Structural insights into how Cas9 targets nucleosomes.

The CRISPR-associated endonuclease Cas9 derived from prokaryotes is used as a genome editing, which targets specific genomic loci by single guide RNAs (sgRNAs). The eukaryotes, the target of genome editing, store their genome DNA in chromatin, in which the nucleosome is a basic unit. Despite previous structural analyses focusing on Cas9 cleaving free DNA, structural insights into Cas9 targeting of DNA within nucleosomes are limited, leading to uncertainties in understanding how Cas9 operates in the eukaryotic genome.

Verification of the interaction between human bitter taste receptor T2R46 and polyphenols; Computational chemistry approach.

Recent studies have indicated that the activation of bitter taste receptors (T2R) expressed in gastrointestinal secretory cells has a regulatory effect on the secretion of gastrointestinal hormones. Polyphenols are known to be ingested at a daily intake of 5 g or more and commonly have a bitter taste. Consequently, the interaction between the bitter taste receptor T2R46 and 490 polyphenols was investigated using simulation techniques.

Structural insights into the agonist selectivity of the adenosine A receptor.

Adenosine receptors play pivotal roles in physiological processes. Adenosine A receptor (AR), the most recently identified adenosine receptor, is expressed in various tissues, exhibiting important roles in neuron, heart, and immune cells, and is often overexpressed in tumors, highlighting the therapeutic potential of AR-selective agents. Recently, we identified RNA-derived N-methyladenosine (mA) as an endogenous agonist for AR, suggesting the relationship between RNA-derived modified adenosine and AR.

Structural mechanisms of potent lysophosphatidic acid receptor 1 activation by nonlipid basic agonists.

Lysophosphatidic acid receptor 1 (LPA) is one of the G protein-coupled receptors activated by the lipid mediator, lysophosphatidic acid (LPA). LPA is associated with a variety of diseases, and LPA agonists have potential therapeutic value for treating obesity and depression. Although potent nonlipid LPA agonists have recently been identified, the mechanisms of nonlipid molecule-mediated LPA activation remain unclear.

Exploring RNA-guided DNA scissors in eukaryotes: Are Fanzors counterparts of CRISPR-Cas12s?

Fanzors are recently characterized RNA-guided DNA endonucleases found in eukaryotic organisms. In this issue of Cell, Xu, Saito et al. reveal the structural diversity of Fanzors and identify key features shared with TnpB and Cas12 proteins, providing a comprehensive perspective on their molecular function and evolution.

The high-light-sensitivity mechanism and optogenetic properties of the bacteriorhodopsin-like channelrhodopsin GtCCR4.

Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization.

Molecular mechanism of the endothelin receptor type B interactions with Gs, Gi, and Gq.

The endothelin receptor type B (ET) exhibits promiscuous coupling with various heterotrimeric G protein subtypes including Gs, Gi/o, Gq/11, and G12/13. Recent fluorescence and structural studies have raised questions regarding the coupling efficiencies and determinants of these G protein subtypes. Herein, by utilizing an integrative approach, combining hydrogen/deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cellular systems, we investigated conformational changes of Gs, Gi, and Gq triggered by ET activation.

Structure and engineering of Brevibacillus laterosporus Cas9.

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets complementary to an RNA guide, and is widely used as a powerful genome-editing tool. Here, we report the crystal structure of Brevibacillus laterosporus Cas9 (BlCas9, also known as BlatCas9), in complex with a guide RNA and its target DNA at 2.4-Å resolution.

PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics.

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.

Optimization of α-amido boronic acids via cryo-electron microscopy analysis: Discovery of a novel highly selective immunoproteasome subunit LMP7 (β5i)/LMP2 (β1i) dual inhibitor.

The immunoproteasome subunit LMP7 (β5i)/LMP2 (β1i) dual blockade has been reported to suppress B cell differentiation and activation, suggesting that the dual inhibition of LMP7/LMP2 is a promising approach for treating autoimmune diseases. In contrast, the inhibition of the constitutive proteasome subunit β5c correlates with cytotoxicity against non-immune cells. Therefore, LMP7/LMP2 dual inhibitors with high selectivity over β5c may be desirable for treating autoimmune diseases.

Structure and dynamics of the pyroglutamylated RF-amide peptide QRFP receptor GPR103.

Pyroglutamylated RF-amide peptide (QRFP) is a peptide hormone with a C-terminal RF-amide motif. QRFP selectively activates a class A G-protein-coupled receptor (GPCR) GPR103 to exert various physiological functions such as energy metabolism and appetite regulation. Here, we report the cryo-electron microscopy structure of the QRFP26-GPR103-G complex at 3.

Structural basis for pegRNA-guided reverse transcription by a prime editor.

The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing guide RNA (pegRNA) to facilitate a wide variety of precise genome edits in living cells. However, owing to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood. Here we present cryo-electron microscopy structures of the SpCas9-M-MLV RTΔRNaseH-pegRNA-target DNA complex in multiple states.

Cryo-EM structure of I domain-containing integrin αEβ7.

The integrin family is a transmembrane receptor that plays critical roles in the cell-cell and cell-extracellular matrix adhesion, signal transduction such as cell cycle regulation, organization of the intracellular cytoskeleton, and immune responses. Consequently, dysfunction of integrins is associated with a wide range of human diseases, including cancer and immune diseases, which makes integrins therapeutic targets for drug discovery. Here we report the cryo-EM structure of the human α-I domain-containing full-length integrin αEβ7, which is expressed in the leukocytes of the immune system and a drug target for inflammatory bowel disease (IBD).

Melatonin receptor structure and signaling.

Melatonin (5-methoxy-N-acetyltryptamine) binds with high affinity and specificity to membrane receptors. Several receptor subtypes exist in different species, of which the mammalian MT and MT receptors are the best-characterized. They are members of the G protein-coupled receptor superfamily, preferentially coupling to G proteins but also to other G proteins in a cell-context-depending manner.

Structure of the human Bre1 complex bound to the nucleosome.

Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the heterodimeric Bre1 complex composed of Bre1A/RNF20 and Bre1B/RNF40. The Bre1 proteins generally function as tumor suppressors, while in certain cancers, they facilitate cancer cell proliferation.

Cryo-EM advances in GPCR structure determination.

G-protein-coupled receptors (GPCRs) constitute a prominent superfamily in humans and are categorized into six classes (A-F) that play indispensable roles in cellular communication and therapeutics. Nonetheless, their structural comprehension has been limited by challenges in high-resolution data acquisition. This review highlights the transformative impact of cryogenic electron microscopy (cryo-EM) on the structural determinations of GPCR-G-protein complexes.

Structure of full-length ERGIC-53 in complex with MCFD2 for cargo transport.

ERGIC-53 transports certain subsets of newly synthesized secretory proteins and membrane proteins from the endoplasmic reticulum to the Golgi apparatus. Despite numerous structural and functional studies since its identification, the overall architecture and mechanism of action of ERGIC-53 remain unclear. Here we present cryo-EM structures of full-length ERGIC-53 in complex with its functional partner MCFD2.

Bicarbonate signalling via G protein-coupled receptor regulates ischaemia-reperfusion injury.

Homoeostatic regulation of the acid-base balance is essential for cellular functional integrity. However, little is known about the molecular mechanism through which the acid-base balance regulates cellular responses. Here, we report that bicarbonate ions activate a G protein-coupled receptor (GPCR), i.

Structural basis for lysophosphatidylserine recognition by GPR34.

GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-G complex bound with one of two ligands bound: either the LysoPS analogue S3E-LysoPS, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. The ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity.

Molecular insights into atypical modes of β-arrestin interaction with seven transmembrane receptors.

β-arrestins (βarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of βarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the βarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of βarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of βarr2 from a β strand to an α helix upon activation by D6R.