Cohesin is a multisubunit protein complex that links sister chromatids from replication until segregation 1, 2. The lack of obvious cohesin-targeting-specific sequences on DNA 3–5, as well as ...cohesin's molecular arrangement as a large ring 6–8, has led to the working hypothesis that cohesin acts as a direct topological linker 9, 10. To preserve the identity of sister chromatids, such a linkage would need to stably persist throughout the entire S and G2 phases of the cell cycle. Unexpectedly, cohesin binds chromatin already in telophase, and a large fraction dissociates from chromosomes during prophase in a phosphorylation-dependent manner 11–17, whereas initiation of anaphase requires proteolytic cleavage of only a small fraction of cohesin 18, 19. These observations raised the question of how and when cohesin interacts with chromatin during the cell cycle. Here, we report a cell-cycle dependence in the stability of cohesin binding to chromatin. Using photobleaching and quantitative live-cell imaging, we identified several cohesin pools with different chromatin binding stabilities. Although all chromatin bound cohesin dissociated after a mean residence time of less than 25 min before replication, about one-third of cohesin was bound much more stably after S phase and persisted until metaphase, consistent with long-lived links mediating cohesion between sister chromatids.
IREM‐1 is an inhibitory receptor involved in the functional regulation of myeloid cells. The expression, in vitro folding, purification, crystallization and X‐ray data collection of the Ig‐V like ...domain of IREM‐1 are reported. X‐ray data were collected from a microcrystal (300 × 10 × 10 µm) at 100 K and a diffraction pattern was obtained to 2.6 Å resolution on microfocus beamline ID23‐2 at the ESRF. The crystal belongs to space group P3121, with unit‐cell parameters a = b = 54.23, c = 72.02 Å, α = γ = 90, β = 120°. Assuming the presence of one molecule per asymmetric unit, VM (the Matthews coefficient) was calculated to be 1.96 Å3 Da−1 and the solvent content was estimated to be 37.27%. Determination of the IREM‐1 structure will provide insights into its structural requirements for ligand discrimination and binding.
NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and ...signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae , can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.
▶ Plants have a family of 14 intracellular ABA receptors, named PYR/PYL/RCAR. ▶ These receptors are able to inhibit the activity of clade A PP2Cs in an ABA-dependent manner. ▶ The structures of three ...receptors, PYR1, PYL1 and PYL2, and two receptor-ABA-phosphatase complexes, PYL1-ABI1 and PYL2-HAB1, have been elucidated. ▶ The gain-of-function mutations abi1
G180D, abi2
G168D and hab1
G246D are immune to receptor-mediated inhibition.
Abscisic acid (ABA) plays an essential function in plant physiology since it is required for biotic and abiotic stress responses as well as control of plant growth and development. A new family of soluble ABA receptors, named PYR/PYL/RCAR, has emerged as ABA sensors able to inhibit the activity of specific protein phosphatases type-2C (PP2Cs) in an ABA-dependent manner. The structural and functional mechanism by which ABA is perceived by these receptors and consequently leads to inhibition of the PP2Cs has been recently elucidated. The module PYR/PYL/RCAR-ABA-PP2C offers an elegant and unprecedented mechanism to control phosphorylation signaling cascades in a ligand-dependent manner. The knowledge of their three-dimensional structures paves the way to the design of ABA agonists able to modulate the plant stress response.
Tabun is a warfare agent that inhibits human acetylcholinesterase (hAChE) by rapid phosphylation of the catalytic serine. A time-dependent reaction occurs on the tabun adduct, leading to an “aged” ...enzyme, resistant to oxime reactivators. The aging reaction may proceed via either dealkylation or deamidation, depending on the stereochemistry of the phosphoramidyl adduct. We solved the X-ray structure of aged tabun−hAChE complexed with fasciculin II, and we show that aging proceeds through O-dealkylation, in agreement with the aging mechanism that we determined for tabun-inhibited human butyrylcholinesterase and mouse acetylcholinesterase. Noteworthy, aging and binding of fasciculin II lead to an improved thermostability, resulting from additional stabilizing interactions between the two subdomains that face each other across the active site gorge. This first structure of hAChE inhibited by a nerve agent provides structural insight into the inhibition and aging mechanisms and a structural template for the design of molecules capable of reactivating aged hAChE.
Although strawberries are highly appreciated fruits, their intake can induce allergic reactions in atopic patients. These reactions can be due to the patient’s previous sensitization to the major ...birch pollen allergen Bet v 1, by which IgE generated in response to Bet v 1 cross-reacts with the structurally related strawberry Fra a 1 protein family. Fra a 1.02 is the most expressed paralog in ripe strawberries and is highly allergenic. To better understand the molecular mechanisms regulating this allergic response, we have determined the three-dimensional structure of Fra a 1.02 and four site-directed mutants that were designed based on their positions in potential epitopes. Fra a 1.02 and mutants conform to the START fold. We show that the cross-reactivity of all the mutant variants to IgE from patients allergic to Bet v 1 was significantly reduced without altering the conserved structural fold, so that they could potentially be used as hypoallergenic Fra a 1 variants for the generation of vaccines against strawberry allergy in atopic patients.
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