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A structural model of OATP1B1. The model was generated by Phyre2 and presented as a cartoon. Y640 and Y645 are highlighted in blue and the image was produced with PyMOL.

Purpose:OATP1B1 (SLCO1B1) is the most abundant and pharmacologically relevant uptake transporter in the liver and a key mediator of xenobiotic clearance. However, the regulatory mechanisms that determine OATP1B1 activity remain uncertain, and as a result, unexpected drug–drug interactions involving OATP1B1 substrates continue to be reported, including several involving tyrosine kinase inhibitors (TKI).Experimental Design:OATP1B1-mediated activity in overexpressing HEK293 cells and hepatocytes was assessed in the presence of FDA-approved TKIs, while rosuvastatin pharmacokinetics in the presence of an OATP1B1 inhibiting TKI were measured in vivo. Tyrosine phosphorylation of OATP1B1 was determined by LC/MS-MS–based proteomics and transport function was measured following exposure to siRNAs targeting 779 different kinases.Results:Twenty-nine of 46 FDA-approved TKIs studied significantly inhibit OATP1B1 function. Inhibition of OATP1B1 by TKIs, such as nilotinib, is predominantly noncompetitive, can increase systemic concentrations of rosuvastatin in vivo, and is associated with reduced phosphorylation of OATP1B1 at tyrosine residue 645. Using genetic screens and functional validation studies, the Src kinase LYN was identified as a potential regulator of OATP1B1 activity that is highly sensitive to inhibition by various TKIs at clinically relevant concentrations.Conclusions:A novel kinase-dependent posttranslational mechanism of OATP1B1 activation was identified and interference with this process by TKIs can influence the elimination of a broad range of xenobiotic substrates.

Cellular viability and uptake of 8FcA by OATP1B1 following reverse transfection with 25 nM siRNA for various protein kinases. Percent uptake was determined relative to OATP1B1 uptake with control siRNA.

Purpose: OATP1B1 (SLCO1B1) is the most abundant and pharmacologically relevant uptake transporter in the liver and a key mediator of xenobiotic clearance. However, the regulatory mechanisms that determine OATP1B1 activity remain uncertain, and as a result, unexpected drug–drug interactions involving OATP1B1 substrates continue to be reported, including several involving tyrosine kinase inhibitors (TKI). Experimental Design: OATP1B1-mediated activity in overexpressing HEK293 cells and hepatocytes was assessed in the presence of FDA-approved TKIs, while rosuvastatin pharmacokinetics in the presence of an OATP1B1 inhibiting TKI were measured in vivo. Tyrosine phosphorylation of OATP1B1 was determined by LC/MS-MS–based proteomics and transport function was measured following exposure to siRNAs targeting 779 different kinases. Results: Twenty-nine of 46 FDA-approved TKIs studied significantly inhibit OATP1B1 function. Inhibition of OATP1B1 by TKIs, such as nilotinib, is predominantly noncompetitive, can increase systemic concentrations of rosuvastatin in vivo, and is associated with reduced phosphorylation of OATP1B1 at tyrosine residue 645. Using genetic screens and functional validation studies, the Src kinase LYN was identified as a potential regulator of OATP1B1 activity that is highly sensitive to inhibition by various TKIs at clinically relevant concentrations. Conclusions: A novel kinase-dependent posttranslational mechanism of OATP1B1 activation was identified and interference with this process by TKIs can influence the elimination of a broad range of xenobiotic substrates.

Significance Site-directed fluorometry was used to understand conformational changes of the Na+/glucose symporter. SGLT1 functions by a mechanism where the substrate-binding site alternates between the two faces of the membrane, but little is known about the underlying conformational changes. Rhodamines were covalently inserted into the substrate cavity, and changes of fluorescence were measured in real time with the opening and closing of the outer gate as SGLT1 was driven between inward and outward conformations using voltage jumps. Structural modeling indicated that the quenching with gating opening was due to an increased solvation of rhodamine and an increase in polar residues lining the wall of the cavity. This experimental approach will lead to a better understanding of the mechanism of membrane transport., In the human sodium glucose cotransporter (hSGLT1) cycle, the protein undergoes conformational changes where the sugar-binding site alternatively faces the external and internal surfaces. Functional site-directed fluorometry was used to probe the conformational changes at the sugar-binding site. Residues (Y290, T287, H83, and N78) were mutated to cysteines. The mutants were expressed in Xenopus laevis oocytes and tagged with environmentally sensitive fluorescent rhodamines [e.g., tetramethylrhodamine (TMR)-thiols]. The fluorescence intensity was recorded as the mutants were driven into different conformations using voltage jumps. Sugar binding and transport by the fluorophore-tagged mutants were blocked, but Na+ binding and the voltage-dependent conformational transitions were unaffected. Structural models indicated that external Na+ binding opened a large aqueous vestibule (600 Å3) leading to the sugar-binding site. The fluorescence of TMR covalently linked to Y290C, T287C, and H83C decreased as the mutant proteins were driven from the inward to the outward open Na+-bound conformation. The time courses of fluorescence changes (milliseconds) were close to the SGLT1 capacitive charge movements. The quench in rhodamine fluorescence indicated that the environment of the chromophores became more polar with opening of the external gates as the protein transitioned from the inward to outward facing state. Structural analyses showed an increase in polar side chains and a decrease in hydrophobic side chains lining the vestibule, and this was reflected in solvation of the chromophore. The results demonstrate the opening and closing of external gates in real time, with the accompanying changes of polarity of the sugar vestibule.

International audience; The voltage-dependent anion channel (VDAC) mediates the flow of metabolites and ions across the outer mitochondrial membrane of all eukaryotic cells. The open channel passes millions of ATP molecules per second, whereas the closed state exhibits no detectable ATP flux. High-resolution structures of VDAC1 revealed a 19-stranded β-barrel with an α-helix partially occupying the central pore. To understand ATP permeation through VDAC, we solved the crystal structure of mouse VDAC1 (mVDAC1) in the presence of ATP, revealing a low-affinity binding site. Guided by these coordinates, we initiated hundreds of molecular dynamics simulations to construct a Markov state model of ATP permeation. These simulations indicate that ATP flows through VDAC through multiple pathways, in agreement with our structural data and experimentally determined physiological rates.

The photosensitizer, methylene blue (MB), generates singlet oxygen ((1)O2) that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark MB inhibits reversibly, binding being accompanied by a bathochromic shift that can be used to show its displacement by other reversible inhibitors binding to the catalytic 'anionic' subsite (CAS), the peripheral 'anionic' subsite (PAS), or bridging them. Data concerning both reversible and irreversible inhibition are here reviewed. MB protects TcAChE from thermal denaturation, and differential scanning calorimetry reveals a ~8 °C increase in the denaturation temperature. The crystal structure of the MB/TcAChE complex reveals a single MB stacked against W279 in the PAS, pointing down the gorge towards the CAS. The intrinsic fluorescence of the irreversibly inhibited enzyme displays new emission bands that can be ascribed to N'-formylkynurenine (NFK); this was indeed confirmed using anti-NFK antibodies. Mass spectroscopy revealed that two Trp residues, Trp84 in the CAS, and Trp279 in the PAS, were the only Trp residues, out of a total of 14, significantly modified by photo-oxidation, both being converted to NFK. In the presence of competitive inhibitors that displace MB from the gorge, their modification is completely prevented. Thus, photo-oxidative damage caused by MB involves targeted release of (1)O2 by the bound photosensitizer within the aqueous milieu of the active-site gorge.

More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human genome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, and fusion of vesicles. We showed thatCRACR2A(Ca(2+) release-activated Ca(2+) channel regulator 2A) encodes a lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand motifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site. Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role for CRACR2A in the activation of the Ca(2+) and c-Jun N-terminal kinase signaling pathways in response to T cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi to the immunological synapse formed between a T cell and a cognate antigen-presenting cell to activate these signaling pathways. The interaction between the PRD of CRACR2A and the guanidine nucleotide exchange factor Vav1 was required for the accumulation of these vesicles at the immunological synapse. Furthermore, we demonstrated that GTP binding and prenylation of CRACR2A were associated with its localization near the Golgi and its stability. Our findings reveal a previously uncharacterized function of a large Rab GTPase and vesicles near the Golgi in TCR signaling. Other GTPases with similar domain architectures may have similar functions in T cells.

The flagellar motor is one type of propulsion device of motile bacteria. The cytoplasmic ring (C-ring) of the motor interacts with the stator to generate torque in clockwise and counterclockwise directions. The C-ring is composed of three proteins, FliM, FliN, and FliG. Together they form the "switch complex" and regulate switching and torque generation. Here we report the crystal structure of the middle domain of FliM in complex with the middle and C-terminal domains of FliG that shows the interaction surface and orientations of the proteins. In the complex, FliG assumes a compact conformation in which the middle and C-terminal domains (FliG(MC)) collapse and stack together similarly to the recently published structure of a mutant of FliG(MC) with a clockwise rotational bias. This intramolecular stacking of the domains is distinct from the intermolecular stacking seen in other structures of FliG. We fit the complex structure into the three-dimensional reconstructions of the motor and propose that the cytoplasmic ring is assembled from 34 FliG and FliM molecules in a 1:1 fashion.

Neuroligins act as heterophilic adhesion molecules at neuronal synapses. Their cytoplasmic domains interact with synaptic scaffolding proteins, and have been shown to be intrinsically disordered. Here we report the backbone and side chain H-1, C-13 and N-15 resonance assignments for the cytoplasmic domain of human neuroligin 3.

Amalgam (Ama) is a secreted neuronal adhesion protein that contains three tandem immunoglobulin domains. It has both homophilic and heterophilic cell adhesion properties, and is required for axon guidance and fasciculation during early stages of Drosophila development. Here, we report its biophysical characterization and use small-angle x-ray scattering to determine its low-resolution structure in solution. The biophysical studies revealed that Ama forms dimers in solution, and that its secondary and tertiary structures are typical for the immunoglobulin superfamily. Ab initio and rigid-body modeling by small-angle x-ray scattering revealed a distinct V-shaped dimer in which the two monomer chains are aligned parallel to each other, with the dimerization interface being formed by domain 1. These data provide a structural basis for the dual adhesion characteristics of Ama. Thus, the dimeric structure explains its homophilic adhesion properties. Its V shape suggests a mechanism for its interaction with its receptor, the single-pass transmembrane adhesion protein neurotactin, in which each “arm” of Ama binds to the extracellular domain of neurotactin, thus promoting its clustering on the outer face of the plasma membrane.

The biennial CASP experiment is a crucial way to evaluate, in an unbiased way, the progress in predicting novel 3D protein structures. In this article, we assess the quality of prediction of template free models, that is, ab initio prediction of 3D structures of proteins based solely on the amino acid sequences, that is, proteins that did not have significant sequence identity to any protein in the Protein Data Bank. There were 13 targets in this category and 102 groups submitted predictions. Analysis was based on the GDT_TS analysis, which has been used in previous CASP experiments, together with a newly developed method, the OK_Rank, as well as by visual inspection. There is no doubt that in recent years many obstacles have been removed on the long and elusive way to deciphering the protein-folding problem. Out of the 13 targets, six were predicted well by a number of groups. On the other hand, it must be stressed that for four targets, none of the models were judged to be satisfactory. Thus, for template free model prediction, as evaluated in this CASP, successes have been achieved for most targets; however, a great deal of research is still required, both in improving the existing methods and in development of new approaches. Proteins 2009. © 2009 Wiley-Liss, Inc.

Cholinesterase-like adhesion molecules (CLAMs) are a family of neuronal cell adhesion molecules with important roles in synaptogenesis, and in maintaining structural and functional integrity of the nervous system. Our earlier study on the cytoplasmic domain of one of these CLAMs, the Drosophila protein, gliotactin, showed that it is intrinsically unstructured in vitro. Bioinformatic analysis suggested that the cytoplasmic domains of other CLAMs are also intrinsically unstructured, even though they bear no sequence homology to each other or to any known protein. In this study, we overexpress and purify the cytoplasmic domain of human neuroligin 3, notwithstanding its high sensitivity to the Escherichia coli endogenous proteases that cause its rapid degradation. Using bioinformatic analysis, sensitivity to proteases, size exclusion chromatography, fluorescence correlation spectroscopy, analytical ultracentrifugation, small angle x-ray scattering, circular dichroism, electron spin resonance, and nuclear magnetic resonance, we show that the cytoplasmic domain of human neuroligin 3 is intrinsically unstructured. However, several of these techniques indicate that it is not fully extended, but becomes significantly more extended under denaturing conditions.

Intrinsically unstructured proteins (IUPs), also known as natively unfolded proteins, lack well-defined secondary and tertiary structure under physiological conditions. In recent years, growing experimental and theoretical evidence has accumulated, indicating that many entire proteins and protein sequences are unstructured under physiological conditions, and that they play significant roles in diverse cellular processes. Bioinformatic algorithms have been developed to identify such sequences in proteins for which structural data are lacking, but still generate substantial numbers of false positives and negatives. We describe here a simple and reliable in vitro assay for identifying IUP sequences based on their susceptibility to 20S proteasomal degradation. We show that 20S proteasomes digest IUP sequences, under conditions in which native, and even molten globule states, are resistant. Furthermore, we show that protein-protein interactions can protect IUPs against 20S proteasomal action. Taken together, our results thus suggest that the 20S proteasome degradation assay provides a powerful system for operational definition of IUPs.

Sodium-dependent glucose transporters (SGLTs) are members of the large solute carrier (SLC) family of proteins that exploit the sodium ion concentration gradient to transport a myriad of small molecules across the plasma membrane. In humans, there are six SGLT subtypes labeled 1-6 that are expressed widely in the small intestine, kidney, lung, muscle, and brain. Due to their role in sugar reabsorption, SGLTs are currently exploited as drug targets for the treatment of type 2 diabetes, especially hSGLT2, which is responsible for 98% of glucose reabsorption in the kidneys. Current inhibitors are chemical derivatives of the naturally occurring small molecule phlorizin, which is expressed in the bark of fruit trees, such as apple and pear. The structural basis of binding is not known, in part, because high-resolution structures of mammalian SGLTs do not exist. However, the inward-facing structure of the bacterial homologue from vibrio parahaemolyticus (vSGLT) has been solved both in apo and in complex with galactose, and our collaborators recently solved the outward-facing structure of a closely related homologue (unpublished). Here we combine homology modeling, virtual screening techniques and molecular dynamics simulations to achieve two goals: 1) model the outward-facing state of SGLTs, and 2) predict the binding mode of phlorizin and its derivatives hSGLT1 and 2. As a result, the spectroscopic data (double electron-electron resonance) probing outward facing state of SGLTs validate our homology model and mutagenesis studies testing binding to hSGLT1 and 2 are in agreement with our predicted binding modes.

In recent years, there has been a vast increase in structural and functional understanding of VDAC1, but VDAC2 and -3 have been understudied despite having many unique phenotypes. One reason for the paucity of structural and biochemical characterization of the VDAC2 and -3 isoforms stems from the inability of obtaining purified, functional protein. Here we demonstrate the expression, isolation, and basic characterization of zebrafish VDAC2 (zfVDAC2). Further, we resolved the structure of zfVDAC2 at 2.8 A resolution, revealing a crystallographic dimer. The dimer orientation was confirmed in solution by double electron-electron resonance spectroscopy and by cross-linking experiments disclosing a dimer population of ∼20% in lauryldimethine amine oxide detergent micelles, whereas in lipidic bicelles a higher population of dimeric and higher order oligomers species were observed. The present study allows for a more accurate structural comparison between VDAC2 and its better-studied counterpart VDAC1.

Secondary active transporters couple electrochemical energy to the transport of solutes across biological membrane. The sodium solute symporters (SSS) comprise a large family of secondary active transporters fundamental to the homeostasis of a variety of solutes including sugars, inositols, anions, short chain fatty acids, choline, and vitamins. This chapter will explicate the structural framework of the sodium galactose transporter from Vibrio parahaemolyticus (vSGLT) and focus on mechanistic hypothesis drawn from these structures, some of which were directly transferable to the human homologues of vSGLT, which are members of the medically important SLC5 family. Compiling functional, structural, and bioinformatics data generated from these studies and incorporating unique conformations from structurally similar proteins, we propose a modified mechanism for sodium-dependent transport.

Dietary sugars have an immense importance in the metabolism of all cells. As such, it is vital to understand their cellular entry and exit pathways through specific transporters. Our research focuses on the sodium dependent sugar transporter from Vibrio parahemolyticus (vSGLT). Currently, this is the best structural model system for the mammalian transporters, which are responsible for sugar absorption in the intestine and reabsorption in the kidney. Recently, this transporter has been the target of various drugs that lower blood-sugar levels by inhibiting sugar reabsorption in the kidney.SGLTs are highly dynamic and switch between a number of conformations to facilitate sugar passage across the cell membrane. There are currently two available x-ray structures for vSGLT in an inward-occluded1 and an inward-open2 conformation. These two distinct conformations have fundamentally advanced our understanding of sugar transport to an atomic level, but did not encapsulate the dynamics of transport, nor the nature of the outward-open conformation.In order to gain further insight into the ligand dependent dynamics of vSGLT, we have conducted double electron-electron resonance (DEER) and wide-angle x-ray scattering (WAXS) studies under three conditions, each favoring different conformers in the protein: in the absence of sodium and galactose, in the presence of sodium only and in the presence of both sodium and galactose.We will discuss the overall insights gained by these two approaches, the similarities and differences with other transporters that share the Leu-T fold and the structural refinement that included combined energy terms from DEER and WAXS.1. Faham et al., (2008) Science 321(5890).2. Watanabe et al., (2010) Nature 468.

The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic “anionic” subsite (CAS), the peripheral “anionic” subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both “anionic” subsites, but a single site is involved in inhibition. MB also quenches TcAChE's intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an ∼8° increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.

β-Secretase (β-site amyloid precursor protein-cleaving enzyme 1; BACE1) is a transmembrane aspartic protease that cleaves the β-amyloid precursor protein en route to generation of the amyloid β-peptide (Aβ) that is believed to be responsible for the Alzheimer's disease amyloid cascade. It is thus a prime target for the development of inhibitors which may serve as drugs in the treatment and/or prevention of Alzheimer's disease. In the following determination of the crystal structures of both apo and complexed BACE1, structural analysis of all crystal structures of BACE1 deposited in the PDB and molecular dynamics (MD) simulations of monomeric and `dimeric' BACE1 were used to study conformational changes in the active-site region of the enzyme. It was observed that a flap able to cover the active site is the most flexible region, adopting multiple conformational states in the various crystal structures. Both the presence or absence of an inhibitor within the active site and the crystal packing are shown to influence the flap's conformation. An open conformation of the flap is mostly observed in the apo structures, while direct hydrogen-bonding interaction between main-chain atoms of the flap and the inhibitor is a prerequisite for the flap to adopt a closed conformation in the crystal structures of complexes. Thus, a systematic study of the conformational flexibility of the enzyme may not only contribute to structure-based drug design of BACE1 inhibitors and of other targets with flexible conformations, but may also help to better understand the mechanistic events associated with the binding of substrates and inhibitors to the enzyme.

Voltage-dependent anion channels (VDAC) mediate the transfer of metabolites such as ATP, ADP, and NADH across the outer mitochondrial membrane of all eukaryotic cells. The open state is selective for anions, while the partially closed state is selective for cations. The x-ray crystal structure of VDAC from mouse (mVDAC1) (Ujwal et al. PNAS 2008) made it possible to study the molecular workings of this channel in unprecedented detail.Previously, we used continuum electrostatic calculations to show that mVDAC1 is anion selective suggesting that the x-ray crystal structure represents the open state of the channel (Choudhary et al. JMB 2010). However, the hallmark of the open state is high ATP flux. The open channel passes millions of ATP molecules per second, while the closed state exhibits no detectable ATP flux. Therefore, we determined the conduction state of the channel with regard to the ATP permeation. To understand ATP permeation through VDAC, we solved the structure of murine VDAC1 (mVDAC1) in the presence of ATP revealing a low-occupancy binding site. Guided by these coordinates, we initiated hundreds of molecular dynamics (MD) simulations to construct a Markov State Model (MSM) of ATP permeation using the software (Beauchamp et al. JCTC 2011). These simulations show a high ATP flux generated from multiple pathways through the channel, consistent with our structural data and previously reported physiological permeation rates.

A bis-(-)-nor-meptazinol derivative in which the two meptazinol rings are linked by a nonamethylene spacer is a novel acetylcholinesterase inhibitor that inhibits both catalytic activity and Abeta peptide aggregation. The crystal structure of its complex with Torpedo californica acetylcholinesterase was determined to 2.7 A resolution. The ligand spans the active-site gorge, with one nor-meptazinol moiety bound at the "anionic" subsite of the active site, disrupting the catalytic triad by forming a hydrogen bond with His440N(epsilon2), which is hydrogen-bonded to Ser200O(gamma) in the native enzyme. The second nor-meptazinol binds at the peripheral "anionic" site at the gorge entrance. A number of GOLD models of the complex, using both native TcAChE and the protein template from the crystal structure of the bis-(-)-nor-meptazinol/TcAChE complex, bear higher similarity to the X-ray structure than a previous model obtained using the mouse enzyme structure. These findings may facilitate rational design of new meptazinol-based acetylcholinesterase inhibitors.

Amalgam, a multi-domain member of the immunoglobulin superfamily, possesses homophilic and heterophilic cell adhesion properties. It is required for axon guidance during Drosophila development in which it interacts with the extracellular domain of the transmembrane protein, neurotactin, to promote adhesion. Amalgam was heterologously expressed in Pichia pastoris, and the secreted protein product, bearing an NH(2)-terminal His(6)Tag, was purified from the growth medium by metal affinity chromatography. Size exclusion chromatography separated the purified protein into two fractions: a major, multimeric fraction and a minor, dimeric one. Two protocols to reduce the percentage of multimers were tested. In one, protein induction was performed in the presence of the zwitterionic detergent CHAPS, yielding primarily the dimeric form of amalgam. In a second protocol, agitation was gradually reduced during the course of the induction and antifoam was added daily to reduce the air/liquid interfacial foam area. This latter protocol lowered the percentage of multimer 2-fold, compared to constant agitation. Circular dichroism measurements showed that the dimeric fraction had a high beta-sheet content, as expected for a protein with an immunoglobulin fold. Dynamic light scattering and sedimentation velocity measurements showed that the multimeric fraction displays a monodisperse distribution, with R(H)=16 nm. When co-expressed together with amalgam the ectodomain of neurotactin copurified with it. Furthermore, both purified fractions of amalgam were shown to interact with Torpedo californica acetylcholinesterase, a structural homolog of neurotactin.

Calcium ion (Ca2+) signaling is essential to many cellular processes, from fertilization to apoptosis (cell death). Stable Ca2+ levels are achieved through tightly regulated and coordinated cellular processes. Alterations in Ca2+ levels are involved in many pathological conditions, including stroke and heart failure ( 1 ). After Ca2+ stimulation, the Na+/Ca2+ exchanger (NCX)—a ubiquitous Ca2+ export protein residing in the plasma membrane—participates in the restoration of resting levels of Ca2+ in excitable cells. On page 686 of this issue, Liao et al. ( 2 ) report the structure of a prokaryotic homolog of NCX, revealing a symmetrical protein scaffold and ion-binding pocket that provides detailed insights into the Na+/Ca2+ exchange process.