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| Open AccessNanoparticles and photochemistry for native-like transmembrane protein footprinting
The intrinsic flexibility of membranes proteins still poses a challenge in determining their active structure. Here the authors describe the development of a method that combines chemical footprinting and mass spectrometry to assist in determining the structure of native membrane proteins and their dynamics.
- Jie Sun
- , Xiaoran Roger Liu
- & Michael L. Gross
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Article
| Open AccessTime-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP
EF-G drives ribosomal translocation along mRNA. Time-resolved cryo-EM captured translocation with EF-G•GTP—without inhibitors—revealing how EF-G uses ribosome fluctuations to drive translocation and GTP hydrolysis to leave at the right moment.
- Christine E. Carbone
- , Anna B. Loveland
- & Andrei A. Korostelev
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Article
| Open AccessHow to build a ribosome from RNA fragments in Chlamydomonas mitochondria
Mitoribosomes are remarkably diverse in their structures and compositions. Here the authors combine biochemistry, genetics, single particle cryo-electron microscopy and in situ cryo-electron tomography to reveal the mitochondrial ribosome of Chlamydomonas reinhardtii as an extreme example of evolution and species-specific adaptation.
- Florent Waltz
- , Thalia Salinas-Giegé
- & Yaser Hashem
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Article
| Open AccessStructural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch
Many RNA viruses employ programmed –1 ribosomal frameshifting (PRF) to expand their coding capacity and optimize production of viral proteins. Here, the authors report structural and biophysical analysis of protein 2A from a cardiovirus, with insights into the mechanism of its PRF-stimulatory function.
- Chris H. Hill
- , Lukas Pekarek
- & Ian Brierley
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Article
| Open AccessHedgehog-Interacting Protein is a multimodal antagonist of Hedgehog signalling
Hedgehog-Interacting Protein (HHIP) is the only reported secreted inhibitor of Sonic Hedgehog (SHH) signalling. Here, the authors report structures of the HHIP N- and C-terminal domains, both in complexes with glycosaminoglycans, providing insights into the molecular basis for SHH sequestration and inhibition.
- Samuel C. Griffiths
- , Rebekka A. Schwab
- & Christian Siebold
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Article
| Open AccessBiphasic activation of β-arrestin 1 upon interaction with a GPCR revealed by methyl-TROSY NMR
β-arrestins commonly bind to two distinct elements in GPCRs: the phosphorylated carboxyl terminal tail (C tail) and the cytoplasmic face of the transmembrane region (TM core). Here, the authors use methyl-TROSY NMR measurements to characterise the interactions between β-arrestin 1 (βarr1) and a GPCR and observe that C tail-mediated interaction with a GPCR alone induces the partial activation of βarr1, whereas the TM core- and C tail-mediated interactions together stabilize the activated conformation of βarr1.
- Yutaro Shiraishi
- , Yutaka Kofuku
- & Ichio Shimada
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Article
| Open AccessSmall molecule modulation of the Drosophila Slo channel elucidated by cryo-EM
Slowpoke (Slo) channels are voltage-gated potassium channels that are activated by high intracellular Ca2+ concentrations, and they are targets for insecticides and antiparasitic drugs. Here, the authors present the cryo-EM structures of the Drosophila melanogaster Slo channel in the Ca2+-bound and Ca2+-free conformations, as well as in complex with the fungal neurotoxin verruculogen and the anthelmintic drug emodepside and discuss the mechanisms by which they affect the activity of Slo.
- Tobias Raisch
- , Andreas Brockmann
- & Stefan Raunser
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Article
| Open AccessStructural basis for transthyretin amyloid formation in vitreous body of the eye
Systemic ATTR amyloidosis causes the abnormal accumulation of ATTR fibrils formed from the human plasma protein transthyretin (TTR) in multiple organs including the eye. Here, the authors present a 3.2 Å cryo-EM structure of an ATTR fibril isolated from the vitreous body of an ATTR patient’s eye and discuss the mechanism for the structural conversion of TTR into a fibrillar form.
- Irina Iakovleva
- , Michael Hall
- & A. Elisabeth Sauer-Eriksson
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Article
| Open AccessSliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket – targeting P-pocket by fragment screening
Here the authors observe a transient P-pocket created when HIV reverse transcriptase slides over DNA substrate, identify fragments targeting this pocket, and develop a cryo-EM platform for lead optimization.
- Abhimanyu K. Singh
- , Sergio E. Martinez
- & Kalyan Das
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Article
| Open AccessPlasticity within the barrel domain of BamA mediates a hybrid-barrel mechanism by BAM
The β-barrel assembly machinery (BAM) assists the folding and membrane insertion of bacterial outer membrane proteins. Here, the authors report structural characterization of BAM in lipid environment and in complex with the client protein EspP integrated into the barrel of BamA, providing insight into BAM mechanism of function.
- Runrun Wu
- , Jeremy W. Bakelar
- & Nicholas Noinaj
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Article
| Open AccessMolecular basis for redox control by the human cystine/glutamate antiporter system xc−
System xc- is a cystine transporter that is expressed in the plasma membrane and imports cystine in exchange for intracellular glutamate. Here, the authors present the cryo-EM structure of human system xc- both in the apo form and the glutamate bound state, and further supported by molecular dynamics and cell-based assays they discuss its cystine transport mechanism.
- Joanne L. Parker
- , Justin C. Deme
- & Simon Newstead
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Article
| Open AccesseIF2B-capturing viral protein NSs suppresses the integrated stress response
Here the authors show that a viral protein interferes with the binding of phosphorylated eIF2 to eIF2B, thereby suppressing the host integrated stress response (ISR). This suppression of the ISR abrogates translational changes of the host and ameliorates neurite degradation under stress.
- Kazuhiro Kashiwagi
- , Yuichi Shichino
- & Takuhiro Ito
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Article
| Open AccessStructural basis of reactivation of oncogenic p53 mutants by a small molecule: methylene quinuclidinone (MQ)
The tumor suppressor p53 is mutated in more than half of human cancers and the compound methylene quinuclidinone (MQ) was shown to reactivate p53 mutants by binding covalently to cysteine residues. Here, the authors present crystal structures of wild-type and cancer related p53 mutant core domains bound to MQ alone and in complex with their DNA response elements and observe that MQ is bound to several cysteine residues located at the surface of the core domain.
- Oksana Degtjarik
- , Dmitrij Golovenko
- & Zippora Shakked
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Article
| Open AccessDeepRank: a deep learning framework for data mining 3D protein-protein interfaces
The authors present DeepRank, a deep learning framework for the data mining of large sets of 3D protein-protein interfaces (PPI). They use DeepRank to address two challenges in structural biology: distinguishing biological versus crystallographic PPIs in crystal structures, and secondly the ranking of docking models.
- Nicolas Renaud
- , Cunliang Geng
- & Li C. Xue
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Article
| Open AccessMechanism of phosphate sensing and signaling revealed by rice SPX1-PHR2 complex structure
SPX proteins sense phosphate levels in plant cells by binding to inositol polyphosphates (InsP) and suppressing the activity of PHR transcription factors. Here the authors show that when bound to InsP6, the rice SPX1 protein inhibits the activity of PHR2 by attenuating both its dimerization and DNA binding activity.
- Jia Zhou
- , Qinli Hu
- & Weiman Xing
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Article
| Open AccessRegulation of the EphA2 receptor intracellular region by phosphomimetic negative charges in the kinase-SAM linker
Eph receptor tyrosine kinases and their ephrin ligands mediate cell-cell communication. Here, the authors assess the structure and dynamics of the EphA2 intracellular region and uncover complex effects of phosphorylation within the linker region between EphA2 kinase and SAM domains.
- Bernhard C. Lechtenberg
- , Marina P. Gehring
- & Elena B. Pasquale
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Article
| Open AccessDiscovery of an exosite on the SOCS2-SH2 domain that enhances SH2 binding to phosphorylated ligands
SOCS2 is a key regulator of growth hormone and cytokine signaling, which recognizes phosphotyrosine (pTyr)-modified targets via a central SH2 domain. Here, the authors discover and characterize an exosite on this SH2 domain that can bind a non-phosphorylated peptide to enhance SOCS2:pTyr affinity.
- Edmond M. Linossi
- , Kunlun Li
- & Sandra E. Nicholson
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Article
| Open AccessCatalytic flexibility of rice glycosyltransferase OsUGT91C1 for the production of palatable steviol glycosides
Steviol glycosides from the plant Stevia rebaudiana are already used as lowcalorie sweeteners, but the most abundant naturally occurring compounds have a bitter aftertaste. Here, the authors characterize and engineer rice glycosyltransferase OsUGT91C1 to facilitate the large-scale production of naturally rare but palatable glycosides Reb D and Reb M
- Jinzhu Zhang
- , Minghai Tang
- & Wei Cheng
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Article
| Open AccessConformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity
The L protein of segmented, negative strand RNA viruses contains the RNA-dependent RNA polymerase essential for virus amplification. Here, the authors report cryoEM structures of the Lassa virus L protein in active, RNA-bound states, and provide mechanistic insights.
- Tomas Kouba
- , Dominik Vogel
- & Stephen Cusack
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Article
| Open AccessKinetic and structural mechanism for DNA unwinding by a non-hexameric helicase
UvrD is a model helicase from the non-hexameric Superfamily 1. Here, the authors use optical tweezers to measure directly the stepwise translocation of UvrD along a DNA hairpin, and propose a mechanism in which UvrD moves one base pair at a time, but sequesters the nascent single strands, releasing them after a variable number of ATP hydrolysis cycles.
- Sean P. Carney
- , Wen Ma
- & Yann R. Chemla
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Article
| Open AccessMechanism of Rad26-assisted rescue of stalled RNA polymerase II in transcription-coupled repair
Here the authors provide models of RNA polymerase II bound to the yeast CSB ortholog Rad26 in different nucleotide states; explain how Rad26 domain motions help the polymerase progress past DNA lesions; and interpret the effects of CSB-associated disease mutations.
- Chunli Yan
- , Thomas Dodd
- & Ivaylo Ivanov
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Article
| Open AccessMounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin
Rearrangement hot spots (Rhs) proteins are bacterial polymorphic toxin systems. Here, the authors show that Rhs1 forms a complex with the Type VI secretion system (T6SS) spike protein VgrG and the EagR chaperone. They also present the cryo-EM structure of the Rhs1-EagR complex and propose a model for Rhs loading and delivery by the T6SS.
- Dukas Jurėnas
- , Leonardo Talachia Rosa
- & Eric Cascales
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Article
| Open AccessStructural basis for the E3 ligase activity enhancement of yeast Nse2 by SUMO-interacting motifs
Nse2 is a SUMO E3 ligase component of the Smc5/6 multisubunit complex involved in the DNA repair and chromosome integrity. Here, the structure of the Nse2 in complex with an E2-SUMO thioester mimetic reveals the combined action of two SIM motifs during the E3- dependent conjugation reaction.
- Nathalia Varejão
- , Jara Lascorz
- & David Reverter
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Article
| Open AccessCrystal structures of phosphatidyl serine synthase PSS reveal the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases
CDP-diacylglycerol (CDP-DAG) alcohol O-phosphatidyl transferases (CDP-APs) are conserved in archaea, bacteria, and eukaryotes and catalyze the de novo synthesis of phospho-lipids from the precursor CDP-DAG and an alcohol. Here, the authors present the crystal structures of the Methanocaldococcus jannaschii phosphatidyl serine synthase (MjPSS) in four different states and suggest a model for its catalytic mechanism.
- Martin Centola
- , Katharina van Pee
- & Özkan Yildiz
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Article
| Open AccessStructural basis of Ty3 retrotransposon integration at RNA Polymerase III-transcribed genes
Ty3 retrotransposon integrates with an exquisite specificity upstream of RNA Polymerase III-transcribed genes, such as transfer RNAs. Here the authors resolve a cryo-EM structure of an active Ty3 intasome in complex with a TFIIIB-bound tRNA promoter, shedding light into the molecular determinants of harmless retrotransposition.
- Guillermo Abascal-Palacios
- , Laura Jochem
- & Alessandro Vannini
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Article
| Open AccessMolecular architecture of black widow spider neurotoxins
The venom of Latrodectus spiders contains seven Latrotoxins (LaTXs), among them α-latrocrustatoxin (LCT) and δ- latroinsectotoxins δ-LIT. LaTXs bind to specific receptors on the surface of neuronal cells and target the molecular exocytosis machinery. Here, the authors present the cryo-EM structure of the α-LCT monomer and the δ-LIT dimer, which reveal that LaTXs are organized in four domains and they discuss the potential oligomerisation mechanism that takes place before LaTXs membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions.
- Minghao Chen
- , Daniel Blum
- & Christos Gatsogiannis
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Article
| Open AccessStructural insights into proteolytic activation of the human Dispatched1 transporter for Hedgehog morphogen release
Dispatched (Disp) RND transporter, activated by Furin-mediated proteolytic cleavage, mediates the release of the lipid-modified Hedgehog (Hh) ligands. Here, the authors report structures of human Disp1 (hDisp1) before and after cleavage, and in complex with lipid-modified Sonic hedgehog (Shh), with insights into the mechanisms of hDisp1 activation and function.
- Wanqiu Li
- , Linlin Wang
- & Xin Gong
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Article
| Open AccessStructural evolution of a DNA repair self-resistance mechanism targeting genotoxic secondary metabolites
Microbial DNA glycosylases associated with the biosynthesis of DNA-damaging antibiotics have evolved self-resistance for their cognate natural products. Here, the authors provide evidence that cellular self-resistance is enabled by reduced affinity of the glycosylases for the excision products of the corresponding DNA lesions.
- Elwood A. Mullins
- , Jonathan Dorival
- & Brandt F. Eichman
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Article
| Open AccessHelicobacter pylori FabX contains a [4Fe-4S] cluster essential for unsaturated fatty acid synthesis
Helicobacter pylori FabX, a dehydrogenase/isomerase flavoprotein, is required for unsaturated fatty acid synthesis. Here, the authors characterize FabX substrate recognition and catalytic mechanism, and reveal that it contains an atypical [4Fe-4S] cluster, which is essential and participates in the catalytic cycle.
- Jiashen Zhou
- , Lin Zhang
- & Liang Zhang
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Article
| Open AccessCryo-EM snapshots of a native lysate provide structural insights into a metabolon-embedded transacetylase reaction
How is acetyl-CoA produced in the context of the endogenous, eukaryotic pyruvate dehydrogenase complex metabolon? Here the authors dissect the embedded transacetylase reaction through biochemical, cryo-EM, HADDOCKing and molecular dynamics methods.
- Christian Tüting
- , Fotis L. Kyrilis
- & Panagiotis L. Kastritis
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Article
| Open AccessStructures of tweety homolog proteins TTYH2 and TTYH3 reveal a Ca2+-dependent switch from intra- to intermembrane dimerization
Tweety Homologs (TTYHs) are highly conserved membrane proteins, whose functions remain poorly understood. Here, the authors present the cryo-EM structures of murine TTYH2 and TTYH3 that form cis-dimers in the presence of Ca2+, whereas in the absence of Ca2+ TTYH2 adopts monomeric and trans-dimeric structures. The presented structures lack ion conducting pathways, which is consistent with results from electrophysiology measurements.
- Baobin Li
- , Christopher M. Hoel
- & Stephen G. Brohawn
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Article
| Open AccessStructure of HIV-1 Vpr in complex with the human nucleotide excision repair protein hHR23A
Vpr is a HIV-1 accessory virulence factor that also interacts with the human DNA repair protein hHR23A. Here, the authors present the structure of Vpr in complex with the C-terminal half of hHR23A comprising the XPC-binding and ubiquitin-associated domains, which reveals that hHR23A interacts with the DCAF1-binding and not the substrate-binding Vpr surface and further illustrates how Vpr acts as a versatile structural adapter that targets diverse DNA repair pathways.
- In-Ja L. Byeon
- , Guillermo Calero
- & Angela M. Gronenborn
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Article
| Open AccessStructural and functional analysis of the promiscuous AcrB and AdeB efflux pumps suggests different drug binding mechanisms
Resistance-nodulation-cell division (RND)-type tripartite efflux pumps confer multidrug resistance to Gram-negative bacteria. Here, structural and functional analyses of AdeB from Acinetobacter baumannii and AcrB from Escherichia coli provide insight into their different drug-binding and conformational drug transport states.
- Alina Ornik-Cha
- , Julia Wilhelm
- & Klaas M. Pos
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Article
| Open AccessA structured RNA motif locks Argonaute2:miR-122 onto the 5’ end of the HCV genome
The RNA genome of the Hepatitis C Virus binds to the liver-specific miR122. Here the authors report the crystal structure of the Ago2:miR122:HCV complex showing that the viral RNA’s structural element traps the Ago2:miR-122 complex on the 5’ end of the viral genome to protect it from degradation.
- Luca F. R. Gebert
- , Mansun Law
- & Ian J. MacRae
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Article
| Open AccessDevelopment of a BCL-xL and BCL-2 dual degrader with improved anti-leukemic activity,
Simultaneous targeting of BCL-xL and BCL-2 is an attractive approach for cancer treatment. Based on information gained by computational structure modelling, the authors develop a PROTAC that induces degradation of both BCL-xL and BCL-2 and effectively targets BCL-xL/2-dependent leukaemia cells.
- Dongwen Lv
- , Pratik Pal
- & Daohong Zhou
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Article
| Open AccessThe structural basis for the phospholipid remodeling by lysophosphatidylcholine acyltransferase 3
During phosphatidylcholine (PC) remodeling re-acylation is catalyzed by lysophosphatidylcholine acyltransferases (LPCAT). Here, the authors present crystal and cryo-EM structures of chicken LPCAT3 in the apo-, acyl donor-bound and acyl receptor-bound states, and based on the structures and further functional analysis they discuss the mechanism of the enzyme.
- Qing Zhang
- , Deqiang Yao
- & Yu Cao
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Article
| Open AccessArchitecture of the outer-membrane core complex from a conjugative type IV secretion system
DNA transfer between two bacterial cells is mediated by the conjugative type 4 secretion systems (T4SSs). Here, the authors report the structure of a complete T4SS outer-membrane core complex (OMCC), revealing distinct C17 and C13 symmetries of its central inner and peripheral outer ring regions, respectively.
- Himani Amin
- , Aravindan Ilangovan
- & Tiago R. D. Costa
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Article
| Open AccessN-terminal tyrosine of ISCU2 triggers [2Fe-2S] cluster synthesis by ISCU2 dimerization
[2Fe-2S] protein cofactors are essential for life and are synthesized on ISCU2 scaffolds. Here, the authors show that hydrophobic interaction of two conserved N-terminal tyrosines induces ISCU2 dimerization and concomitant [2Fe-2S] cluster synthesis.
- Sven-A. Freibert
- , Michal T. Boniecki
- & Roland Lill
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Article
| Open AccessCryo-EM structures of intermediates suggest an alternative catalytic reaction cycle for cytochrome c oxidase
Cytochrome c oxidase is a fundamental enzyme of life and its mechanism is not fully understood yet. Here, the authors present four cryo-EM structures of different intermediate states, which suggest an alternative cytochrome c oxidase reaction cycle.
- F. Kolbe
- , S. Safarian
- & H. Michel
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Article
| Open AccessStructure of the class C orphan GPCR GPR158 in complex with RGS7-Gβ5
The orphan GPR158 receptor belongs to the class C GPCR family and interacts with the regulator of G protein signaling 7 (RGS7)-Gβ5 complex. Here, the authors present the cryo-EM structure of human GPR158, which reveals that the extracellular domain contains a PAS domain, and they also determine the structures of GPR158 in complex with either one or two RGS7-Gβ5 heterodimers and discuss implications for the signaling mechanism.
- Eunyoung Jeong
- , Yoojoong Kim
- & Yunje Cho
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Article
| Open AccessHuman RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis
The pseudokinase MLKL is activated by the upstream kinase RIPK3 in the necroptotic pathway but the structural basis of MLKL activation is not well understood yet. Here, the authors present the crystal structures of the human RIPK3:MLKL complex and human RIPK3 kinase alone, which reveal structural differences between human and murine RIPK3 and they discuss mechanistic implications.
- Yanxiang Meng
- , Katherine A. Davies
- & James M. Murphy
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Article
| Open AccessPhosphorylation activates the yeast small heat shock protein Hsp26 by weakening domain contacts in the oligomer ensemble
Small heat shock proteins (sHsps) form large spherical assemblies and their regulation is not well understood. Here, the authors provide insights into the mechanism of Hsp26 activation by characterising phospho-mimetic mutants of yeast Hsp26. They present cryo-EM structures of the wild-type Hsp26 40mer and its phospho-mimetic mutants that reveal the location of the thermosensor in the oligomer, and the authors also show that the thermosensor domain is targeted by phosphorylation, which relieves the intrinsic inhibition of chaperone activity.
- Moritz Mühlhofer
- , Carsten Peters
- & Johannes Buchner
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Article
| Open AccessHPF1 dynamically controls the PARP1/2 balance between initiating and elongating ADP-ribose modifications
HPF1 controls the ADP-ribosylation activity of PARP1/2 in response to DNA breaks. Here, the authors show that HPF1 regulates the balance between ADP-ribose initiation and elongation through a dynamic interaction that accelerates the initiation rate on serine residues.
- Marie-France Langelier
- , Ramya Billur
- & John M. Pascal
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Article
| Open AccessMDA5 disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA
MDA5 is the primary immune sensor for SARS-CoV-2 and many other viruses. Mutations in MDA5 can cause disease. Here the authors employ CryoEM and biochemical methods to show how steric constraints cause MDA5 to misrecognize endogenous RNA as viral RNA.
- Qin Yu
- , Alba Herrero del Valle
- & Yorgo Modis
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Article
| Open AccessCrystal structures of the elusive Rhizobium etli l-asparaginase reveal a peculiar active site
L-asparaginases catalyse the hydrolysis of L-asparagine to L-aspartic acid and ammonia. Here, the authors present high resolution crystal structures of Rhizobium etli L-asparaginase that contains a Zn2+ binding site without a catalytic role and discuss the catalytic mechanism of the enzyme.
- Joanna I. Loch
- , Barbara Imiolczyk
- & Mariusz Jaskolski
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Article
| Open AccessDistinct conformations of the HIV-1 V3 loop crown are targetable for broad neutralization
The V3-crown of the HIV-1 envelope protein largely elicits non-neutralizing antibodies. Here, the authors show that the V3-crown can be targeted by broadly neutralizing designed ankyrin repeat proteins recognizing two conformations one of which resembles CCR5- bound V3.
- Nikolas Friedrich
- , Emanuel Stiegeler
- & Alexandra Trkola
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Article
| Open AccessStructures of prokaryotic ubiquitin-like protein Pup in complex with depupylase Dop reveal the mechanism of catalytic phosphate formation
Pupylation is a bacterial post-translational protein modification, where the small ubiquitin-like protein Pup is covalently attached to lysine side chains of target proteins, which is a reversible process and depupylation is catalysed by the depupylase enzyme, Dop. Here, the authors present crystal structures of Dop in different functional states, which together with biochemical experiments provide insights into the catalytic mechanism of this enzyme.
- Hengjun Cui
- , Andreas U. Müller
- & Eilika Weber-Ban
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Article
| Open AccessAbemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation
Abemaciclib is a third generation CDK-directed drug used in the treatment of HR + /HER2 negative advanced or metastatic breast cancer. Here the authors demonstrate that members of the Homeodomain-interacting protein kinases (HIPKs) HIPK3 and DYRK1A are also targeted by Abemaciclib.
- Ines H. Kaltheuner
- , Kanchan Anand
- & Matthias Geyer
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Article
| Open AccessMechanism of actin-dependent activation of nucleotidyl cyclase toxins from bacterial human pathogens
The nucleotidyl cyclase toxin exoenzyme Y (ExoY), which is secreted by the human pathogens Pseudomonas aeruginosa and Vibrio vulnificus is activated by actin. Here, the authors present the cryo-EM structures of PaExoY bound to F-actin and VvExoY in complex with G-actin-profilin. These structures together with molecular dynamics simulations and enzymatic assays provide insights into the activation mechanism for both bacterial cyclase toxin families that interact with either F- or G-actin.
- Alexander Belyy
- , Felipe Merino
- & Stefan Raunser