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Arginyl-tRNA-protein transferase 1 (ATE1) is a master regulator of protein homeostasis, stress response, cytoskeleton maintenance, and cell migration. The diverse functions of ATE1 arise from its unique enzymatic activity to covalently attach an arginine onto its protein substrates in a tRNA-dependent manner. However, how ATE1 (and other aminoacyl-tRNA transferases) hijacks tRNA from the highly efficient ribosomal protein synthesis pathways and catalyzes the arginylation reaction remains a mystery. Here, we describe the three-dimensional structures of Saccharomyces cerevisiae ATE1 with and without its tRNA cofactor. Importantly, the putative substrate binding domain of ATE1 adopts a previously uncharacterized fold that contains an atypical zinc-binding site critical for ATE1 stability and function. The unique recognition of tRNA Arg by ATE1 is coordinated through interactions with the major groove of the acceptor arm of tRNA. Binding of tRNA induces conformational changes in ATE1 that helps explain the mechanism of substrate arginylation. ATE1 is a highly specific enzyme hijacking tRNA from ribosomal pathways to install an arginine onto proteins as a post-translational modification. Here, the authors describe the structures of yeast ATE1 with or without its tRNA cofactor. ATE1 recognizes and selects tRNA in a unique mechanism.

The biodiversity and the biogeography are still poorly understood for freshwater invertebrates. The crustacean Chydorus sphaericus-brevilabris complex (Cladocera: Chydoridae) is composed of species that are important components of Holarctic freshwater food webs. Recent morphological and genetic study of the complex has indicated a substantial species diversity in the northern hemisphere. However, we know little of the geographic boundaries of these novel lineages. Moreover, a large section of the Palearctic remains unexamined at the genetic level. Here we attempt to address the biodiversity knowledge gap for the Chydorus sphaericus group in the central Palearctic and assess its diversity and biogeographic boundaries. We sequenced nuclear (ITS-2) and mitochondrial (COI) gene regions of Chydorus specimens across the Palearctic and compared them with already available Holarctic sequences. We detected six main clades in the C. sphaericus group in the Palearctic, of which two of the groups are novel. Three of the more divergent clades are geographically widespread. The central portion of Eurasia (the Yenisey River basin) appears to be a narrow zone of secondary contact for phylogroups that expanded from European and Beringian refugia. As such, the previously unsampled central Palearctic represents an important region for understanding the evolutionary consequences of Pleistocene climatic oscillations on the Chydorus sphaericus group.

Interventions against variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Stable and potent nanobodies (Nbs) that target the receptor binding domain (RBD) of SARS-CoV-2 spike are promising therapeutics. However, it is unknown if Nbs broadly neutralize circulating variants. We found that RBD Nbs are highly resistant to variants of concern (VOCs). High-resolution cryoelectron microscopy determination of eight Nb-bound structures reveals multiple potent neutralizing epitopes clustered into three classes: Class I targets ACE2-binding sites and disrupts host receptor binding. Class II binds highly conserved epitopes and retains activity against VOCs and RBD SARS-CoV . Cass III recognizes unique epitopes that are likely inaccessible to antibodies. Systematic comparisons of neutralizing antibodies and Nbs provided insights into how Nbs target the spike to achieve high-affinity and broadly neutralizing activity. Structure-function analysis of Nbs indicates a variety of antiviral mechanisms. Our study may guide the rational design of pan-coronavirus vaccines and therapeutics. Highly potent neutralizing nanobodies (Nbs) are of great interest as potential COVID-19 therapeutics. Here, the authors show that potent neutralizing Nbs targeting the receptor binding domain (RBD) of the SARS-CoV-2 spike protein are also effective against convergent variants of concern of the virus. They determine eight Nb-bound spike protein cryo-EM structures, classify the binding epitopes of the Nbs and discuss their neutralization mechanisms.

Pond-dwelling cyclic parthenogens are often proposed to be highly vagile. However, the Holarctic biogeography of parthenogens has been hampered by very limited sampling in the eastern Palearctic. Here we examine the geographic boundaries, diversity, and connectivity across the Palearctic for the Daphnia curvirostris complex (Cladocera: Daphniidae). Nuclear (HSP90) and mitochondrial (ND2) sequence data supported the existence of five main clades (most of which corresponded to presumptive species) with one eastern Palearctic clade being novel to this study (the average mitochondrial genetic divergence from known species was 19.2%). D . curvirostris s.s. was geographically widespread in the Palearctic, with a population genetic signature consistent with postglacial expansion. The Eastern Palearctic had local nine endemic species and/or subclades (other Holarctic regions lacked more than one endemic subclade). Even though several endemic species appeared to have survived Pleistocene glaciation in the eastern Palearctic, much of the Palearctic has been recolonized by D . curvirostris s.str. from a Western Palearctic refugium. A disjunct population in Mexico also shared its haplotypes with D . curvirostris s.str., consistent with a recent introduction. The only apparently endemic North American lineage was detected in a thermally disturbed pond system in northwestern Alaska. Our results for pond-dwelling cyclic parthenogens further support the hypothesis that the Eastern Palearctic is a diversity hotspot for freshwater invertebrates.

Serotonin receptors (5-HT 3A R) directly regulate gut movement, and drugs that inhibit 5-HT 3A R function are used to control emetic reflexes associated with gastrointestinal pathologies and cancer therapies. The 5-HT 3A R function involves a finely tuned orchestration of three domain movements that include the ligand-binding domain, the pore domain, and the intracellular domain. Here, we present the structure from the full-length 5-HT 3A R channel in the apo-state determined by single-particle cryo-electron microscopy at a nominal resolution of 4.3 Å. In this conformation, the ligand-binding domain adopts a conformation reminiscent of the unliganded state with the pore domain captured in a closed conformation. In comparison to the 5-HT 3A R crystal structure, the full-length channel in the apo-conformation adopts a more expanded conformation of all the three domains with a characteristic twist that is implicated in gating. Serotonin receptor (5-HT 3A R), a pentameric ligand-gated ion channel, regulates numerous gastrointestinal functions. Here the authors provide a cryo-electron microscopic structure from the full-length 5-HT 3A R in the apo-state which corresponds to a resting conformation of the channel.

Binding of precursor tRNAs (ptRNAs) by bacterial ribonuclease P (RNase P) involves an encounter complex (ES) that isomerizes to a catalytic conformation (ES*). However, the structures of intermediates and the conformational changes that occur during binding are poorly understood. Here, we show that pairing between the 5′ leader and 3′RCCA extending the acceptor stem of ptRNA inhibits ES* formation. Cryo-electron microscopy single particle analysis reveals a dynamic enzyme that becomes ordered upon formation of ES* in which extended acceptor stem pairing is unwound. Comparisons of structures with alternative ptRNAs reveals that once unwinding is completed RNase P primarily uses stacking interactions and shape complementarity to accommodate alternative sequences at its cleavage site. Our study reveals active site interactions and conformational changes that drive molecular recognition by RNase P and lays the foundation for understanding how binding interactions are linked to helix unwinding and catalysis. Ribonuclease P efficiently processes all tRNA precursors despite sequence variation at the site of cleavage. Here, authors use high-throughput enzymology and cryoEM to reveal conformational changes that drive recognition by bacterial RNase P.

15-prostaglandin dehydrogenase (15-PGDH) is a negative regulator of tissue stem cells that acts via enzymatic activity of oxidizing and degrading PGE2, and related eicosanoids, that support stem cells during tissue repair. Indeed, inhibiting 15-PGDH markedly accelerates tissue repair in multiple organs. Here we have used cryo-electron microscopy to solve the solution structure of native 15-PGDH and of 15-PGDH individually complexed with two distinct chemical inhibitors. These structures identify key 15-PGDH residues that mediate binding to both classes of inhibitors. Moreover, we identify a dynamic 15-PGDH lid domain that closes around the inhibitors, and that is likely fundamental to the physiologic 15-PGDH enzymatic mechanism. We furthermore identify two key residues, F185 and Y217, that act as hinges to regulate lid closing, and which both inhibitors exploit to capture the lid in the closed conformation, thus explaining their sub-nanomolar binding affinities. These findings provide the basis for further development of 15-PGDH targeted drugs as therapeutics for regenerative medicine. Inhibition of 15-prostaglandin dehydrogenase (15-PGDH) is a promising therapeutic target for regenerative medicine. We report the structure of 15-PGDH in complex with two different inhibitors. Unexpectedly, access to the binding pocket is regulated by a dynamic “lid” of the enzyme.

Organisms that live at the freshwater surface layer (the neuston) occupy a high energy habitat that is threatened by human activities. Daphniids of the genera Scapholeberis and Megafenestra are adapted to the neuston but are poorly studied for biogeography and diversity. Here we assess the global phylogeography of neustonic daphniids. We obtained 402 new multigene sequences from the 12S rRNA, 16S rRNA, and tRNA (val) regions of the mitochondrial genomes of daphniids from 186 global sites. We assessed the intercontinental origins and boundaries of mitochondrial lineages and the relative rates of evolution in neustonic and planktonic daphniids. We identified 17 divergent lineages in the neustonic daphniids that were associated with biogeographic regions. Six of these lineages had intercontinental ranges – four of these were Transberingian. Patagonian populations of Scapholeberis rammneri were monophyletic and nested within a closely related clade of western North American haplotypes, suggesting an introduction from the Western Nearctic to South America. The Eastern Palearctic was more diverse than other regions, containing eight of the major lineages detected in the Scapholeberinae. The Genus Scapholeberis had high levels of divergence compared to non-neustonic daphniids. Neustonic daphniids have more divergent biogeographic lineages than previously appreciated.

Mitochondrial fission is a critical cellular event to maintain organelle function. This multistep process is initiated by the enhanced recruitment and oligomerization of dynamin-related protein 1 (Drp1) at the surface of mitochondria. As such, Drp1 is essential for inducing mitochondrial division in mammalian cells, and homologous proteins are found in all eukaryotes. As a member of the dynamin superfamily of proteins (DSPs), controlled Drp1 self-assembly into large helical polymers stimulates its GTPase activity to promote membrane constriction. Still, little is known about the mechanisms that regulate correct spatial and temporal assembly of the fission machinery. Here we present a cryo-EM structure of a full-length Drp1 dimer in an auto-inhibited state. This dimer reveals two key conformational rearrangements that must be unlocked through intramolecular rearrangements to achieve the assembly-competent state observed in previous structures. This structural insight provides understanding into the mechanism for regulated self-assembly of the mitochondrial fission machinery. Structural and functional studies highlight the molecular regulation of assembling the mitochondrial division machinery. The core unit is closed, and specific interactions open this unit to facilitate assembly at the right place and time in cells.

The arginyl-transferase ATE1 is a tRNA-dependent enzyme that covalently attaches an arginine molecule to a protein substrate. Conserved from yeast to humans, ATE1 deficiency in mice correlates with defects in cardiovascular development and angiogenesis and results in embryonic lethality, while conditional knockouts exhibit reproductive, developmental, and neurological deficiencies. Despite the recent revelation of the tRNA binding mechanism and the catalytic cycle of yeast ATE1, the structure-function relationship of ATE1 in higher organisms is not well understood. In this study, we present the three-dimensional structure of human ATE1 in an apo-state and in complex with its tRNA cofactor and a peptide substrate. In contrast to its yeast counterpart, human ATE1 forms a symmetric homodimer, which dissociates upon binding of a substrate. Furthermore, human ATE1 includes a unique and extended loop that wraps around tRNA Arg , creating extensive contacts with the T-arm of the tRNA cofactor. Substituting key residues identified in the substrate binding site of ATE1 abolishes enzymatic activity and results in the accumulation of ATE1 substrates in cells. ATE1 is an essential gene in mammals and is recognized as a master regulator of cells. Here, the authors describe structural insights into human ATE1, revealing mechanisms that regulate arginylation activity in cells.