CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems ...contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.
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•Structures of the smallest type VI CRISPR effector in guide and target-bound states•Mechanistic insights into guide RNA and target RNA recognition•Insights into apo Cas13d structural dynamics through cryo-EM and HDX-MS•Rational engineering of Cas13d for minimal coding sequence
Cryo-EM structures and biochemical analysis of CRISPR-Cas13d in apo, guide-bound, and target-bound states offer insight for engineering this RNA-targeting system.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Irisin is secreted by muscle, increases with exercise, and mediates certain favorable effects of physical activity. In particular, irisin has been shown to have beneficial effects in adipose tissues, ...brain, and bone. However, the skeletal response to exercise is less clear, and the receptor for irisin has not been identified. Here we show that irisin binds to proteins of the αV class of integrins, and biophysical studies identify interacting surfaces between irisin and αV/β5 integrin. Chemical inhibition of the αV integrins blocks signaling and function by irisin in osteocytes and fat cells. Irisin increases both osteocytic survival and production of sclerostin, a local modulator of bone remodeling. Genetic ablation of FNDC5 (or irisin) completely blocks osteocytic osteolysis induced by ovariectomy, preventing bone loss and supporting an important role of irisin in skeletal remodeling. Identification of the irisin receptor should greatly facilitate our understanding of irisin’s function in exercise and human health.
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•The αV class of integrins are irisin receptors in osteocytes and adipose tissues•Irisin increases sclerostin expression in osteocytes to induce bone resorption•Genetic deletion of FNDC5 (or Irisin) completely blocks OVX-induced trabecular bone loss
Irisin, through its integrin receptor, promotes skeletal remodeling with implications for stemming bone loss.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Carbonic anhydrase (CA) is one of nature’s fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to ...accelerate carbon capture is limited by the enzyme’s sensitivity to the harsh process conditions. Using directed evolution, the properties of a β-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO ₂ absorption 25-fold compared with the noncatalyzed reaction.
Significance It is clear that to address climate change, the amount of CO ₂ released into the atmosphere by industrial processes has to be reduced. Carbonic anhydrase regulates CO ₂ in nearly every single living organism and is one of the most efficient enzymes in nature. To leverage that efficiency, a β-class carbonic anhydrase was engineered using directed evolution to withstand some of the harshest conditions associated with an industrial carbon capture process. The approach laid out can be generally applied in the development of natural enzymes for their use in industrial applications.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Circularized nandiscs (cNDs) exhibit superb monodispersity and have the potential to transform functional and structural studies of membrane proteins. In particular, cNDs can stabilize large patches ...of lipid bilayers for the reconstitution of complex membrane biochemical reactions, enabling the capture of crucial intermediates involved in synaptic transmission and viral entry. However, previous methods for building cNDs require multiple steps and suffer from low yields. We herein introduce a simple, one-step approach to ease the construction of cNDs using the SpyCatcher-SpyTag technology. This approach increases the yield of cNDs by over 10-fold and is able to rapidly generates cNDs with diameters ranging from 11 to over 100 nm. We demonstrate the utility of these cNDs for mechanistic interrogations of vesicle fusion and protein-lipid interactions that are unattainable using small nanodiscs. Together, the remarkable performance of SpyCatcher-SpyTag in nanodisc circularization paves the way for the use of cNDs in membrane biochemistry and structural biology.
The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain ...activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.
Somatic mutations in the estrogen receptor alpha (ERα) gene (ESR1), especially Y537S and D538G, have been linked to acquired resistance to endocrine therapies. Cell-based studies demonstrated that ...these mutants confer ERα constitutive activity and antiestrogen resistance and suggest that ligand-binding domain dysfunction leads to endocrine therapy resistance. Here, we integrate biophysical and structural biology data to reveal how these mutations lead to a constitutively active and antiestrogen-resistant ERα. We show that these mutant ERs recruit coactivator in the absence of hormone while their affinities for estrogen agonist (estradiol) and antagonist (4-hydroxytamoxifen) are reduced. Further, they confer antiestrogen resistance by altering the conformational dynamics of the loop connecting Helix 11 and Helix 12 in the ligand-binding domain of ERα, which leads to a stabilized agonist state and an altered antagonist state that resists inhibition.
An amine transaminase was engineered for the efficient production of a chiral precursor to sacubitril, (2R,4S)-5-(1,1′-biphenyl-4-yl)-4-amino-2-methylpentanoic acid, a key component in the ...blockbuster heart failure drug Entresto. Starting from an enzyme with trace activity and preference for the undesired diastereoisomer, 11 rounds of enzyme evolution were performed. The resultant variant, CDX-043, showed high productivity giving 90% conversion at 75 g/L substrate concentration with 1% enzyme loading with respect to the substrate in 24 h and without the use of an organic cosolvent. The product diastereomeric purity toward the desired (2R,4S)-stereoisomer was >99.9:0.1 d.r. This variant also exhibited high process robustness and could tolerate reaction temperatures up to 65 °C, isopropylamine concentrations of at least 2 M, and reaction times of at least 5 days. A structural analysis of the enzyme variants gave insight into how the mutations affected activity and selectivity. This enzyme variant allows for the efficient and cost-effective production of sacubitril at large scale.
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IJS, KILJ, NUK, PNG, UL, UM
How to catch a dynamic state
AMP-activated protein kinase (AMPK) is a key sensor of energy status in eukaryotes. Its dynamic structure is regulated by allosteric factors including phosphorylation and ...binding of nucleotides and metabolites. Yan
et al.
developed conformation-specific antibodies that trap AMPK in a fully inactive state that has experienced a large, domain-level rotation. Biophysical experiments in cells and in vitro are consistent with the structural work and support a model in which the activation loop is fully exposed in the completely inactive, dephosphorylated state. These structures inform our understanding of the complex allosteric behavior in this crucial metabolic regulator.
Science
, abe7565, this issue p.
413
Conformation-specific antibodies trap adenosine monophosphate–activated protein kinase in a fully inactive state.
Adenosine monophosphate (AMP)–activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo–electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.
The vitamin D receptor/retinoid X receptor-α heterodimer (VDRRXRα) regulates bone mineralization via transcriptional control of osteocalcin (BGLAP) gene and is the receptor for 1α,25-dihydroxyvitamin ...D
(1,25D3). However, supra-physiological levels of 1,25D3 activates the calcium-regulating gene TRPV6 leading to hypercalcemia. An approach to attenuate this adverse effect is to develop selective VDR modulators (VDRMs) that differentially activate BGLAP but not TRPV6. Here we present structural insight for the action of a VDRM compared with agonists by employing hydrogen/deuterium exchange. Agonist binding directs crosstalk between co-receptors upon DNA binding, stabilizing the activation function 2 (AF2) surfaces of both receptors driving steroid receptor co-activator-1 (SRC1) interaction. In contrast, AF2 of VDR within VDRM:BGLAP bound heterodimer is more vulnerable for large stabilization upon SRC1 interaction compared with VDRM:TRPV6 bound heterodimer. These results reveal that the combination of ligand structure and DNA sequence tailor the transcriptional activity of VDR toward specific target genes.The vitamin D receptor/retinoid X receptor-α heterodimer (VDRRXRα) regulates bone mineralization. Here the authors employ hydrogen/deuterium exchange (HDX) mass spectrometry to study the conformational dynamics of VDRRXRα and give mechanistic insights into how VDRRXRα controls the transcriptional activity of specific genes.
Retinoic acid inducible gene-I (RIG-I) ensures immune surveillance of viral RNAs bearing a 5'-triphosphate (5'ppp) moiety. Mutations in RIG-I (C268F and E373A) lead to impaired ATPase activity, ...thereby driving hyperactive signaling associated with autoimmune diseases. Here we report, using hydrogen/deuterium exchange, mechanistic models for dysregulated RIG-I proofreading that ultimately result in the improper recognition of cellular RNAs bearing 7-methylguanosine and N
-2'-O-methylation (Cap1) on the 5' end. Cap1-RNA compromises its ability to stabilize RIG-I helicase and blunts caspase activation and recruitment domains (CARD) partial opening by threefold. RIG-I H830A mutation restores Cap1-helicase engagement as well as CARDs partial opening event to a level comparable to that of 5'ppp. However, E373A RIG-I locks the receptor in an ATP-bound state, resulting in enhanced Cap1-helicase engagement and a sequential CARDs stimulation. C268F mutation renders a more tethered ring architecture and results in constitutive CARDs signaling in an ATP-independent manner.
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