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Human APOBEC3G (A3G) is a virus restriction factor that inhibits HIV-1 replication and triggers lethal hypermutation on viral reverse transcripts. HIV-1 viral infectivity factor (Vif) breaches this host A3G immunity by hijacking a cellular E3 ubiquitin ligase complex to target A3G for ubiquitination and degradation. The molecular mechanism of A3G targeting by Vif-E3 ligase is unknown, limiting the antiviral efforts targeting this host-pathogen interaction crucial for HIV-1 infection. Here, we report the cryo–electron microscopy structures of A3G bound to HIV-1 Vif in complex with T cell transcription cofactor CBF-β and multiple components of the Cullin-5 RING E3 ubiquitin ligase. The structures reveal unexpected RNA-mediated interactions of Vif with A3G primarily through A3G’s noncatalytic domain, while A3G’s catalytic domain is poised for ubiquitin transfer. These structures elucidate the molecular mechanism by which HIV-1 Vif hijacks the host ubiquitin ligase to specifically target A3G to establish infection and offer structural information for the rational development of antiretroviral therapeutics.

The past fifty years have been marked by the surge of neurodegenerative diseases. Unfortunately, current treatments are only symptomatic. Hence, the search for new and innovative therapeutic targets for curative treatments becomes a major challenge. Among these targets, the adenosine A

Type VI CRISPR-Cas systems are the only CRISPR variety that cleaves exclusively RNA1,2. In addition to the CRISPR RNA (crRNA)-guided, sequence-specific binding and cleavage of target RNAs, such as phage transcripts, the type VI effector, Cas13, causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from phage spread3,4. We show here that the principal form of collateral RNA degradation elicited by Cas13a protein from Leptotrichia shahii upon target RNA recognition is the cleavage of anticodons of multiple tRNA species, primarily those with anticodons containing uridines. This tRNA cleavage is necessary and sufficient for bacterial dormancy induction by Cas13a. In addition, Cas13a activates the RNases of bacterial toxin-antitoxin modules, thus indirectly causing mRNA and rRNA cleavage, which could provide a back-up defense mechanism. The identified mode of action of Cas13a resembles that of bacterial anticodon nucleases involved in antiphage defense5, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module6,7 encompassing an anticodon nuclease.

Type VI CRISPR-Cas systems are the only CRISPR variety that cleaves exclusively RNA1,2. In addition to the CRISPR RNA (crRNA)-guided, sequence-specific binding and cleavage of target RNAs, such as phage transcripts, the type VI effector, Cas13, causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from phage spread3,4. We show here that the principal form of collateral RNA degradation elicited by Cas13a protein from Leptotrichia shahii upon target RNA recognition is the cleavage of anticodons of multiple tRNA species, primarily those with anticodons containing uridines. This tRNA cleavage is necessary and sufficient for bacterial dormancy induction by Cas13a. In addition, Cas13a activates the RNases of bacterial toxin-antitoxin modules, thus indirectly causing mRNA and rRNA cleavage, which could provide a back-up defense mechanism. The identified mode of action of Cas13a resembles that of bacterial anticodon nucleases involved in antiphage defense5, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module6,7 encompassing an anticodon nuclease.

CRISPR ( c lustered r egularly i nterspaced s hort p alindromic r epeats)-Cas (CRISPR-associated) systems provide prokaryotes with efficient protection against foreign nucleic acid invaders. We have recently demonstrated the defensive interference function of a CRISPR-Cas system from Clostridioides (Clostridium) difficile, a major human enteropathogen, and showed that it could be harnessed for efficient genome editing in this bacterium. However, molecular details are still missing on CRISPR-Cas function for adaptation and sequence requirements for both interference and new spacer acquisition in this pathogen. Despite accumulating knowledge on the individual CRISPR-Cas systems in various prokaryotes, no data are available on the adaptation process in bacterial type I-B CRISPR-Cas systems. Here, we report the first experimental evidence that the C. difficile type I-B CRISPR-Cas system acquires new spacers upon overexpression of its adaptation module. The majority of new spacers are derived from a plasmid expressing Cas proteins required for adaptation or from regions of the C. difficile genome where generation of free DNA termini is expected. Results from p rotospacer- a djacent m otif (PAM) library experiments and plasmid conjugation efficiency assays indicate that C. difficile CRISPR-Cas requires the YCN consensus PAM for efficient interference. We revealed a functional link between the adaptation and interference machineries, since newly adapted spacers are derived from sequences associated with a CCN PAM, which fits the interference consensus. The definition of functional PAMs and establishment of relative activity levels of each of the multiple C. difficile CRISPR arrays in present study are necessary for further CRISPR-based biotechnological and medical applications involving this organism. IMPORTANCE CRISPR-Cas systems provide prokaryotes with adaptive immunity for defense against foreign nucleic acid invaders, such as viruses or phages and plasmids. The CRISPR-Cas systems are highly diverse, and detailed studies of individual CRISPR-Cas subtypes are important for our understanding of various aspects of microbial adaptation strategies and for the potential applications. The significance of our work is in providing the first experimental evidence for type I-B CRISPR-Cas system adaptation in the emerging human enteropathogen Clostridioides difficile. This bacterium needs to survive in phage-rich gut communities, and its active CRISPR-Cas system might provide efficient antiphage defense by acquiring new spacers that constitute memory for further invader elimination. Our study also reveals a functional link between the adaptation and interference CRISPR machineries. The definition of all possible functional trinucleotide motifs upstream protospacers within foreign nucleic acid sequences is important for CRISPR-based genome editing in this pathogen and for developing new drugs against C. difficile infections.

G Protein-Coupled Receptors (GPCRs), or 7-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without signaling partners bound requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here we show that the structure of a human A2A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by cryoFIB milling. We determined the structure of the A2A receptor to 2.8 Å resolution and resolved an antagonist in its orthosteric ligand-binding site as well as 4 cholesterol molecules bound to the receptor. This study lays the groundwork for future GPCR structural studies using single microcrystals that would otherwise be impossible by other crystallographic methods.One sentence summaryFIB milled LCP-GPCR structure determined by MicroED

Prostaglandin D(2) (PGD(2)) signals through the G protein–coupled receptor (GPCR) CRTH2 to mediate various inflammatory responses. CRTH2 is the only member of the prostanoid receptor family that is phylogenetically distant from others, implying a nonconserved mechanism of lipid action on CRTH2. Here, we report a crystal structure of human CRTH2 bound to a PGD(2) derivative, 15R-methyl-PGD(2) (15mPGD(2)), by serial femtosecond crystallography. The structure revealed a “polar group in”–binding mode of 15mPGD(2) contrasting the “polar group out”–binding mode of PGE(2) in its receptor EP3. Structural comparison analysis suggested that these two lipid-binding modes, associated with distinct charge distributions of ligand-binding pockets, may apply to other lipid GPCRs. Molecular dynamics simulations together with mutagenesis studies also identified charged residues at the ligand entry port that function to capture lipid ligands of CRTH2 from the lipid bilayer. Together, our studies suggest critical roles of charge environment in lipid recognition by GPCRs.

A method is presented for efficient co-crystal structure determination for G protein-coupled receptors taking advantage of serial femtosecond crystallography., Rational structure-based drug design (SBDD) relies on the availability of a large number of co-crystal structures to map the ligand-binding pocket of the target protein and use this information for lead-compound optimization via an iterative process. While SBDD has proven successful for many drug-discovery projects, its application to G protein-coupled receptors (GPCRs) has been limited owing to extreme difficulties with their crystallization. Here, a method is presented for the rapid determination of multiple co-crystal structures for a target GPCR in complex with various ligands, taking advantage of the serial femtosecond crystallography approach, which obviates the need for large crystals and requires only submilligram quantities of purified protein. The method was applied to the human β2-adrenergic receptor, resulting in eight room-temperature co-crystal structures with six different ligands, including previously unreported structures with carvedilol and propranolol. The generality of the proposed method was tested with three other receptors. This approach has the potential to enable SBDD for GPCRs and other difficult-to-crystallize membrane proteins.

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.

Significance Primed adaptation allows rapid acquisition of protective spacers derived from foreign mobile genetic elements into CRISPR arrays of the host. Primed adaptation requires ongoing CRISPR interference that destroys foreign genetic elements, but the nature of this requirement is unknown. Using the Escherichia coli I-E CRISPR-Cas as a model, we show that prespacers, short fragments of foreign DNA on their way to become incorporated into CRISPR arrays as spacers, are associated with both the adaptation integrase Cas1 and the interference nuclease Cas3, implying physical association of the interference and adaptation machineries during priming., For Type I CRISPR-Cas systems, a mode of CRISPR adaptation named priming has been described. Priming allows specific and highly efficient acquisition of new spacers from DNA recognized (primed) by the Cascade-crRNA (CRISPR RNA) effector complex. Recognition of the priming protospacer by Cascade-crRNA serves as a signal for engaging the Cas3 nuclease–helicase required for both interference and primed adaptation, suggesting the existence of a primed adaptation complex (PAC) containing the Cas1–Cas2 adaptation integrase and Cas3. To detect this complex in vivo, we here performed chromatin immunoprecipitation with Cas3-specific and Cas1-specific antibodies using cells undergoing primed adaptation. We found that prespacers are bound by both Cas1 (presumably, as part of the Cas1–Cas2 integrase) and Cas3, implying direct physical association of the interference and adaptation machineries as part of PAC.

CRISPR-associated proteins 1 and 2 (Cas1-2) are necessary and sufficient for new spacer acquisition in some CRISPR-Cas systems (e.g., type I-E), but adaptation in other systems (e.g., type II-A) involves the crRNA-guided surveillance complex. Here we show that the type I-F Cas1-2/3 proteins are necessary and sufficient to produce low levels of spacer acquisition, but the presence of the type I-F crRNA-guided surveillance complex (Csy) improves the efficiency of adaptation and significantly increases the fidelity of protospacer adjacent motif selection. Sequences selected for integration are preferentially derived from specific regions of extrachromosomal DNA, and patterns of spacer selection are highly reproducible between independent biological replicates. This work helps define the role of the Csy complex in I-F adaptation and reveals that actively replicating mobile genetic elements have antigenic signatures that facilitate their integration during CRISPR adaptation.

Two distinct antagonist-bound structures of CysLT1R reveal unique ligand-binding modes and signaling mechanisms., The G protein–coupled cysteinyl leukotriene receptor CysLT1R mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, and cancer. Selective CysLT1R antagonists are widely prescribed as antiasthmatic drugs; however, these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects. To gain deeper understanding into the functional mechanisms of CysLTRs, we determined the crystal structures of CysLT1R bound to two chemically distinct antagonists, zafirlukast and pranlukast. The structures reveal unique ligand-binding modes and signaling mechanisms, including lateral ligand access to the orthosteric pocket between transmembrane helices TM4 and TM5, an atypical pattern of microswitches, and a distinct four-residue–coordinated sodium site. These results provide important insights and structural templates for rational discovery of safer and more effective drugs.

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms

Change history: In this Letter, the rotation signs around 90°, 135° and 15° were missing and in the HTML, Extended Data Tables 2 and 3 were the wrong tables; these errors have been corrected online.

Background. A lot of neurodegenerative diseases are coursed by amiloidization of proteins in nerve tissues. In the patients brains suffered from Alzheimer’s disease the high fraction of the nerve cells with abnormal chromosome amount was revealed. There are some data showing that prion form of protein PrP may prevent chromosome segregation in mitosis. But the direct association of prionisation and genome stability was not revealed. Materials and methods. We compared the yeast S. cerevisiae strain bearing the prion form of the termination translation factor Sup35, and the strain with non-prionized Sup35 in the alpha-test system. The model of the alpha-test is based on the mechanism of mating type switching in heterothallic yeast strains. The MAT locus that controls the mating type of yeast cell can be presented by two idiomorphs: the MATalpha and MATa that determine the alpha and a cell types, correspondingly. Only two cells with opposite mating types (alpha × a) could copulate. In the mixture of two yeast strains with alpha mating type the hybrids appears only if one of the parent cells had changed its mating type alpha → a. The mating type switching could course the following genetic events: the loss of the chromosome, gene conversion, recombination, loss of the arm of the chromosome, gene mutations and temporary lesions. These events could be distinguished by using the specially constructed alpha-test system. Results. The [PSI+] strain has showed 2-times decreased frequency of «illegitimate» hybridization in the alpha-test compared to [psi-] strain. But [PSI+] doesn’t influence the frequency of «legitimate» hybridization in the alpha × a crossing. The prion [PSI+] also 2-times reduces the frequency of chromosome loss and gene mutations and increases gene conversion 5-times. This results are also confirmed by the canavanine test. Conclusion. We investigated the effect of the Sup35 prionizaion on the genome stability. Unexpectedly in the [PSI+] strain the frequency of «illegitimate» hybridization was 2-times lower, and frequency of gene mutations and chromosome loss was also reduced. The mechanism of this effect is unclear and requires the further investigation.

Study of sorption of uranium onto mineral surfaces plays a key role in controlling its mobility in groundwater. This work focuses on sorption properties of granites of Nizhnekansk massif in order to find optimal conditions for restriction of uranium migration in the environment. Four different types of granites from Itatsky and Kamenny sites were involved in sorption experiments. Distribution coefficients were determined in different conditions and sorption kinetics was analyzed. The results of these experiments are considered to have practical application in searching for the optimal place for efficient geochemical isolation of spent nuclear fuel and high-level radioactive waste.