. 25-hydroxycholesterol (25HC) is an oxysterol produced through the oxidation of cholesterol, a process catalyzed by cholesterol 25-hydroxylase (CH25H), which is an interferon-stimulated gene that is upregulated during viral infections. In this study, we report for the first time that 25HC effectively prevents JEV infection in cells. Our results […]
- by Kane, E. I., Trefs, L. S., Eckert, L., Coelho, S. M., Weir, J. R.Most extant eukaryotic systems share core meiosis-specific genes, suggesting meiosis evolved only once in the last eukaryotic common ancestor (LECA). These genes have been characterized as master regulators of meiotic recombination, ensuring genetically diverse lineages. However, our understanding is limited as eukaryotic organisms beyond the animal, plant, and yeast lineages remain poorly understood. Recently, core meiotic genes have been identified in the genome of the model brown alga Ectocarpus, but currently lack proper characterization. Here, we combine bioinformatic, structural, and […]
- by Xia, B., Kim, A.-R., Liu, F., Han, M., Stoneburner, E., Makdissi, S., Di Cara, F., Mohr, S., Ring, A., Perrimon, N.Nanobodies, single-domain antibodies derived from camelid heavy-chain antibodies, are known for their high affinity, stability, and small size, which make them useful in biological research and therapeutic applications. However, traditional nanobody generation methods rely on camelid immunization, which can be costly and time-consuming, restricting their practical feasibility. In this study, we present a phage-displayed synthetic library for nanobody discovery. To validate this approach, we screened nanobodies targeting various Drosophila secreted proteins. The nanobodies identified were suitable for applications such as […]
- by Mandala, V. S., MacKinnon, R.Gating in voltage-dependent ion channels is regulated by the transmembrane voltage. This form of regulation is enabled by voltage sensing domains (VSDs) that respond to transmembrane voltage differences by changing their conformation and exerting force on the pore to open or close it. Here we use cryogenic electron microscopy to study the neuronal Kv2.1 channel in lipid vesicles with and without a voltage difference across the membrane. Hyperpolarizing voltage differences displace the positively charged S4 helix in the voltage sensor […]
- by Gezelle, J. G., Korn, S. M., McDonald, J. T., Gong, Z., Erickson, A., Huang, C.-H., Yang, F., Cronin, M., Kuo, Y.-W., Wimberly, B. T., Steckelberg, A.-L.Exoribonuclease-resistant RNAs (xrRNAs) are viral RNA structures that block degradation by cellular 5'-3' exoribonucleases to produce subgenomic viral RNAs during infection. Initially discovered in flaviviruses, xrRNAs have since been identified in wide range of RNA viruses, including those that infect plants. High sequence variability among viral xrRNAs raises questions about the shared molecular features that characterize this functional RNA class. Here, we present the first structure of a plant-virus xrRNA in its active exoribonuclease-resistant conformation. The xrRNA forms a 9 […]
- by Yang, L., Pathiranage, V., Zhou, S., Sun, X., Zhang, H., Lai, C., Gu, C., Subach, F. V., Walker, A. R., Piatkevich, K. D.Potassium ion (K+) dynamics are vital for various biological processes. However, the limited availability of detection tools for tracking intracellular and extracellular K+ has impeded a comprehensive understanding of the physiological roles of K+ in intact biological systems. In this study, we developed two novel red genetically encoded potassium indicators (RGEPOs), RGEPO1 and RGEPO2, through a combination of directed evolution in E. coli and subsequent optimization in mammalian cells. RGEPO1, targeted to the extracellular membrane, and RGEPO2, localized in the […]
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