scholarly journals Structural basis of dynamic P5CS filaments

2021 ◽  
Author(s):  
Jiale Zhong ◽  
Chen-Jun Guo ◽  
Xian Zhou ◽  
Chia-Chun Chang ◽  
Boqi Yin ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Essential Role ◽  
Point Mutations ◽  
Underlying Mechanism ◽  
Structural Basis ◽  
Multiple Interfaces ◽  
Cryo Electron Microscopy ◽  
Neurocutaneous Syndrome

AbstractThe bifunctional enzyme Δ1-pyrroline-5-carboxylate synthase (P5CS) is central to the synthesis of proline and ornithine. Pathogenic mutations in P5CS gene (ALDH18A1) lead to neurocutaneous syndrome and skin relaxation connective tissue disease in humans, and P5CS deficiency seriously damages the ability to resist adversity in plants, which has an essential role in agriculture and human health. Recently, P5CS has been demonstrated forming the cytoophidium in vivo and filaments in vitro. However, the underlying mechanism for the function of P5CS filamentation and catalyze the synthesis of P5C is hardly accessible without structural basis. Here, we have succeeded in determining the full-length structures of Drosophila P5CS filament in three states at resolution from 3.1 to 4.3 Å under cryo-electron microscopy, we observed the distinct ligand-binding states and conformational changes for GK and GPR domain separately. These structures show the distinctive spiral filament is assembled by P5CS tetramers and stabilized by multiple interfaces. Point mutations that deplete such interactions disturb P5CS filamentation and greatly reduce the activity. Our findings reveal a previously undescribed mechanism that filamentation is crucial for the coordination between GK and GPR domains, and provide insights into structural basis for catalysis function of P5CS filament.

scholarly journals Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

2020 ◽  
Author(s):  
Tilak Kumar Gupta ◽  
Sven Klumpe ◽  
Karin Gries ◽  
Steffen Heinz ◽  
Wojciech Wietrzynski ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Point Mutations ◽  
Binding Pocket ◽  
Thylakoid Membranes ◽  
Lipid Binding ◽  
Structural Basis ◽  
Amphipathic Helices ◽  
Cryo Electron Microscopy

AbstractVesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-shaping function. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket that is required for VIPP1 oligomerization. Inside the ring’s lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo point mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Our study provides a structural basis for understanding how the oligomerization of VIPP1 drives the biogenesis of thylakoid membranes and protects these life-giving membranes from environmental stress.

Structural basis for pH-dependent retrieval of ER proteins from the Golgi by the KDEL receptor

Science ◽  
2019 ◽  
Vol 363 (6431) ◽  
pp. 1103-1107 ◽  
Author(s):  
Philipp Bräuer ◽  
Joanne L. Parker ◽  
Andreas Gerondopoulos ◽  
Iwan Zimmermann ◽  
Markus A. Seeger ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Secretory Pathway ◽  
Cell Function ◽  
Interaction Network ◽  
Eukaryotic Cell ◽  
Structural Basis ◽  
Ph Dependent ◽  
Kdel Receptor

Selective export and retrieval of proteins between the endoplasmic reticulum (ER) and Golgi apparatus is indispensable for eukaryotic cell function. An essential step in the retrieval of ER luminal proteins from the Golgi is the pH-dependent recognition of a carboxyl-terminal Lys-Asp-Glu-Leu (KDEL) signal by the KDEL receptor. Here, we present crystal structures of the chicken KDEL receptor in the apo ER state, KDEL-bound Golgi state, and in complex with an antagonistic synthetic nanobody (sybody). These structures show a transporter-like architecture that undergoes conformational changes upon KDEL binding and reveal a pH-dependent interaction network crucial for recognition of the carboxyl terminus of the KDEL signal. Complementary in vitro binding and in vivo cell localization data explain how these features create a pH-dependent retrieval system in the secretory pathway.

scholarly journals Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Janine Weber ◽  
Han Bao ◽  
Christoph Hartlmüller ◽  
Zhiqin Wang ◽  
Almut Windhager ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nucleic Acid ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Point Mutations ◽  
Structural Basis ◽  
Binding Modes ◽  
Nucleic Acid Binding

The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

scholarly journals Structural basis of prokaryotic ubiquitin-like protein engagement and translocation by the mycobacterial Mpa-proteasome complex

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Mikhail Kavalchuk ◽  
Ahmad Jomaa ◽  
Andreas U. Müller ◽  
Eilika Weber-Ban
Keyword(s):  
Electron Microscopy ◽  
Mycobacterium Tuberculosis ◽  
Early Stage ◽  
Structural Basis ◽  
Human Pathogen ◽  
Core Particle ◽  
Cryo Electron Microscopy ◽  
Complementary Interactions

AbstractProteasomes are present in eukaryotes, archaea and Actinobacteria, including the human pathogen Mycobacterium tuberculosis, where proteasomal degradation supports persistence inside the host. In mycobacteria and other members of Actinobacteria, prokaryotic ubiquitin-like protein (Pup) serves as a degradation tag post-translationally conjugated to target proteins for their recruitment to the mycobacterial proteasome ATPase (Mpa). Here, we use single-particle cryo-electron microscopy to determine the structure of Mpa in complex with the 20S core particle at an early stage of pupylated substrate recruitment, shedding light on the mechanism of substrate translocation. Two conformational states of Mpa show how substrate is translocated stepwise towards the degradation chamber of the proteasome core particle. We also demonstrate, in vitro and in vivo, the importance of a structural feature in Mpa that allows formation of alternating charge-complementary interactions with the proteasome resulting in radial, rail-guided movements during the ATPase conformational cycle.

Structural basis of a novel activity of bacterial 6-pyruvoyltetrahydropterin synthase homologues distinct from mammalian 6-pyruvoyltetrahydropterin synthase activity

2014 ◽  
Vol 70 (5) ◽  
pp. 1212-1223 ◽  
Author(s):  
Kyung Hye Seo ◽  
Ningning Zhuang ◽  
Young Shik Park ◽  
Ki Hun Park ◽  
Kon Ho Lee
Keyword(s):  
Active Site ◽  
Essential Role ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Side Chain ◽  
Wild Type ◽  
Specific Enzyme ◽  
Aromatic Residues

Escherichia coli6-carboxytetrahydropterin synthase (eCTPS), a homologue of 6-pyruvoyltetrahydropterin synthase (PTPS), possesses a much stronger catalytic activity to cleave the side chain of sepiapterinin vitrocompared with genuine PTPS activity and catalyzes the conversion of dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterinin vivo. Crystal structures of wild-type apo eCTPS and of a Cys27Ala mutant eCTPS complexed with sepiapterin have been determined to 2.3 and 2.5 Å resolution, respectively. The structures are highly conserved at the active site and the Zn2+binding site. However, comparison of the eCTPS structures with those of mammalian PTPS homologues revealed that two specific residues, Trp51 and Phe55, that are not found in mammalian PTPS keep the substrate bound by stacking it with their side chains. Replacement of these two residues by site-directed mutagenesis to the residues Met and Leu, which are only found in mammalian PTPS, converted eCTPS to the mammalian PTPS activity. These studies confirm that these two aromatic residues in eCTPS play an essential role in stabilizing the substrate and in the specific enzyme activity that differs from the original PTPS activity. These aromatic residues Trp51 and Phe55 are a key signature of bacterial PTPS enzymes that distinguish them from mammalian PTPS homologues.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

scholarly journals Compensatory mechanisms in resistant Anopheles gambiae AcerKis and KdrKis neurons modulate insecticide-based mosquito control

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Stéphane Perrier ◽  
Eléonore Moreau ◽  
Caroline Deshayes ◽  
Marine El-Adouzi ◽  
Delphine Goven ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Anopheles Gambiae ◽  
Calcium Concentration ◽  
Mosquito Control ◽  
Point Mutations ◽  
Resting Membrane Potential ◽  
Compensatory Mechanisms ◽  
Pyrethroid Insecticides

AbstractIn the malaria vector Anopheles gambiae, two point mutations in the acetylcholinesterase (ace-1R) and the sodium channel (kdrR) genes confer resistance to organophosphate/carbamate and pyrethroid insecticides, respectively. The mechanisms of compensation that recover the functional alterations associated with these mutations and their role in the modulation of insecticide efficacy are unknown. Using multidisciplinary approaches adapted to neurons isolated from resistant Anopheles gambiae AcerKis and KdrKis strains together with larval bioassays, we demonstrate that nAChRs, and the intracellular calcium concentration represent the key components of an adaptation strategy ensuring neuronal functions maintenance. In AcerKis neurons, the increased effect of acetylcholine related to the reduced acetylcholinesterase activity is compensated by expressing higher density of nAChRs permeable to calcium. In KdrKis neurons, changes in the biophysical properties of the L1014F mutant sodium channel, leading to enhance overlap between activation and inactivation relationships, diminish the resting membrane potential and reduce the fraction of calcium channels available involved in acetylcholine release. Together with the lower intracellular basal calcium concentration observed, these factors increase nAChRs sensitivity to maintain the effect of low concentration of acetylcholine. These results explain the opposite effects of the insecticide clothianidin observed in AcerKis and KdrKis neurons in vitro and in vivo.

scholarly journals GCH1 induces immunosuppression through metabolic reprogramming and IDO1 upregulation in triple-negative breast cancer

2021 ◽  
Vol 9 (7) ◽  
pp. e002383
Author(s):  
Jin-Li Wei ◽  
Si-Yu Wu ◽  
Yun-Song Yang ◽  
Yi Xiao ◽  
Xi Jin ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Molecular Taxonomy ◽  
Underlying Mechanism ◽  
Metabolic Reprogramming ◽  
Sequencing Data ◽  
Aryl Hydrocarbon

PurposeRegulatory T cells (Tregs) heavily infiltrate triple-negative breast cancer (TNBC), and their accumulation is affected by the metabolic reprogramming in cancer cells. In the present study, we sought to identify cancer cell-intrinsic metabolic modulators correlating with Tregs infiltration in TNBC.Experimental designUsing the RNA-sequencing data from our institute (n=360) and the Molecular Taxonomy of Breast Cancer International Consortium TNBC cohort (n=320), we calculated the abundance of Tregs in each sample and evaluated the correlation between gene expression levels and Tregs infiltration. Then, in vivo and in vitro experiments were performed to verify the correlation and explore the underlying mechanism.ResultsWe revealed that GTP cyclohydrolase 1 (GCH1) expression was positively correlated with Tregs infiltration and high GCH1 expression was associated with reduced overall survival in TNBC. In vivo and in vitro experiments showed that GCH1 increased Tregs infiltration, decreased apoptosis, and elevated the programmed cell death-1 (PD-1)-positive fraction. Metabolomics analysis indicated that GCH1 overexpression reprogrammed tryptophan metabolism, resulting in L-5-hydroxytryptophan (5-HTP) accumulation in the cytoplasm accompanied by kynurenine accumulation and tryptophan reduction in the supernatant. Subsequently, aryl hydrocarbon receptor, activated by 5-HTP, bound to the promoter of indoleamine 2,3-dioxygenase 1 (IDO1) and thus enhanced the transcription of IDO1. Furthermore, the inhibition of GCH1 by 2,4-diamino-6-hydroxypyrimidine (DAHP) decreased IDO1 expression, attenuated tumor growth, and enhanced the tumor response to PD-1 blockade immunotherapy.ConclusionsTumor-cell-intrinsic GCH1 induced immunosuppression through metabolic reprogramming and IDO1 upregulation in TNBC. Inhibition of GCH1 by DAHP serves as a potential immunometabolic strategy in TNBC.

scholarly journals Characterization of LDD-2633 as a Novel RET Kinase Inhibitor with Anti-Tumor Effects in Thyroid Cancer

2021 ◽  
Vol 14 (1) ◽  
pp. 38
Author(s):  
Hyo Jeong Lee ◽  
Pyeonghwa Jeong ◽  
Yeongyu Moon ◽  
Jungil Choi ◽  
Jeong Doo Heo ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Thyroid Carcinoma ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Carcinoma Cells ◽  
Medullary Thyroid ◽  
Potent Inhibition ◽  
Kinase Inhibitory Activity

Rearranged during transfection (RET), a receptor tyrosine kinase, is activated by glial cell line-derived neurotrophic factor family ligands. Chromosomal rearrangement or point mutations in RET are observed in patients with papillary thyroid and medullary thyroid carcinomas. Oncogenic alteration of RET results in constitutive activation of RET activity. Therefore, inhibiting RET activity has become a target in thyroid cancer therapy. Here, the anti-tumor activity of a novel RET inhibitor was characterized in medullary thyroid carcinoma cells. The indirubin derivative LDD-2633 was tested for RET kinase inhibitory activity. In vitro, LDD-2633 showed potent inhibition of RET kinase activity, with an IC50 of 4.42 nM. The growth of TT thyroid carcinoma cells harboring an RET mutation was suppressed by LDD-2633 treatment via the proliferation suppression and the induction of apoptosis. The effects of LDD-2633 on the RET signaling pathway were examined; LDD-2633 inhibited the phosphorylation of the RET protein and the downstream molecules Shc and ERK1/2. Oral administration of 20 or 40 mg/kg of LDD-2633 induced dose-dependent suppression of TT cell xenograft tumor growth. The in vivo and in vitro experimental results supported the potential use of LDD-2633 as an anticancer drug for thyroid cancers.

scholarly journals E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

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