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A pioneer in the biochemistry of membrane proteins Jens Christian Skou, a pioneer of biomembrane research and molecular physiology, died on 28 May, just a few months shy of his 100th birthday. The Danish physiologist discovered the enzyme known as the sodium-potassium adenosine triphosphatase (Na + ,K + -ATPase), or sodium-potassium pump, which exports sodium ions from and imports potassium ions into cells. Almost all modern biochemistry, cell biology, and physiology textbooks showcase the sodium-potassium pump, which accounts for about 20 to 30% of the energy used by cells through hydrolysis of ATP in human and animal bodies and up to 70 to 80% in the brain. In recognition of this first discovery of an ion-transporting ATPase, Skou shared the 1997 Nobel Prize in Chemistry with chemists Paul D. Boyer and John E. Walker for their work on the mechanism of ATP synthesis.

Efficient spectrographs at large telescopes have made it possible to obtain high-resolution spectra of stars with high signal-to-noise ratio and advances in model atmosphere analyses have enabled estimates of high-precision differential abundances of the elements from these spectra, i.e. with errors in the range 0.01–0.03 dex for F, G, and K stars. Methods to determine such high-precision abundances together with precise values of effective temperatures and surface gravities from equivalent widths of spectral lines or by spectrum synthesis techniques are outlined, and effects on abundance determinations from using a 3D non-LTE analysis instead of a classical 1D LTE analysis are considered. The determination of high-precision stellar abundances of the elements has led to the discovery of unexpected phenomena and relations with important bearings on the astrophysics of galaxies, stars, and planets, i.e. (i) Existence of discrete stellar populations within each of the main Galactic components (disk, halo, and bulge) providing new constraints on models for the formation of the Milky Way. (ii) Differences in the relation between abundances and elemental condensation temperature for the Sun and solar twins suggesting dust-cleansing effects in proto-planetary disks and/or engulfment of planets by stars; (iii) Differences in chemical composition between binary star components and between members of open or globular clusters showing that star- and cluster-formation processes are more complicated than previously thought; (iv) Tight relations between some abundance ratios and age for solar-like stars providing new constraints on nucleosynthesis and Galactic chemical evolution models as well as the composition of terrestrial exoplanets. We conclude that if stellar abundances with precisions of 0.01–0.03 dex can be achieved in studies of more distant stars and stars on the giant and supergiant branches, many more interesting future applications, of great relevance to stellar and galaxy evolution, are probable. Hence, in planning abundance surveys, it is important to carefully balance the need for large samples of stars against the spectral resolution and signal-to-noise ratio needed to obtain high-precision abundances. Furthermore, it is an advantage to work differentially on stars with similar atmospheric parameters, because then a simple 1D LTE analysis of stellar spectra may be sufficient. However, when determining high-precision absolute abundances or differential abundance between stars having more widely different parameters, e.g. metal-poor stars compared to the Sun or giants to dwarfs, then 3D non-LTE effects must be taken into account.

Model building into experimental maps is a key element of structural biology, but can be both time consuming and error prone for low-resolution maps. Here we present Namdinator , an easy-to-use tool that enables the user to run a molecular dynamics flexible fitting simulation followed by real-space refinement in an automated manner through a pipeline system. Namdinator will modify an atomic model to fit within cryo-EM or crystallography density maps, and can be used advantageously for both the initial fitting of models, and for a geometrical optimization step to correct outliers, clashes and other model problems. We have benchmarked Namdinator against 39 deposited cryo-EM models and maps, and observe model improvements in 34 of these cases (87%). Clashes between atoms were reduced, and the model-to-map fit and overall model geometry were improved, in several cases substantially. We show that Namdinator is able to model large-scale conformational changes compared to the starting model. Namdinator is a fast and easy tool for structural model builders at all skill levels. Namdinator is available as a web service (https://namdinator.au.dk), or it can be run locally as a command-line tool.

The Na ⁺,K ⁺-ATPase maintains electrochemical gradients for Na ⁺ and K ⁺ that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na ⁺,K ⁺-ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na ⁺,K ⁺-ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the β-surface of the steroid core of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg ²⁺, and crystallographic studies indicate that Rb ⁺ and Mn ²⁺, but not Na ⁺, bind to this site. Comparison with the low-affinity [K ₂]E2–MgF ₓ–ouabain structure [Ogawa et al. (2009) Proc Natl Acad Sci USA 106(33):13742–13747) shows that the CTS binding pocket of [Mg]E2P allows deep ouabain binding with possible long-range interactions between its polarized five-membered lactone ring and the Mg ²⁺. K ⁺ binding at the same site unwinds a turn of αM4, dragging residues Ile318–Val325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K ⁺–Mg ²⁺ competition and explain the well-known antagonistic effect of K ⁺ on CTS binding.

Background Cell death triggered by unmitigated endoplasmic reticulum (ER) stress plays an important role in physiology and disease, but the death-inducing signaling mechanisms are incompletely understood. To gain more insight into these mechanisms, the ER stressor thapsigargin (Tg) is an instrumental experimental tool. Additionally, Tg forms the basis for analog prodrugs designed for cell killing in targeted cancer therapy. Tg induces apoptosis via the unfolded protein response (UPR), but how apoptosis is initiated, and how individual effects of the various UPR components are integrated, is unclear. Furthermore, the role of autophagy and autophagy-related (ATG) proteins remains elusive. Methods To systematically address these key questions, we analyzed the effects of Tg and therapeutically relevant Tg analogs in two human cancer cell lines of different origin (LNCaP prostate- and HCT116 colon cancer cells), using RNAi and inhibitory drugs to target death receptors, UPR components and ATG proteins, in combination with measurements of cell death by fluorescence imaging and propidium iodide staining, as well as real-time RT-PCR and western blotting to monitor caspase activity, expression of ATG proteins, UPR components, and downstream ER stress signaling. Results In both cell lines, Tg-induced cell death depended on death receptor 5 and caspase-8. Optimal cytotoxicity involved a non-autophagic function of MAP1LC3B upstream of procaspase-8 cleavage. PERK, ATF4 and CHOP were required for Tg-induced cell death, but surprisingly acted in parallel rather than as a linear pathway; ATF4 and CHOP were independently required for Tg-mediated upregulation of death receptor 5 and MAP1LC3B proteins, whereas PERK acted via other pathways. Interestingly, IRE1 contributed to Tg-induced cell death in a cell type-specific manner. This was linked to an XBP1-dependent activation of c-Jun N-terminal kinase, which was pro-apoptotic in LNCaP but not HCT116 cells. Molecular requirements for cell death induction by therapy-relevant Tg analogs were identical to those observed with Tg. Conclusions Together, our results provide a new, integrated understanding of UPR signaling mechanisms and downstream mediators that induce cell death upon Tg-triggered, unmitigated ER stress. 1yG1LZYrokkFLLYyz3UJEs Video Abstract Graphical abstract

Neurotransmitter:sodium symporters (NSS) are conserved from bacteria to man and serve as targets for drugs, including antidepressants and psychostimulants. Here we report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na + /substrate-bound, inward-facing occluded conformation. To obtain this structure, we were guided by findings from single-molecule fluorescence spectroscopy and molecular dynamics simulations indicating that L -Phe binding and mutation of the conserved N-terminal Trp8 to Ala both promote an inward-facing state. Compared to the outward-facing occluded conformation, our structure reveals a major tilting of the cytoplasmic end of transmembrane segment (TM) 5, which, together with release of the N-terminus but without coupled movement of TM1, opens a wide cavity towards the second Na + binding site. The structure of this key intermediate in the LeuT transport cycle, in the context of other NSS structures, leads to the proposal of an intracellular release mechanism of substrate and ions in NSS proteins. Neurotransmitter:sodium symporters (NSS) serve as targets for drugs including antidepressants and psychostimulants. Here authors report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na + /substrate-bound, inward-facing occluded conformation which is a key intermediate in the LeuT transport cycle.

The Na⁺, K⁺—adenosine triphosphatase (ATPase) maintains the electrochemical gradients of Na⁺ and K⁺ across the plasma membrane—a prerequisite for electrical excitability and secondary transport. Hitherto, structural information has been limited to K⁺-bound or ouabain-blocked forms. We present the crystal structure of a Na⁺-bound Na⁺, K⁺-ATPase as determined at 4.3 Å resolution. Compared with the K⁺-bound form, large conformational changes are observed in the α subunit whereas the β and γ subunit structures are maintained. The locations of the three Na⁺ sites are indicated with the unique site III at the recently suggested IIIb, as further supported by electrophysiological studies on leak currents. Extracellular release of the third Na⁺ from IIIb through IIIa, followed by exchange of Na⁺ for K⁺ at sites I and II, is suggested.

Asymmetric distribution of phospholipids in eukaryotic membranes is essential for cell integrity, signaling pathways, and vesicular trafficking. P4-ATPases, also known as flippases, participate in creating and maintaining this asymmetry through active transport of phospholipids from the exoplasmic to the cytosolic leaflet. Here, we present a total of nine cryo-electron microscopy structures of the human flippase ATP8B1-CDC50A complex at 2.4 to 3.1 Å overall resolution, along with functional and computational studies, addressing the autophosphorylation steps from ATP, substrate recognition and occlusion, as well as a phosphoinositide binding site. We find that the P4-ATPase transport site is occupied by water upon phosphorylation from ATP. Additionally, we identify two different autoinhibited states, a closed and an outward-open conformation. Furthermore, we identify and characterize the PI(3,4,5)P 3 binding site of ATP8B1 in an electropositive pocket between transmembrane segments 5, 7, 8, and 10. Our study also highlights the structural basis of a broad lipid specificity of ATP8B1 and adds phosphatidylinositol as a transport substrate for ATP8B1. We report a critical role of the sn-2 ester bond of glycerophospholipids in substrate recognition by ATP8B1 through conserved S403. These findings provide fundamental insights into ATP8B1 catalytic cycle and regulation, and substrate recognition in P4-ATPases. Asymmetric phospholipid distribution in cell membranes is vital for cellular function. Here, authors reveal how ATP8B1, a P4-ATPase, can transport different lipids, including phosphatidylinositol.

The Na + ,K + -ATPase, or sodium pump, is well known for its role in ion transport across the plasma membrane of animal cells. It carries out the transport of Na + ions out of the cell and of K + ions into the cell and thus maintains electrolyte and fluid balance. In addition to the fundamental ion-pumping function of the Na + ,K + -ATPase, recent work has suggested additional roles for Na + ,K + -ATPase in signal transduction and biomembrane structure. Several signaling pathways have been found to involve Na + ,K + -ATPase, which serves as a docking station for a fast-growing number of protein interaction partners. In this review, we focus on Na + ,K + -ATPase as a signal transducer, but also briefly discuss other Na + ,K + -ATPase protein–protein interactions, providing a comprehensive overview of the diverse signaling functions ascribed to this well-known enzyme.

Key Points Ions are transported across the plasma membrane by molecular pumps to generate chemical gradients and regulate pH or cell growth. P-type ATPases are a family of molecular pumps that transport cations in or outside the cell. Members of this family include the Na + ,K + -ATPase (found in animals) and the H + -ATPase (found in plants and fungi). The Na + ,K + -ATPase exchanges Na + for K + and the H + -ATPase pumps H + out of the cell. P-type ATPases undergo conformational changes as part of their functional cycle, giving rise to two enzymatic states, E1 and E2, with different affinities for the primary transported ions. P-type ATPases contain a cytoplasmic core comprising the phosphorylation, nucleotide-binding and actuator domains. These carry out autophosphatase activities and are responsible for ATP hydrolysis. All P-type ATPases have six transmembrane helices (M1–M6). The Na + ,K + -ATPase and the H + -ATPase have additional transmembrane helices (M7–M10) that may provide specificity or stability in the Na + ,K + -ATPase and the H + -ATPase, respectively. Many P-type ATPases also have regulatory domains that fine-tune their activity in ion pumping. Crystal structures and functional studies of the Na + ,K + -ATPase and the H + -ATPase have provided insight into their mechanisms of action in eukaryotic cells. ATPases transport ions into and out of cells to maintain ion concentration gradients and control aspects of the cellular environment, such as pH. Structural studies of the Na + ,K + -ATPase, which transports Na + and K + , and the H + -ATPase, which transports H + , have provided insights into their functions in eukaryotic cells. Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na + -dependent and proton-dependent secondary transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H + -ATPase maintains a proton gradient in plants and fungi and the Na + ,K + -ATPase maintains a Na + and K + gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps.