- by Yun, J., Yang, S., Kwon, J. H., Vecchietti, L. F., Cha, M., Chung, H. J., Oh, J. E., Kim, H. M.Interferon-{lambda}, a type III interferon that selectively targets epithelial cells, holds strong potential as an intranasal antiviral due to its ability to suppress respiratory virus replication without inducing systemic inflammation. However, clinical translation of human IFN-{lambda} is hindered by limited thermostability, protease susceptibility, and rapid mucosal clearance. In this study, instability-prone elements in hIFN-{lambda} are eliminated through artificial intelligence (AI)-based backbone remodeling and targeted surface hydrophobic patch engineering. A protease-sensitive loop is replaced with a de novo -helix, which shields […]
- by Hauf, S., Nakamura, R., Cellini, B., Yokobayashi, Y., Dindo, M.The emergence of the first catalytically active biopolymers remains a scientific mystery. Some abiotic chemistries for the formation of precursor molecules to biopolymers — such as nucleotides and amino acids — are known. However, abiotic polymerization usually results in short polymers of only a few units in length. This is too short for meaningful information storage or catalytic activity, a limitation known as the Flory Length Problem. Additionally, the first biocatalysts presumably had low activity because they were generated by […]
- by Montagud-Martinez, R., Ruiz, R., Baldanta, S., Delicado-Mateo, R., Rodrigo, G.Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key structural determinants underlying this function. First, we show that open R-loops in the PAM-distal region favor trans-cleavage activity, which occur when targeting short double-stranded or single-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impairs this collateral activity. Third, although trans-cleavage is mediated by the RuvC domain, we show that […]
- by Kar, M.FET (FUS-EWSR1-TAF15) family proteins inherently form mesoscale molecular assemblies, known as clusters, under physiological conditions at concentrations well below the threshold for phase separation. This study demonstrates that adenosine triphosphate (ATP), an amphiphilic molecule and essential cellular metabolite, modulates the size of these sub-saturation mesoscale clusters in a concentration-dependent manner. At low concentrations (1-2 mM), ATP acts as a crosslinker for FET proteins, resulting in larger size clusters. At moderate concentrations (5 mM), the size of the clusters decreases but […]
- by Paraschiakos, T., Yuan, B., Sopelniak, K., Bucher, M., Simon, L., Zonjic, K., Eggers, D., Selle, F., Li, J., Linder, S., Marlovits, T. C., Windhorst, S.Regulation of the actin cytoskeleton by actin binding proteins (ABPs) is essential for cellular homeostasis, and the mode of actin binding determines the activity of ABPs. Here, we discovered a novel Short linear F-actin binding motif (SFM) on the basis of the cryo-EM structure of the ITPKA-F-actin complex. We developed the computational pipeline SLiMFold, which identified 103 human SFM containing-proteins exhibiting diverse cellular functions. The SFM probably developed ex nihilo and remained conserved in eukaryotes, with a binding affinity to […]
- by Ustock, F. I., Huntington, J. A.Thrombin is generated from its precursor prothrombin by sequential cleavage at Arg320 and Arg271 by the prothrombinase complex, composed of factor (f) Xa and fVa on phospholipid (PL) membrane surfaces. The affinity of human fXa for fVa is low in the absence of PL. However, fXa orthologues from the venom of group D snakes bind to fVa with high affinity, forming an active complex without PL. We previously characterized the properties of the fXa orthologue Hopsarin D (HopD) from Hoplocephalus […]
- by Chan, H.-J., Krichel, B., Bandura, L. J., Chapman, E. A., Rogers, H. T., Fischer, M. S., Roberts, D. S., Gao, Z., Wang, M.-D., Wu, J., Uetrecht, C., Jin, S., Ge, Y.AMP-activated protein kinase (AMPK) is a heterotrimeric complex ({beta}{gamma}) that serves as a master regulator of cellular metabolism, making it a prominent drug target for various diseases. Post-translational modifications (PTMs) and ligand binding significantly affect the activity and function of AMPK. However, the dynamic interplay of PTMs, non-covalent interactions, and higher-order structures of the kinase complex remains poorly understood. Herein, we report the structural heterogeneity of the AMPK complex arising from ligand binding and proteoforms–protein products derived from PTMs, alternative […]
- by Schlichthaerle, T., Yang, A., Detraux, D., Johnson, D. E., Peach, C. J., Edman, N. I., Sniezek, C., Williams, C. A., Arora, S., Katiyar, N., Chen, I., Etemadi, A., Favor, A., Lee, D., Kubo, C., Coventry, B., Huang, B., Gerben, S., Ennist, N. M., Milles, L., Sankaran, B., Kang, A., Nguyen, H., Bera, A. K., Negahdari, B., Hamazaki, N., Schweppe, D. K., Stewart, L., Young, J. E., Bunnett, N. W., Ruohola-Baker, H., Mathieu, J., Pattwell, S., Garcia, K. C., Baker, D.The clinical use of Nerve Growth Factor (NGF) for neuronal regeneration has been hampered by pain sensitization side effects. NGF signals through the receptor tyrosine kinase TrkA and the co-receptor p75NTR; pain sensitization is thought to involve p75NTR. We sought to overcome this limitation by de novo design of a TrkA agonist that does not bind p75NTR. We designed homodimeric TrkA engaging constructs that dimerize TrkA subunits in a variety of geometries, and identified those eliciting the strongest signaling. The […]
- by Neidhardt, L., Tung, J., Kuchersky, M., Milczarek, J., Kargas, V., Stott, K., Rosenzweig, R. S., Ron, D., Yan, Y.The endoplasmic reticulum (ER) unfolded protein response (UPR) is tuned by the balance between unfolded proteins and chaperones. While reserve chaperones are known to suppress the UPR transducers via their stress-sensing luminal domains, the underlying structural mechanisms remain unclear. Cellular and biophysical analyses established that the ER chaperone AGR2 forms a repressive complex with the luminal domain of the UPR transducer IRE1{beta}. Structural prediction, X-ray crystallography and NMR spectroscopy identify critical interactions between an AGR2 monomer and a regulatory loop […]
- by Carrizales, M. G., Boulton, A., Koleske, A. J.The TRIO protein contains two guanine exchange factor (GEF) domains, GEF1 and GEF2, which coordinate cytoskeletal rearrangements by activating Rho family GTPases. Rare variants that impact TRIO GEF1 function are associated with autism spectrum disorder, developmental delay, and intellectual disability, but variants are also found throughout the gene. GEF1 promotes GTP exchange on Rac1 and RhoG, while GEF2 activates RhoA. Although GEF1 and GEF2 share a common architecture, the pleckstrin homology (PH) domain in TRIO GEF1 (PH1) assists its activity, […]
- by Sakuma, M., Mahato, D. R., Feixas, F., Jackson, C. J., Nakata, E., Osuna, S., Tokuriki, N.Enzymes achieve catalysis by dynamically sampling diverse conformational states. Beyond this plasticity, individual enzyme molecules occupy metastable substates, forming an ensemble of functional substates within a population. Since shifts in functional substate dynamics drive phenotypic variation, their evolutionary trajectories are central to the emergence of new functions. However, the challenge of measuring functional substates has hindered our understanding of their role in enzyme evolution and the optimization of conformational substates. Here, we address this gap by investigating how functional and […]
- by Fernandes, M. V., Antunes, J. M. A., Salgueiro, C. A., Morgado, L.Electroactive bacteria mediate electron exchange with external surfaces through a process known as extracellular electron transfer (EET). A key step in EET is the transfer of electrons from the menaquinone pool to inner membrane-associated quinone-cytochrome c oxidoreductase complexes, which subsequently relay electrons to periplasmic redox partners. Gene knockout and proteomic analyses have identified several critical components involved in EET in Geobacter sulfurreducens, including six inner membrane oxidoreductase gene clusters. Of these, three – CbcL, ImcH, and CbcBA – have been […]
- by Barentsen, R., Kronborg, K., Brodersen, D. E., ZHANG, Y. E.Understanding how bacteria rapidly adapt their metabolism in response to external stimuli is key to addressing the present crisis of antibiotics-resistant infections. In the Gram-negative bacterium Escherichia coli, the universal stringent response is elicited in response to some antibiotics and involves production of the global alarmones, (p)ppGpp, which bind directly to many cellular targets. The nucleosidase PpnN that cleaves nucleotides into 5-phosphate ribose and nucleobase, was shown to be a target of (p)ppGpp and control the delicate balance of bacterial […]
- by Wang, F., He, Q., O'Donnell, M. E., Li, H.Significance StatementReplicative DNA polymerases (Pol) function with a sliding clamp and their proofreading exonuclease provides high fidelity. Thus, study of the proofreading mechanism must both contain the Pol-clamp complex and generate the mismatch in situ to ensure the mismatched primer follows a physiological route from the pol to exo site. Despite numerous previous studies, none satisfy both criteria. This study on human Pol{varepsilon}-PCNA meets both criteria. Cryo-EM analysis captured proofreading intermediates that reveal an unexpected proofreading process. The primer is […]
- by Zhu, B., Van, R., Wang, H., Kuang, S., Jia, Y., Leon, E. C., Yang, F., Zhang, J., Yang, J., Hong, H., Fleur Marie, L., Yu, A., Wang, J., Tanzi, R. E., Zhang, C. M., Mao, X. M., Shao, Y., Ran, C.Visualizing misfolded proteins would greatly facilitate early diagnosis, etiology elucidation, and therapy monitoring of neurodegeneration. Although several probes have been reported, simple and versatile detection in vivo is still challenging. We demonstrated that both generic and precise detection of misfolded proteins could be achieved with a chemiluminescence probe, ADLumin-1. For the generic aspect, ADLumin-1 was highly sensitive to various misfolded proteins, showing up to 127.73-fold higher signal-to-noise ratio than Thioflavin T. ADLumin-1 could also non-invasively visualize misfolded proteins in mouse […]
- by Paredes, A., Singh, H., Hull, M. T., Greene, D., Northrup, A. J., Brown, J. B., Chacon, K. N., Patrauchan, M. A., Smith, A. T.Pseudomonas aeruginosa is a ubiquitous, Gram-negative bacterium that forms biofilms and is responsible for antibiotic-resistant nosocomial infections in humans. The P. aeruginosa BqsRS two-component system (TCS) regulates biofilm formation and dispersal by sensing extracytoplasmic Fe2+, but the mechanistic details of this process are poorly understood. In this work, we report the crystal and solution structures of the PaBqsR response regulator (RR) receiver domain (RD), comprising a ({beta})5 response regulator assembly, and DNA-binding domain (DBD), comprising a winged helix-turn-helix motif. Consistent […]
- by Dickson, C. J.The KRasG12D mutant is an attractive target in oncology and can now be drugged via the switch-II allosteric pocket. Non-covalent ligands typically bind in the presence of a conserved structural water, which interacts with Thr58 and Gly10. In this work, we use a dataset of published non-covalent KRasG12D inhibitors to evaluate free energy methods for the interaction with or displacement of this conserved water molecule. We find that the Thr58 water and a second proximal water site are predicted to […]
- by Karimian Shamsabadi, M., Stoneham, C., De-leon, A., Fares, T., Guatelli, J., Jia, X.Human transmembrane proteins Serinc3 and Serinc5 are antiviral restriction factors that inhibit HIV-1 infectivity. In the absence of viral antagonism, Serinc3 and Serinc5 incorporate into the envelopes of nascent virions and inhibit the fusion of virions to the target cells. The HIV-1 virus counteracts the restriction of Serinc3 by downregulating it from the cell surface and thus excluding it from budding virions. This is orchestrated by the viral accessory protein Nef and involves hijacking of the clathrin adaptor protein complex […]
- by Romenskaia, I., Mastick, C. C., Coster, A.In mammalian fat and muscle cells, insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular storage compartments to the plasma membrane in adipocytes and muscle cells, significantly increasing glucose uptake. In unstimulated (basal) cells, GLUT4 is sequestered in non-cycling/very slowly cycling compartments. Insulin mobilizes GLUT4 by releasing it from sequestration, enabling its exocytosis and continuous cycling between the plasma membrane and endosomes. Upon insulin withdrawal, GLUT4 is rapidly internalized and re-sequestered internally for future activation. Dynamic studies using […]
- by Warrick, J. E., Attili, D., van Eeuwen, T., Hoffmann-Weitsman, S. E., Forsyth, N. C., Barmada, S. J., Kearse, M. G.The loss and mutation of Topoisomerase 3{beta} (TOP3B), the only known eukaryotic topoisomerase with the ability to catalyze RNA strand passage reactions, is linked to schizophrenia, autism, and intellectual disability. Uniquely, TOP3B primarily localizes to the cytoplasm and has been shown to regulate translation and stability of a subset of mRNA transcripts. Three neurological disease-linked de novo TOP3B point mutations outside of the active site have been identified but their impact on TOP3B activity in cells remains poorly understood. Upon […]
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