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| Open AccessRegiodivergent biosynthesis of bridged bicyclononanes
Hypericum plants contain complex compounds with promising medicinal properties. Here, the authors report a pair of enzymes catalyzing prenylation and regiodivergent cyclization. The forged scaffolds are characteristic of hyperforin analogs.
- Lukas Ernst
- , Hui Lyu
- & Benye Liu
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Article
| Open AccessPreserving ester-linked modifications reveals glutamate and aspartate mono-ADP-ribosylation by PARP1 and its reversal by PARG
Ester-linked modifications are common but difficult to detect. Here, the authors present methods based on ester preservation and a sensitive antibody to reveal DNA damage-induced mono-ADP-ribosylation on aspartate and glutamate. This signal, part of the first wave of PARP1 signaling, is removed by PARG.
- Edoardo José Longarini
- & Ivan Matić
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Article
| Open AccessCryo-EM structures of the human Elongator complex at work
Here the authors determined several cryo-EM structures of the human Elongator complex, which modifies anticodons of tRNAs. The structural work is complemented by functional analyses to understand this clinically relevant cellular machine at the molecular level.
- Nour-el-Hana Abbassi
- , Marcin Jaciuk
- & Sebastian Glatt
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Article
| Open AccessThe structural basis for 2′−5′/3′−5′-cGAMP synthesis by cGAS
Here, the authors provide structural and biochemical data describing the mechanism through which cGAS synthesizes 2′−5′/3′−5′-cGAMP.
- Shuai Wu
- , Sandra B. Gabelli
- & Jungsan Sohn
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Article
| Open AccessStructural basis of human NOX5 activation
NADPH oxidase 5 (NOX5) is activated by Ca2+ signaling, catalyzing superoxide production by transferring electrons from intracellular NADPH to extracellular oxygen. Here the authors uncover the molecular basis of NOX5 activation and electron transfer.
- Chenxi Cui
- , Meiqin Jiang
- & Ji Sun
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Article
| Open AccessMutational dissection of a hole hopping route in a lytic polysaccharide monooxygenase (LPMO)
Lytic polysaccharide monooxygenases (LPMOs) are mono copper enzymes with outstanding industrial applicability. Here, the authors investigate the “hole hopping” mechanism in a bacterial LPMO and show that a strictly conserved tryptophan is critical for radical formation and hole transference, as well as reveal a correlation between the efficiency of hole transference and enzyme performance under oxidative stress.
- Iván Ayuso-Fernández
- , Tom Z. Emrich-Mills
- & Vincent G. H. Eijsink
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Article
| Open AccessGenetic and functional diversity of β-N-acetylgalactosamine-targeting glycosidases expanded by deep-sea metagenome analysis
Four β-N-acetylgalactosaminidase gene families were identified via deep-sea metagenome analysis. The biochemical and structural characterization of the aforementioned enzymes revealed their functional diversity and monophyletic evolutionary history.
- Tomomi Sumida
- , Satoshi Hiraoka
- & Takuro Nunoura
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Article
| Open AccessInterplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis
Enzymes involve structural flexibility in their function, but understanding enzyme catalysis as connected to protein motions is a major challenge. Here, the authors obtain energetic description of C-H activation in nicotinamide coenzyme-dependent UDP-glucuronic acid C4 epimerase based on temperature kinetic studies and isotope effect measurements.
- Christian Rapp
- , Annika Borg
- & Bernd Nidetzky
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Article
| Open AccessActive site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms
Here the authors show mutants of isocitrate dehydrogenase 1 (IDH1), an enzyme implicated in various cancers, have distinct catalytic and structural features that drive their ability to generate an oncometabolite.
- Matthew Mealka
- , Nicole A. Sierra
- & Christal D. Sohl
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Article
| Open AccessPlatform-directed allostery and quaternary structure dynamics of SAMHD1 catalysis
SAMHD1 is a regulator of dNTP homeostasis and an HIV restriction factor. The authors use time-resolved cryo-EM to visualise dynamic conformational changes that drive the catalytic cycle and allosteric regulation of this multi-subunit enzyme.
- Oliver J. Acton
- , Devon Sheppard
- & Ian A. Taylor
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Article
| Open AccessUnraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9
CRISPR-Cas9 is a powerful tool, but the strict requirement for an “NGG” protospacer-adjacent motif (PAM) sequence limits the number of editable genes. Here the authors combine enzyme kinetics, cryo-EM, and single-molecule imaging to determine how SpRY interrogates DNA and recognises target sites for cleavage.
- Grace N. Hibshman
- , Jack P. K. Bravo
- & David W. Taylor
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Article
| Open AccessMechanism of DNA unwinding by MCM8-9 in complex with HROB
MCM8-9 and HROB function together in DNA damage response. Here, the authors describe the mechanism of DNA unwinding by MCM8-9 and its activation by HROB. HROB makes direct contacts with both MCM8 and MCM9 and promotes DNA unwinding downstream of MCM8-9 loading and hexameric ring formation on DNA.
- Ananya Acharya
- , Hélène Bret
- & Petr Cejka
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Article
| Open AccessNMR characterization of RNA binding property of the DEAD-box RNA helicase DDX3X and its implications for helicase activity
DDX3X is a member of the RNA helicase family, which remodels RNA structures. Using solution NMR, here the authors show that DDX3X preferentially binds to single-stranded RNA, which underlies its unwinding activity toward various structured RNA substrates.
- Yuki Toyama
- & Ichio Shimada
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Article
| Open AccessRNA targeting and cleavage by the type III-Dv CRISPR effector complex
Here, Schwartz, Bravo, and Ahsan et al. show how multi-subunit fusion proteins are arranged around a crRNA in a type III CRISPR-Cas effector to cleave target RNA. Structures and molecular dynamics of this complex show three distinct active sites that can be used for programmable RNA cleavage.
- Evan A. Schwartz
- , Jack P. K. Bravo
- & David W. Taylor
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Article
| Open AccessThe DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis
Cruz et al. describe the role of Dbp10/DDX54 in remodeling rRNA structure within the immature eukaryotic peptidyl transferase center of the ribosome, coupling energy-dependent catalysis to a post-catalytic role in factor exchange during 60S ribosomal subunit assembly.
- Victor E. Cruz
- , Christine S. Weirich
- & Jan P. Erzberger
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Article
| Open AccessInsights into the inhibition of protospacer integration via direct interaction between Cas2 and AcrVA5
Here, the authors characterize an anti-CRISPR protein that prevents protospacer integration by Cas1-Cas2, providing structural insights that may benefit CRISPR-Cas systems research.
- Mingfang Bi
- , Wenjing Su
- & Xiaobing Mo
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Article
| Open AccessDynamic inter-domain transformations mediate the allosteric regulation of human 5, 10-methylenetetrahydrofolate reductase
Here the authors present the cryo-EM structure of active and inhibited human MTHFR, revealing a dynamic inhibitory mechanism dependent on dual SAM binding. The resulting closed conformation features an autoinhibitory element effectively blocking enzymatic activity.
- Linnea K. M. Blomgren
- , Melanie Huber
- & Thomas J. McCorvie
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Article
| Open AccessProtein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis
Vascular protein disulfide isomerase (PDI) regulates thrombosis and targeting extracellular PDI remains a promising antithrombotic approach. Here, the authors show that PDI cleaves allosteric disulfides on histidine-rich glycoprotein to influence its functions during coagulation and thus fine-tune the kinetics of thrombus formation.
- Keyu Lv
- , Shuai Chen
- & Chao Fang
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Article
| Open AccessDiscovery of a small-molecule inhibitor that traps Polθ on DNA and synergizes with PARP inhibitors
Here the authors discover a small-molecule that inhibits DNA polymerase theta by trapping the enzyme on DNA in the closed conformation. The inhibitor selectively kills BRCA-mutant cells and exhibits strong synergistic activity with PARP inhibitors.
- William Fried
- , Mrityunjay Tyagi
- & Richard T. Pomerantz
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Article
| Open AccessDevelopment and crystal structures of a potent second-generation dual degrader of BCL-2 and BCL-xL
Here, the authors have determined structures of 753b PROTAC, BCL-xL/BCL-2 and VHL E3 ligase ternary complexes which reveal the basis for the dual degrader activity of 753b. The structures and subsequent functional analyses facilitated design of WH244 PROTAC, with enhanced degrader activity in cells.
- Digant Nayak
- , Dongwen Lv
- & Shaun K. Olsen
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Article
| Open AccessIdentifying a key spot for electron mediator-interaction to tailor CO dehydrogenase’s affinity
Carbon monoxide dehydrogenases (CODH) employ artificial electron mediators like viologens for biocatalysis, but little is known about the interaction between the mediators and the enzyme. Here, the authors discover the critical site for viologen interactions at the D-cluster of Carboxydothermus hydrogenoformans CODH2 via alanine mutations and crystallography, and report variants with increased ethyl viologen affinity.
- Suk Min Kim
- , Sung Heuck Kang
- & Yong Hwan Kim
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Article
| Open AccessMethyl transfer in psilocybin biosynthesis
The natural hallucinogen psilocybin — produced by so-called magic mushrooms — holds promise for the treatment of depression and other mental health conditions. Here, the authors provide a structural and biochemical analysis of the Psilocybe methyl transferase PsiM that provides mechanistic insight into the last step of psilocybin biosynthesis.
- Jesse Hudspeth
- , Kai Rogge
- & Sebastiaan Werten
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Article
| Open AccessActivation and friction in enzymatic loop opening and closing dynamics
Enzymes present loops around active sites whose closing and opening dynamics are essential for its activity. Here the authors unveil the mechanism governing loop motion, showing that it involves an activated conformational rearrangement around a couple of torsional angles taking place under the strong friction exerted by the rest of loop torsions.
- Kirill Zinovjev
- , Paul Guénon
- & Iñaki Tuñón
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Article
| Open AccessLegionella metaeffector MavL reverses ubiquitin ADP-ribosylation via a conserved arginine-specific macrodomain
The pathogen Legionella pneumophila mediates NAD+-dependent ubiquitination pathways upon infection. Here, the authors show the Legionella effector MavL reverses ubiquitin ADP-ribosylation to regulate these pathways. MavL represents a new macrodomain class specific for reversal of arginine ADP-ribosylation with distinct ADP-ribose binding features.
- Zhengrui Zhang
- , Jiaqi Fu
- & Chittaranjan Das
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Article
| Open AccessTwo DOT1 enzymes cooperatively mediate efficient ubiquitin-independent histone H3 lysine 76 tri-methylation in kinetoplastids
Trypanosoma brucei DOT1A and DOT1B methylate H3K76 without H2B-ubiquitin. Based on structural and enzymatic data, Frisbie et al. reveal a mechanism of how these enzymes cooperatively and efficiently tri-methylate H3K76 in a ubiquitin-independent way.
- Victoria S. Frisbie
- , Hideharu Hashimoto
- & Erik W. Debler
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Article
| Open AccessMesoscale DNA features impact APOBEC3A and APOBEC3B deaminase activity and shape tumor mutational landscapes
Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer. This study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their substrate selectivity.
- Ambrocio Sanchez
- , Pedro Ortega
- & Rémi Buisson
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Article
| Open AccessStructural and mechanistic insights into activation of the human RNA ligase RTCB by Archease
RTCB-type RNA ligases play important roles in tRNA splicing, the unfolded protein response and RNA repair. Here, Gerber et al. present structural snapshots of RTCB’s reaction cycle, and show how an activation complex with Archease primes RTCB for ligation.
- Janina Lara Gerber
- , Suria Itzel Morales Guzmán
- & Jirka Peschek
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Article
| Open AccessSubstrate recognition mechanism of the endoplasmic reticulum-associated ubiquitin ligase Doa10
Doa10/MARCHF6 is a conserved E3 ubiquitin ligase in the endoplasmic reticulum (ER) membrane in eukaryotes, but its molecular mechanism was unknown. The authors combine cryo-EM, computational and biochemical analyses to reveal how Doa10 recognizes its substrate proteins for ER-associated degradation.
- Kevin Wu
- , Samuel Itskanov
- & Eunyong Park
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Article
| Open AccessA three-level regulatory mechanism of the aldo-keto reductase subfamily AKR12D
Here, the authors characterise an aldo-keto reductase AKRtyl, which belongs to a previously unidentified subfamily AKR12D. They uncover a complex mechanism of allosteric regulation that is mediated by 3 distinct states.
- Zhihong Xiao
- , Jinyin Zha
- & Shaobo Dai
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Article
| Open AccessDimerization-dependent serine protease activity of FAM111A prevents replication fork stalling at topoisomerase 1 cleavage complexes
FAM111A is a serine protease important for DNA replication and antiviral defense. Here, the authors report that the FAM111A dimerization is crucial for its proteolytic activity and for promoting DNA replication at trapped topoisomerase I.
- Sowmiya Palani
- , Yuka Machida
- & Yuichi J. Machida
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Article
| Open AccessDJ-1 protects proteins from acylation by catalyzing the hydrolysis of highly reactive cyclic 3-phosphoglyceric anhydride
Human protein DJ-1 displays neuroprotective properties. Here, the authors demonstrate that DJ-1 hydrolyzes cyclic 3-phosphoglyceric anhydride (cPGA), thereby protecting proteins from acylation by this highly reactive metabolite spontaneously forming in glycolysis.
- Aizhan Akhmadi
- , Adilkhan Yeskendir
- & Darkhan Utepbergenov
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Article
| Open AccessA non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme
The authors previously showed that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita Baylis-Hillman (MBH) reaction. Here, they report another efficient MBHase that employs a non-canonical Nδ-methylhistidine nucleophile paired with a catalytic glutamate, providing an alternative mechanistic solution for MBH catalysis.
- Amy E. Hutton
- , Jake Foster
- & Anthony P. Green
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Article
| Open AccessFilament formation drives catalysis by glutaminase enzymes important in cancer progression
Mitochondrial enzymes, collectively known as glutaminase, satisfy the metabolic requirements of cancer cells. Here the authors show that glutaminases form filamentous structures necessary for their catalytic activity.
- Shi Feng
- , Cody Aplin
- & Richard A. Cerione
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Article
| Open AccessStructural insight into an Arl1–ArfGEF complex involved in Golgi recruitment of a GRIP-domain golgin
Arl1 is a GTP-binding protein that interacts with the guanine nucleotide exchange factor Gea2 to recruit the golgin Imh1 to the Golgi. Here, the authors report structures of the full-length Gea2 and the Arl1–Gea2 complex, with insights into the mechanism of their function in membrane trafficking.
- H. Diessel Duan
- , Bhawik K. Jain
- & Huilin Li
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Article
| Open AccessSubstrate binding and catalytic mechanism of the Se-glycosyltransferase SenB in the biosynthesis of selenoneine
SenB is a Se-glycosyltransferase in the microbial biosynthesis pathway of selenoneine. Here, the authors perform the structure-function investigation, providing mechanistic insights into a two-step catalytic reaction of SenB.
- Wei Huang
- , Jun Song
- & Feng Long
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Article
| Open AccessDistinct functional constraints driving conservation of the cofilin N-terminal regulatory tail
Here the authors screen a saturation mutagenesis library of the disordered N-terminal tail of the actin severing protein cofilin. Their results reveal how a key phosphorylation site can balance competing sequence constraints on function and regulation.
- Joel A. Sexton
- , Tony Potchernikov
- & Benjamin E. Turk
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Article
| Open AccessPoly-γ-glutamylation of biomolecules
Poly-γ-glutamate tails are a distinctive feature of folate and F420 cofactors, but it was unclear how these tails elongate while maintaining substrate specificity. Here, the authors discover that folylpolyglutamate synthase and γ-glutamyl ligase enzymes add successive L-glutamates to the termini of the growing γ-glutamyl chain in a processive mechanism.
- Ghader Bashiri
- , Esther M. M. Bulloch
- & Christopher J. Squire
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Article
| Open AccessStructure-guided engineering enables E3 ligase-free and versatile protein ubiquitination via UBE2E1
Ubiquitin E3 ligases are key to accessing ubiquitinated proteins, but only a few substrates have defined E3 ligases. Here, the authors reveal the mechanism of naturally occurring E3-independent ubiquitination and develop an E3-free enzymatic strategy for the versatile generation of ubiquitinated proteins.
- Xiangwei Wu
- , Yunxiang Du
- & Lei Liu
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Article
| Open AccessSystems engineering of Escherichia coli for high-level glutarate production from glucose
Glutarate is a platform chemical widely used in the production of polyesters and polyamindes. Here, the authors design the shortest and thermodynamically favorable pathway, and increase glutarate production from glucose through systematic engineering of E. coli.
- Zhilan Zhang
- , Ruyin Chu
- & Cong Gao
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Article
| Open AccessAsymmetric nucleosome PARylation at DNA breaks mediates directional nucleosome sliding by ALC1
Bacic et al. demonstrate that PARP1/HPF1 preferentially modify histone tails closest to the DNA break, directing ALC1-catalyzed nucleosome sliding. These findings suggest a mechanism for rendering DNA breaks more accessible to repair factors.
- Luka Bacic
- , Guillaume Gaullier
- & Sebastian Deindl
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Article
| Open AccessReaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase
New antimalarials are urgently needed. Here, the authors identify Open Source Malaria compound, OSMS-106, as a reaction hijacking inhibitor of the malaria parasite protein synthesis machinery, with potential use for treatment and prophylaxis.
- Stanley C. Xie
- , Yinuo Wang
- & Leann Tilley
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Article
| Open AccessElucidation of unusual biosynthesis and DnaN-targeting mode of action of potent anti-tuberculosis antibiotics Mycoplanecins
Mycoplanecins show promising activity against tuberculosis. Here, the authors identify and study mycoplanecins’ biosynthesis, antibacterial effects, and binding mechanism to DnaN, suggesting potential for fighting multidrug-resistant tuberculosis.
- Chengzhang Fu
- , Yunkun Liu
- & Rolf Müller
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Article
| Open AccessMassively parallel profiling of RNA-targeting CRISPR-Cas13d
Systematic understanding of CRISPR enzyme RNA binding specificity and cleavage is lacking. Here the authors report RNA chip-hybridised association-mapping platform (RNA-CHAMP), a workflow that repurposes next generation DNA sequencing chips to measure the binding affinity for RNA targets.
- Hung-Che Kuo
- , Joshua Prupes
- & Ilya J. Finkelstein
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Article
| Open AccessStructural basis of antiphage immunity generated by a prokaryotic Argonaute-associated SPARSA system
Short prokaryotic Argonaute and Sir2 proteins function as an antivirus system. Here the authors describe structures of SPARSA (a heterodimer of Sir2-APAZ and prokaryotic Argonaute) with and without template DNA and guide RNA, providing structural basis of its assembly and activation by the recognition of the invading virus.
- Xiangkai Zhen
- , Xiaolong Xu
- & Songying Ouyang
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Article
| Open AccessMultiple redox switches of the SARS-CoV-2 main protease in vitro provide opportunities for drug design
Here the authors demonstrate that the SARS-CoV-2 main protease (Mpro) is subject to redox regulation in vitro, reversibly switching between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues.
- Lisa-Marie Funk
- , Gereon Poschmann
- & Kai Tittmann
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Article
| Open AccessCovalent PARylation of DNA base excision repair proteins regulates DNA demethylation
The PARylation activity of PARP recruits DNA repair proteins to damaged DNA, most likely via non-covalent protein-PAR interactions. Here, the authors show that PARP1 covalently PARylates base excision repair proteins to modulate their DNA transactions and thus promote active BER DNA demethylation.
- Simon D. Schwarz
- , Jianming Xu
- & Roland Steinacher
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Article
| Open AccessMolecular basis of the inositol deacylase PGAP1 involved in quality control of GPI-AP biogenesis
The inositol deacylase PGAP1 initiates glycolipid remodeling required for GPI-AP sorting and secretion. Here, authors capture three PGAP1 states in a lipid environment and with products, revealing mechanisms for substrate selectivity and catalysis.
- Jingjing Hong
- , Tingting Li
- & Dianfan Li
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Article
| Open AccessMonovalent metal ion binding promotes the first transesterification reaction in the spliceosome
Hybrid QM/MM molecular dynamics simulations reveal that the kinetics and thermodynamics of the first splicing step are regulated by a K+ ion that facilitates optimal positioning of reactive moieties.
- Jana Aupič
- , Jure Borišek
- & Alessandra Magistrato
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Article
| Open AccessInsights into the ISG15 transfer cascade by the UBE1L activating enzyme
Interferon stimulated gene 15 (ISG15) is a ubiquitin-like protein with critical roles in the innate immune response. Here, the authors present a Cryo-EM structure of ISG15 in complex with its E1 and E2 enzymes, providing insights into the specificity determinants of this pathway.
- Iona Wallace
- , Kheewoong Baek
- & Kirby N. Swatek