Direct evidence of lipid transport by the Drs2-Cdc50 flippase upon truncation of its terminal regions.

P4-ATPases in complex with Cdc50 subunits are lipid flippases that couple ATP hydrolysis with lipid transport to the cytoplasmic leaflet of membranes to create lipid asymmetry. Such vectorial transport has been shown to contribute to vesicle formation in the late secretory pathway. Some flippases are regulated by autoinhibitory regions that can be destabilized by protein kinase-mediated phosphorylation and possibly by binding of cytosolic proteins. In addition, the binding of lipids to flippases may also induce conformational changes required for the activity of these transporters. Here, we address the role of phosphatidylinositol-4-phosphate (PI4P) and the terminal autoinhibitory tails on the lipid flipping activity of the yeast lipid flippase Drs2-Cdc50. By functionally reconstituting the full-length and truncated forms of Drs2 in a 1:1 complex with the Cdc50 subunit, we provide compelling evidence that lipid flippase activity is exclusively detected for the truncated Drs2 variant and is dependent on the presence of the phosphoinositide PI4P. These findings highlight the critical role of phosphoinositides as lipid co-factors in the regulation of lipid transport by the Drs2-Cdc50 flippase.

Activation and substrate specificity of the human P4-ATPase ATP8B1.

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)P3 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.

Monomeric α-synuclein activates the plasma membrane calcium pump.

Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well known for pathological aggregation in neurodegeneration. However, the physiological function of aSN is disputed. Pull-down experiments have pointed to plasma membrane Ca2+ -ATPase (PMCA) as a potential interaction partner. From proximity ligation assays, we find that aSN and PMCA colocalize at neuronal synapses, and we show that calcium expulsion is activated by aSN and PMCA. We further show that soluble, monomeric aSN activates PMCA at par with calmodulin, but independent of the autoinhibitory domain of PMCA, and highly dependent on acidic phospholipids and membrane-anchoring properties of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop connecting the cytosolic A domain and transmembrane segment 3. Our studies point toward a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.

Technical Comment on "Inhibition mechanism of NKCC1 involves the carboxyl terminus and long-range conformational coupling".

Flygaard, Habeck and Nissen question claims on bumetanide and furosemide binding to sodium-potassium-chloride cotransporter NKCC1.

AraC interacts with p75NTR transmembrane domain to induce cell death of mature neurons.

Cytosine arabinoside (AraC) is one of the main therapeutic treatments for several types of cancer, including acute myeloid leukaemia. However, after a high-dose AraC chemotherapy regime, patients develop severe neurotoxicity and cell death in the central nervous system leading to cerebellar ataxia, dysarthria, nystagmus, somnolence and drowsiness. AraC induces apoptosis in dividing cells. However, the mechanism by which it leads to neurite degeneration and cell death in mature neurons remains unclear. We hypothesise that the upregulation of the death receptor p75NTR is responsible for AraC-mediated neurodegeneration and cell death in leukaemia patients undergoing AraC treatment. To determine the role of AraC-p75NTR signalling in the cell death of mature neurons, we used mature cerebellar granule neurons' primary cultures from p75NTR knockout and p75NTRCys259 mice. Evaluation of neurite degeneration, cell death and p75NTR signalling was done by immunohistochemistry and immunoblotting. To assess the interaction between AraC and p75NTR, we performed cellular thermal shift and AraTM assays as well as Homo-FRET anisotropy imaging. We show that AraC induces neurite degeneration and programmed cell death of mature cerebellar granule neurons in a p75NTR-dependent manner. Mechanistically, Proline 252 and Cysteine 256 residues facilitate AraC interaction with the transmembrane domain of p75NTR resulting in uncoupling of p75NTR from the NFκB survival pathway. This, in turn, exacerbates the activation of the cell death/JNK pathway by recruitment of TRAF6 to p75NTR. Our findings identify p75NTR as a novel molecular target to develop treatments for counteract AraC-mediated cell death of mature neurons.

Fast-forward on P-type ATPases: recent advances on structure and function.

P-type ATPase are present in nearly all organisms. They maintain electrochemical gradients for many solutes, in particular ions, they control membrane lipid asymmetry, and are crucial components of intricate signaling networks. All P-type ATPases share a common topology with a transmembrane and three cytoplasmic domains and their transport cycle follows a general scheme - the Post-Albers-cycle. Recently, P-type ATPase research has been advanced most significantly by the technological advancements in cryo-EM analysis, which has elucidated many new P-type ATPase structures and mechanisms and revealed several new ways of regulation. In this review, we highlight the progress of the field and focus on special features that are present in the five subfamilies. Hence, we outline the new intersubunit transport model of KdpFABC, the ways in which heavy metal pumps have evolved to accommodate various substrates, the strategies Ca2+ pumps utilize to adapt to different environmental needs, the intricate molecular builds of the ion binding sites in Na,K- and H,K-ATPases, the remarkable hexameric assembly of fungal proton pumps, the many ways in which P4-ATPase lipid flippases are regulated, and finally the deorphanization of P5 pumps. Interestingly many of the described features are found in more than one of the five subfamilies, and mixed and matched together to provide optimal function and precise regulation.

eGFP as an All-in-One Tag for Purification of Membrane Proteins.

Within the last decade, cryo-electron microscopy has revolutionized our understanding of membrane proteins, but they still represent challenging targets for biochemical and structural studies. The first obstacle is often to obtain high production levels of correctly folded target protein. In these cases, the use of eGFP tags is an efficient strategy, as it allows rapid screenings of expression systems, constructs, and detergents for solubilization. Additionally, eGFP tags can now be used for affinity purification with recently developed nanobodies. Here we present a series of methods based on enhanced green fluorescent protein (eGFP) fluorescence to efficiently screen for production and stabilization of detergent-solubilized eGFP-tagged membrane proteins produced in S. cerevisiae via in-gel fluorescence SDS-PAGE and fluorescence-detection size-exclusion chromatography (FSEC). Additionally, we present a protocol describing the production of affinity resin based on eGFP-binding nanobodies produced in E. coli. We showcase the purification of human ATP7B, a copper transporting P-type ATPase, as an example of the applicability of the methods.

Activation and inhibition of the C-terminal kinase domain of p90 ribosomal S6 kinases.

The p90 ribosomal S6 kinases (RSKs) contain two distinct catalytic kinase domains, the N-terminal and C-terminal kinase domains (NTKD and CTKD, respectively). The activation of CTKD is regulated by phosphorylation by extracellular signal-regulated kinase (ERK1/2) and an autoinhibitory αL helix. Through a mutational series in vitro of the RSK CTKDs, we found a complex mechanism lifting autoinhibition that led us to design constitutively active RSK CTKDs. These are based on a phosphomimetic mutation and a C-terminal truncation (e.g., RSK2 T577E D694*) where a high activity in absence of ERK phosphorylation is obtained. Using these constructs, we characterize IC50 values of ATP-competitive inhibitors and provide a setup for determining specificity constants (kinact/Ki) of covalent CTKD inhibitors.

Cryo-EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity.

The sodium-potassium-chloride transporter NKCC1 of the SLC12 family performs Na+ -dependent Cl- - and K+ -ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo-electron microscopy structure of human NKCC1 in the substrate-loaded (Na+ , K+ , and 2 Cl- ) and occluded, inward-facing state that has also been observed for the SLC6-type transporters MhsT and LeuT. Cl- binding at the Cl1 site together with the nearby K+ ion provides a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl- -ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl- -sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na+ release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.

Variations on a theme: crystal forms of the amino-acid transporter MhsT.

The bacterial amino-acid transporter MhsT from the SLC6A family has been crystallized in complex with different substrates in order to understand the determinants of the substrate specificity of the transporter. Surprisingly, crystals of the different MhsT-substrate complexes showed interrelated but different crystal-packing arrangements. Space-group assignment and structure determination of these different crystal forms present challenging combinations of pseudosymmetry, twinning and translational noncrystallographic symmetry.

The Na+,K+-ATPase in complex with beryllium fluoride mimics an ATPase phosphorylated state.

The Na+,K+-ATPase generates electrochemical gradients of Na+ and K+ across the plasma membrane via a functional cycle that includes various phosphoenzyme intermediates. However, the structure and function of these intermediates and how metal fluorides mimick them require further investigation. Here, we describe a 4.0 Å resolution crystal structure and functional properties of the pig kidney Na+,K+-ATPase stabilized by the inhibitor beryllium fluoride (denoted E2-BeFx). E2-BeFx is expected to mimic properties of the E2P phosphoenzyme, yet with unknown characteristics of ion and ligand binding. The structure resembles the E2P form obtained by phosphorylation from inorganic phosphate (Pi) and stabilized by cardiotonic steroids, including a low-affinity Mg2+ site near ion binding site II. Our anomalous Fourier analysis of the crystals soaked in Rb+ (a K+ congener) followed by a low-resolution rigid-body refinement (6.9-7.5 Å) revealed preocclusion transitions leading to activation of the dephosphorylation reaction. We show that the Mg2+ location indicates a site of initial K+ recognition and acceptance upon binding to the outward-open E2P state after Na+ release. Furthermore, using binding and activity studies, we find that the BeFx-inhibited enzyme is also able to bind ADP/ATP and Na+. These results relate the E2-BeFx complex to a transient K+- and ADP-sensitive E∗P intermediate of the functional cycle of the Na+,K+-ATPase, prior to E2P.

Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders.

P4-ATPases flip lipids from the exoplasmic to the cytosolic leaflet, thus maintaining lipid asymmetry in eukaryotic cell membranes. Mutations in several human P4-ATPase genes are associated with severe diseases, for example in ATP8B1 causing progressive familial intrahepatic cholestasis, a rare inherited disorder progressing toward liver failure. ATP8B1 forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report functional studies and the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 Å resolution. We find that ATP8B1 is autoinhibited by its N- and C-terminal tails, which form extensive interactions with the catalytic sites and flexible domain interfaces. Consistently, ATP hydrolysis is unleashed by truncation of the C-terminus, but also requires phosphoinositides, most markedly phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), and removal of both N- and C-termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminal segment further suggests molecular communication between N- and C-termini in the autoinhibition and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A recurring (G/A)(Y/F)AFS motif of the C-terminal segment suggests that this mechanism is employed widely across P4-ATPase lipid flippases in plasma membrane and endomembranes.

Structural Investigations of Full-Length Insulin Receptor Dynamics and Signalling.

Insulin regulates glucose homeostasis via binding and activation of the insulin receptor dimer at two distinct pairs of binding sites 1 and 2. Here, we present cryo-EM studies of full-length human insulin receptor (hIR) in an active state obtained at non-saturating, physiologically relevant insulin conditions. Insulin binds asymmetrically to the receptor under these conditions, occupying up to three of the four possible binding sites. Deletion analysis of the receptor together with site specific peptides and insulin analogs used in binding studies show that both sites 1 and 2 are required for high insulin affinity. We identify a homotypic interaction of the fibronectin type III domain (FnIII-3) of IR resulting in tight interaction of membrane proximal domains of the active, asymmetric receptor dimer. Our results show how insulin binding at two distinct types of sites disrupts the autoinhibited apo-IR dimer and stabilizes the active dimer. We propose an insulin binding and activation mechanism, which is sequential, exhibits negative cooperativity, and is based on asymmetry at physiological insulin concentrations with one to three insulin molecules activating IR.

Structure and Function of Na,K-ATPase-The Sodium-Potassium Pump.

Na,K-ATPase is an ubiquitous enzyme actively transporting Na-ions out of the cell in exchange for K-ions, thereby maintaining their concentration gradients across the cell membrane. Since its discovery more than six decades ago the Na-pump has been studied extensively and its vital physiological role in essentially every cell has been established. This article aims at providing an overview of well-established biochemical properties with a focus on Na,K-ATPase isoforms, its transport mechanism and principle conformations, inhibitors, and insights gained from crystal structures. © 2021 American Physiological Society. Compr Physiol 11:1-21, 2021.

Mind the Gap: Molecular Architecture of the Axon Initial Segment - From Fold Prediction to a Mechanistic Model of Function?

The axon initial segment (AIS) is a distinct neuronal domain, which is responsible for initiating action potentials, and therefore of key importance to neuronal signaling. To determine how it functions, it is necessary to establish which proteins reside there, how they are organized, and what the dynamic features are. Great strides have been made in recent years, and it is now clear that several AIS cytoskeletal and membrane proteins interact to form a higher-order periodic structure. Here we briefly describe AIS function, protein composition and molecular architecture, and discuss perspectives for future structural characterization, and if structure predictions will be able to model complex higher-order assemblies.

Transport unplugged: KCCs are regulated through an N-terminal plug of the ion pathway.

The ability to regulate transmembrane ion transport in response to various cues is vital to any living cell. In neurons, one key example of critical ion control relates to the extrusion of chloride mediated by the potassium-chloride-cotransporters (KCC1-4). In a recent hallmark study, Chi et␣al (2021) report cryo-EM structures of human KCC1 and KCC3b, delineating in detail how regulation by phosphorylation inhibits the transport activity. The authors also identify a stabilizing binding site for nucleotides and speculate on its functional role.

Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase.

P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na+/K+-ATPase or Ca2+-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.

The Crystal Structure of the Ca2+-ATPase 1 from Listeria monocytogenes reveals a Pump Primed for Dephosphorylation.

Many bacteria export intracellular calcium using active transporters homologous to the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). Here we present three crystal structures of Ca2+-ATPase 1 from Listeria monocytogenes (LMCA1). Structures with BeF3- mimicking a phosphoenzyme state reveal a closed state, which is intermediate between the outward-open E2P and the proton-occluded E2-P* conformations known for SERCA. It suggests that LMCA1 in the E2P state is pre-organized for dephosphorylation upon Ca2+ release, consistent with the rapid dephosphorylation observed in single-molecule studies. An arginine side-chain occupies the position equivalent to calcium binding site I in SERCA, leaving a single Ca2+ binding site in LMCA1, corresponding to SERCA site II. Observing no putative transport pathways dedicated to protons, we infer a direct proton counter transport through the Ca2+ exchange pathways. The LMCA1 structures provide insight into the evolutionary divergence and conserved features of this important class of ion transporters.

From Plant to Patient: Thapsigargin, a Tool for Understanding Natural Product Chemistry, Total Syntheses, Biosynthesis, Taxonomy, ATPases, Cell Death, and Drug Development.

Thapsigargin, the first representative of the hexaoxygenated guaianolides, was isolated 40 years ago in order to understand the skin-irritant principles of the resin of the umbelliferous plant Thapsia garganica. The pronounced cytotoxicity of thapsigargin is caused by highly selective inhibition of the intracellular sarco-endoplasmic Ca2+-ATPase (SERCA) situated on the membrane of the endo- or sarcoplasmic reticulum. Thapsigargin is selective to the SERCA pump and to a minor extent the secretory pathway Ca2+/Mn2+ ATPase (SPCA) pump. Thapsigargin has become a tool for investigation of the importance of SERCA in intracellular calcium homeostasis. In addition, complex formation of thapsigargin with SERCA has enabled crystallization and structure determination of calcium-free states by X-ray crystallography. These results led to descriptions of the mechanism of action and kinetic properties of SERCA and other ATPases. Inhibition of SERCA depletes Ca2+ from the sarco- and endoplasmic reticulum provoking the unfolded protein response, and thereby has enabled new studies on the mechanism of cell death. Development of protocols for selective transformation of thapsigargin disclosed the chemistry and facilitated total synthesis of the molecule. Conversion of trilobolide into thapsigargin offered an economically feasible sustainable source of thapsigargin, which enables a future drug production. Principles for prodrug development were used by conjugating a payload derived from thapsigargin with a hydrophilic peptide selectively cleaved by proteases in the tumor. Mipsagargin was developed in order to obtain a drug for treatment of cancer diseases characterized by the presence of prostate specific membrane antigen (PSMA) in the neovascular tissue of the tumors. Even though mipsagargin showed interesting clinical effects the results did not encourage funding and consequently the attempt to register the drug has been abandoned. In spite of this disappointing fact, the research performed to develop the drug has resulted in important scientific discoveries concerning the chemistry, biosynthesis and biochemistry of sesquiterpene lactones, the mechanism of action of ATPases including SERCA, mechanisms for cell death caused by the unfolded protein response, and the use of prodrugs for cancer-targeting cytotoxins. The presence of toxins in only some species belonging to Thapsia also led to a major revision of the taxonomy of the genus.