The role of the C-terminal tail region as a plug to regulate XKR8 lipid scramblase.

XK-related 8 (XKR8), in complex with the transmembrane glycoprotein basigin, functions as a phospholipid scramblase activated by the caspase-mediated cleavage or phosphorylation of its C-terminal tail. It carries a putative phospholipid translocation path of multiple hydrophobic and charged residues in the transmembrane region. It also has a crucial tryptophan at the exoplasmic end of the path that regulates its scrambling activity. We herein investigated the tertiary structure of the human XKR8-basigin complex embedded in lipid nanodiscs at an overall resolution of 3.66 Å. We found that the C-terminal tail engaged in intricate polar and van der Waals interactions with a groove at the cytoplasmic surface of XKR8. These interactions maintained the inactive state of XKR8. Point mutations to disrupt these interactions strongly enhanced the scrambling activity of XKR8, suggesting that the activation of XKR8 is mediated by releasing the C-terminal tail from the cytoplasmic groove. We speculate that the cytoplasmic tail region of XKR8 functions as a plug to prevent the scrambling of phospholipids.

Crystal structures of Na+ ,K+ -ATPase reveal the mechanism that converts the K+ -bound form to Na+ -bound form and opens and closes the cytoplasmic gate.

Na+ ,K+ -ATPase (NKA) plays a pivotal role in establishing electrochemical gradients for Na+ and K+ across the cell membrane by alternating between the E1 (showing high affinity for Na+ and low affinity for K+ ) and E2 (low affinity to Na+ and high affinity to K+ ) forms. Presented here are two crystal structures of NKA in E1·Mg2+ and E1·3Na+ states at 2.9 and 2.8 Å resolution, respectively. These two E1 structures fill a gap in our description of the NKA reaction cycle based on the atomic structures. We describe how NKA converts the K+ -bound E2·2K+ form to an E1 (E1·Mg2+ ) form, which allows high-affinity Na+ binding, eventually closing the cytoplasmic gate (in E1 ~ P·ADP·3Na+ ) after binding three Na+ , while keeping the extracellular ion pathway sealed. We now understand previously unknown functional roles for several parts of NKA and that NKA uses even the lipid bilayer for gating the ion pathway.

Structural Determination, Total Synthesis, and Biological Activity of Iezoside, a Highly Potent Ca2+-ATPase Inhibitor from the Marine Cyanobacterium Leptochromothrix valpauliae.

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a membrane protein on the endoplasmic reticulum (ER) that transports Ca2+ from the cytosol into the ER. As its function is associated with various biological phenomena, SERCA has been recognized as a promising druggable target. Here, we report the second-strongest SERCA-inhibitory compound known to date, which we isolated from the marine cyanobacterium Leptochromothrix valpauliae and named iezoside (1). The structure of iezoside (1) is fundamentally different from that of any other SERCA inhibitor, and its potency is the strongest among marine natural products (Ki 7.1 nM). In this article, we report our comprehensive analysis of iezoside (1), which covers its isolation, structural characterization supported by density functional theory (DFT) calculations and statistical analysis, total synthesis, and clarification of the mode of action of its potent antiproliferative activity (IC50 6.7 ± 0.4 nM against HeLa cells).

Cryo-electron microscopy of Na+ ,K+ -ATPase reveals how the extracellular gate locks in the E2·2K+ state.

Na+ ,K+ -ATPase (NKA) is one of the most important members of the P-type ion-translocating ATPases and plays a pivotal role in establishing electrochemical gradients for Na+ and K+ across the cell membrane. Presented here is a 3.3 Å resolution structure of NKA in the E2·2K+ state solved by cryo-electron microscopy. It is a stable state with two occluded K+ after transferring three Na+ into the extracellular medium and releasing inorganic phosphate bound to the cytoplasmic P domain. We describe how the extracellular ion pathway wide open in the E2P state becomes closed and locked in E2·2K+ , linked to events at the phosphorylation site more than 50 Å away. We also show, although at low resolution, how ATP binding to NKA in E2·2K+ relaxes the gating machinery and thereby accelerates the transition into the next step, that is, the release of K+ into the cytoplasm, more than 100 times.

Cryoelectron microscopy of Na+,K+-ATPase in the two E2P states with and without cardiotonic steroids.

Cryoelectron microscopy (cryo-EM) was applied to Na+,K+-ATPase (NKA) to determine the structures of two E2P states, one (E2PATP) formed by ATP and Mg2+ in the forward reaction, and the other (E2PPi) formed by inorganic phosphate (Pi) and Mg2+ in the backward reaction, with and without ouabain or istaroxime, representatives of classical and new-generation cardiotonic steroids (CTSs). These two E2P states exhibit different biochemical properties. In particular, K+-sensitive acceleration of the dephosphorylation reaction is not observed with E2PPi, attributed to the presence of a Mg2+ ion in the transmembrane cation binding sites. The cryo-EM structures of NKA demonstrate that the two E2P structures are nearly identical but Mg2+ in the transmembrane binding cavity is identified only in E2PPi, corroborating the idea that it should be denoted as E2PPi·Mg2+. We can now explain why the absence of transmembrane Mg2+ in E2PATP confers the K+ sensitivity in dephosphorylation. In addition, we show that ATP bridges the actuator (A) and nucleotide binding (N) domains, stabilizing the E2PATP state; CTS binding causes hardly any changes in the structure of NKA, both in E2PATP and E2PPi·Mg2+, indicating that the binding mechanism is conformational selection; and istaroxime binds to NKA, extending its aminoalkyloxime group deep into the cation binding site. This orientation is upside down compared to that of classical CTSs with respect to the steroid ring. Notably, mobile parts of NKA are resolved substantially better in the electron microscopy (EM) maps than in previous X-ray structures, including sugars sticking out from the ß-subunit and many phospholipid molecules.

The tertiary structure of the human Xkr8-Basigin complex that scrambles phospholipids at plasma membranes.

Xkr8-Basigin is a plasma membrane phospholipid scramblase activated by kinases or caspases. We combined cryo-EM and X-ray crystallography to investigate its structure at an overall resolution of 3.8 Å. Its membrane-spanning region carrying 22 charged amino acids adopts a cuboid-like structure stabilized by salt bridges between hydrophilic residues in transmembrane helices. Phosphatidylcholine binding was observed in a hydrophobic cleft on the surface exposed to the outer leaflet of the plasma membrane. Six charged residues placed from top to bottom inside the molecule were essential for scrambling phospholipids in inward and outward directions, apparently providing a pathway for their translocation. A tryptophan residue was present between the head group of phosphatidylcholine and the extracellular end of the path. Its mutation to alanine made the Xkr8-Basigin complex constitutively active, indicating that it plays a vital role in regulating its scramblase activity. The structure of Xkr8-Basigin provides insights into the molecular mechanisms underlying phospholipid scrambling.

Binding of cardiotonic steroids to Na+,K+-ATPase in the E2P state.

The sodium pump (Na+, K+-ATPase, NKA) is vital for animal cells, as it actively maintains Na+ and K+ electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF3- complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K+-antagonism and suggest a route to isoform specific CTSs.

What ATP binding does to the Ca2+ pump and how nonproductive phosphoryl transfer is prevented in the absence of Ca2.

Under physiological conditions, most Ca2+-ATPase (SERCA) molecules bind ATP before binding the Ca2+ transported. SERCA has a high affinity for ATP even in the absence of Ca2+, and ATP accelerates Ca2+ binding at pH values lower than 7, where SERCA is in the E2 state with low-affinity Ca2+-binding sites. Here we describe the crystal structure of SERCA2a, the isoform predominant in cardiac muscle, in the E2·ATP state at 3.0-Å resolution. In the crystal structure, the arrangement of the cytoplasmic domains is distinctly different from that in canonical E2. The A-domain now takes an E1 position, and the N-domain occupies exactly the same position as that in the E1·ATP·2Ca2+ state relative to the P-domain. As a result, ATP is properly delivered to the phosphorylation site. Yet phosphoryl transfer never takes place without the filling of the two transmembrane Ca2+-binding sites. The present crystal structure explains what ATP binding itself does to SERCA and how nonproductive phosphorylation is prevented in E2.

Mechanism of the E2 to E1 transition in Ca2+ pump revealed by crystal structures of gating residue mutants.

Ca2+-ATPase of sarcoplasmic reticulum (SERCA1a) pumps two Ca2+ per ATP hydrolyzed from the cytoplasm and two or three protons in the opposite direction. In the E2 state, after transferring Ca2+ into the lumen of sarcoplasmic reticulum, all of the acidic residues that coordinate Ca2+ are thought to be protonated, including the gating residue Glu309. Therefore a Glu309Gln substitution is not expected to significantly perturb the structure. Here we report crystal structures of the Glu309Gln and Glu309Ala mutants of SERCA1a under E2 conditions. The Glu309Gln mutant exhibits, unexpectedly, large structural rearrangements in both the cytoplasmic and transmembrane domains, apparently uncoupling them. However, the structure definitely represents E2 and, together with the help of quantum chemical calculations, allows us to postulate a mechanism for the E2 → E1 transition triggered by deprotonation of Glu309.

Suppressor mutation analysis combined with 3D modeling explains cohesin's capacity to hold and release DNA.

Cohesin is a fundamental protein complex that holds sister chromatids together. Separase protease cleaves a cohesin subunit Rad21/SCC1, causing the release of cohesin from DNA to allow chromosome segregation. To understand the functional organization of cohesin, we employed next-generation whole-genome sequencing and identified numerous extragenic suppressors that overcome either inactive separase/Cut1 or defective cohesin in the fission yeast Schizosaccharomyces pombe Unexpectedly, Cut1 is dispensable if suppressor mutations cause disorders of interfaces among essential cohesin subunits Psm1/SMC1, Psm3/SMC3, Rad21/SCC1, and Mis4/SCC2, the crystal structures of which suggest physical and functional impairment at the interfaces of Psm1/3 hinge, Psm1 head-Rad21, or Psm3 coiled coil-Rad21. Molecular-dynamics analysis indicates that the intermolecular ß-sheets in the cohesin hinge of cut1 suppressor mutants remain intact, but a large mobility change occurs at the coiled coil bound to the hinge. In contrast, suppressors of rad21-K1 occur in either the head ATPase domains or the Psm3 coiled coil that interacts with Rad21. Suppressors of mis4-G1326E reside in the head of Psm3/1 or the intragenic domain of Mis4. These may restore the binding of cohesin to DNA. Evidence is provided that the head and hinge of SMC subunits are proximal, and that they coordinate to form arched coils that can hold or release DNA by altering the angles made by the arched coiled coils. By combining molecular modeling with suppressor sequence analysis, we propose a cohesin structure designated the "hold-and-release" model, which may be considered as an alternative to the prevailing "ring" model.

Distinct pH dependencies of Na+/K+ selectivity at the two faces of Na,K-ATPase.

The sodium pump (Na,K-ATPase) in animal cells is vital for actively maintaining ATP hydrolysis-powered Na+ and K+ electrochemical gradients across the cell membrane. These ion gradients drive co- and countertransport and are critical for establishing the membrane potential. It has been an enigma how Na,K-ATPase discriminates between Na+ and K+, despite the pumped ion on each side being at a lower concentration than the other ion. Recent crystal structures of analogs of the intermediate conformations E2·Pi·2K+ and Na+-bound E1∼P·ADP suggest that the dimensions of the respective binding sites in Na,K-ATPase are crucial in determining its selectivity. Here, we found that the selectivity at each membrane face is pH-dependent and that this dependence is unique for each face. Most notable was a strong increase in the specific affinity for K+ at the extracellular face (i.e. E2 conformation) as the pH is lowered from 7.5 to 5. We also observed a smaller increase in affinity for K+ on the cytoplasmic side (E1 conformation), which reduced the selectivity for Na+ Theoretical analysis of the pKa values of ion-coordinating acidic amino acid residues suggested that the face-specific pH dependences and Na+/K+ selectivities may arise from the protonation or ionization of key residues. The increase in K+ selectivity at low pH on the cytoplasmic face, for instance, appeared to be associated with Asp808 protonation. We conclude that changes in the ionization state of coordinating residues in Na,K-ATPase could contribute to altering face-specific ion selectivity.

Protein-phospholipid interplay revealed with crystals of a calcium pump.

The lipid bilayer has so far eluded visualization by conventional crystallographic methods, severely limiting our understanding of phospholipid- and protein-phospholipid interactions. Here we describe electron density maps for crystals of Ca2+-ATPase in four different states obtained by X-ray solvent contrast modulation. These maps resolve the entire first layer of phospholipids surrounding the transmembrane helices, although less than half of them are hydrogen-bonded to protein residues. Phospholipids follow the movements of associated residues, causing local distortions and changes in thickness of the bilayer. Unexpectedly, the entire protein tilts during the reaction cycle, governed primarily by a belt of Trp residues, to minimize energy costs accompanying the large perpendicular movements of the transmembrane helices. A class of Arg residues extend their side chains through the cytoplasm to exploit phospholipids as anchors for conformational switching. Thus, phospholipid-Arg/Lys and phospholipid-Trp interactions have distinct functional roles in the dynamics of ion pumps and, presumably, membrane proteins in general.

Mn(2+) transport by Ca(2+) -ATPase of sarcoplasmic reticulum.

Ca(2+) -ATPase of sarcoplasmic reticulum is known to pump Mn(2+) in addition to Ca(2+) , but whether its transport mechanism is identical to that of Ca(2+) is ambiguous. To clarify, we examined, by atomic absorption spectroscopy, competition between Mn(2+) and Ca(2+) in active transport using vesicles of sarcoplasmic reticulum (SR). Here, we demonstrate that Ca(2+) -ATPase transports Ca(2+) and Mn(2+) concomitantly but has a much lower affinity for Mn(2+) (apparent Kd ~ 0.5 mm). Stoichiometries of transported ions per ATP hydrolysed, Vmax values and activation energies are very similar. Altogether, Ca(2+) -ATPase appears to use the same mechanism for transporting Mn(2+) as that for Ca(2+) .

Sequential substitution of K(+) bound to Na(+),K(+)-ATPase visualized by X-ray crystallography.

Na(+),K(+)-ATPase transfers three Na(+) from the cytoplasm into the extracellular medium and two K(+) in the opposite direction per ATP hydrolysed. The binding and release of Na(+) and K(+) are all thought to occur sequentially. Here we demonstrate by X-ray crystallography of the ATPase in E2·MgF4(2-)·2K(+), a state analogous to E2·Pi·2K(+), combined with isotopic measurements, that the substitution of the two K(+) with congeners in the extracellular medium indeed occurs at different rates, substantially faster at site II. An analysis of thermal movements of protein atoms in the crystal shows that the M3-M4E helix pair opens and closes the ion pathway leading to the extracellular medium, allowing K(+) at site II to be substituted first. Taken together, these results indicate that site I K(+) is the first cation to bind to the empty cation-binding sites after releasing three Na(+).

Functional analysis of SERCA1b, a highly expressed SERCA1 variant in myotonic dystrophy type 1 muscle.

Myotonic dystrophy type 1 (DM1) is a genetic disorder in which multiple genes are aberrantly spliced. Sarco/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1) is one of these genes, and it encodes a P-type ATPase. SERCA1 transports Ca(2+) from the cytosol to the lumen, and is involved in muscular relaxation. It has two splice variants (SERCA1a and SERCA1b) that differ in the last eight amino acids, and the contribution of these variants to DM1 pathology is unclear. Here, we show that SERCA1b protein is highly expressed in DM1 muscle tissue, mainly localised at fast twitch fibres. Additionally, when SERCA1a and SERCA1b were overexpressed in cells, we found that the ATPase and Ca(2+) uptake activity of SERCA1a was almost double that of SERCA1b. Although the affinity for both ATP and Ca(2+) was similar between the two variants, SERCA1b was more sensitive to the inner microsomal environment. Thus, we hypothesise that aberrant expression of SERCA1b in DM1 patients is the cause of abnormal intracellular Ca(2+) homeostasis.

Biselyngbyasides, cytotoxic marine macrolides, are novel and potent inhibitors of the Ca(2+) pumps with a unique mode of binding.

Biselyngbyasides (BLSs), macrolides from a marine cyanobacterium, are cytotoxic natural products whose target molecule is unknown. Here we report that BLSs are high affinity (Ki∼10 nM) inhibitors of Ca(2+)-pumps with a unique binding mode. The crystal structures of the Ca(2+)-pump in complex with BLSs at 3.2-3.5 Å-resolution show that BLSs bind to the pump near the cytoplasmic surface of the transmembrane region. The crystal structures and activity measurement of BLS analogs allow us to identify the structural features that confer high potency to BLSs as inhibitors of the pump.

Electron crystallography of ultrathin 3D protein crystals: atomic model with charges.

Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca(2+)-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca(2+)-binding sites of Ca(2+)-ATPase and that of the iron atom in the heme in catalase.

Design, synthesis and structure-activity relationship studies of novel sirtuin 2 (SIRT2) inhibitors with a benzamide skeleton.

Human sirtuin 2 (SIRT2) is an attractive target molecule for development of drugs to treat neurodegenerative diseases and cancer, because SIRT2 inhibitors have a protective effect against neurodegeneration and an anti-proliferative effect on cancer stem cells. We designed and synthesized a series of benzamide derivatives as SIRT2 inhibitor candidates. Among them, compound 17k showed the most potent SIRT2-inhibitory activity (IC50=0.60µM), with more than 150-fold selectivity over SIRT1 and SIRT3 isoforms (IC50 >100µM).

Stimulation, inhibition, or stabilization of Na,K-ATPase caused by specific lipid interactions at distinct sites.

The activity of membrane proteins such as Na,K-ATPase depends strongly on the surrounding lipid environment. Interactions can be annular, depending on the physical properties of the membrane, or specific with lipids bound in pockets between transmembrane domains. This paper describes three specific lipid-protein interactions using purified recombinant Na,K-ATPase. (a) Thermal stability of the Na,K-ATPase depends crucially on a specific interaction with 18:0/18:1 phosphatidylserine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine; SOPS) and cholesterol, which strongly amplifies stabilization. We show here that cholesterol associates with SOPS, FXYD1, and the α subunit between trans-membrane segments αTM8 and -10 to stabilize the protein. (b) Polyunsaturated neutral lipids stimulate Na,K-ATPase turnover by >60%. A screen of the lipid specificity showed that 18:0/20:4 and 18:0/22:6 phosphatidylethanolamine (PE) are the optimal phospholipids for this effect. (c) Saturated phosphatidylcholine and sphingomyelin, but not saturated phosphatidylserine or PE, inhibit Na,K-ATPase activity by 70-80%. This effect depends strongly on the presence of cholesterol. Analysis of the Na,K-ATPase activity and E1-E2 conformational transitions reveals the kinetic mechanisms of these effects. Both stimulatory and inhibitory lipids poise the conformational equilibrium toward E2, but their detailed mechanisms of action are different. PE accelerates the rate of E1 → E2P but does not affect E2(2K)ATP → E13NaATP, whereas sphingomyelin inhibits the rate of E2(2K)ATP → E13NaATP, with very little effect on E1 → E2P. We discuss these lipid effects in relation to recent crystal structures of Na,K-ATPase and propose that there are three separate sites for the specific lipid interactions, with potential physiological roles to regulate activity and stability of the pump.