April Goehring

Structure and mechanism of a na+-independent amino Acid transporter.

APC Transporter Structure Amino acid, polyamine, and organocation (APC) transporters that move a range of organic molecules across the cell membrane are important in many cellular processes. Shaffer et al. (p. 1010; published online 16 July) report the crystal structure of apoApcT, a proton-dependent APC transporter. The structure shows similarity to the sodium-coupled amino acid transporter LeuT and has an amino group from a lysine in an equivalent position to a sodium ion in LeuT. This suggests common principles between proton- and sodium-coupled transporters. The structure of the transporter ApcT reveals common architectural principles between proton- and sodium-coupled transporters. Amino acid, polyamine, and organocation (APC) transporters are secondary transporters that play essential roles in nutrient uptake, neurotransmitter recycling, ionic homeostasis, and regulation of cell volume. Here, we present the crystal structure of apo-ApcT, a proton-coupled broad-specificity amino acid transporter, at 2.35 angstrom resolution. The structure contains 12 transmembrane helices, with the first 10 consisting of an inverted structural repeat of 5 transmembrane helices like the leucine transporter LeuT. The ApcT structure reveals an inward-facing, apo state and an amine moiety of lysine-158 located in a position equivalent to the sodium ion site Na2 of LeuT. We propose that lysine-158 is central to proton-coupled transport and that the amine group serves the same functional role as the Na2 ion in LeuT, thus demonstrating common principles among proton- and sodium-coupled transporters.

NMDA receptor structures reveal subunit arrangement and pore architecture.

N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present X-ray crystal structures of the Xenopus laevis GluN1–GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains. The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

X-Ray Structure of Acid-Sensing Ion Channel 1–Snake Toxin Complex Reveals Open State of a Na+-Selective Channel

Acid-sensing ion channels (ASICs) detect extracellular protons produced during inflammation or ischemic injury and belong to the superfamily of degenerin/epithelial sodium channels. Here, we determine the cocrystal structure of chicken ASIC1a with MitTx, a pain-inducing toxin from the Texas coral snake, to define the structure of the open state of ASIC1a. In the MitTx-bound open state and in the previously determined low-pH desensitized state, TM2 is a discontinuous α helix in which the Gly-Ala-Ser selectivity filter adopts an extended, belt-like conformation, swapping the cytoplasmic one-third of TM2 with an adjacent subunit. Gly 443 residues of the selectivity filter provide a ring of three carbonyl oxygen atoms with a radius of ∼3.6 Å, presenting an energetic barrier for hydrated ions. The ASIC1a-MitTx complex illuminates the mechanism of MitTx action, defines the structure of the selectivity filter of voltage-independent, sodium-selective ion channels, and captures the open state of an ASIC.

Screening and large-scale expression of membrane proteins in mammalian cells for structural studies

Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8–tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI− (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4–6 weeks.

Structural basis for action by diverse antidepressants on biogenic amine transporters.

The biogenic amine transporters (BATs) regulate endogenous neurotransmitter concentrations and are targets for a broad range of therapeutic agents including selective serotonin reuptake inhibitors (SSRIs), serotonin–noradrenaline reuptake inhibitors (SNRIs) and tricyclic antidepressants (TCAs). Because eukaryotic BATs are recalcitrant to crystallographic analysis, our understanding of the mechanism of these inhibitors and antidepressants is limited. LeuT is a bacterial homologue of BATs and has proven to be a valuable paradigm for understanding relationships between their structure and function. However, because only approximately 25% of the amino acid sequence of LeuT is in common with that of BATs, and as LeuT is a promiscuous amino acid transporter, it does not recapitulate the pharmacological properties of BATs. Indeed, SSRIs and TCAs bind in the extracellular vestibule of LeuT and act as non-competitive inhibitors of transport. By contrast, multiple studies demonstrate that both TCAs and SSRIs are competitive inhibitors for eukaryotic BATs and bind to the primary binding pocket. Here we engineered LeuT to harbour human BAT-like pharmacology by mutating key residues around the primary binding pocket. The final LeuBAT mutant binds the SSRI sertraline with a binding constant of 18 nM and displays high-affinity binding to a range of SSRIs, SNRIs and a TCA. We determined 12 crystal structures of LeuBAT in complex with four classes of antidepressants. The chemically diverse inhibitors have a remarkably similar mode of binding in which they straddle transmembrane helix (TM) 3, wedge between TM3/TM8 and TM1/TM6, and lock the transporter in a sodium- and chloride-bound outward-facing open conformation. Together, these studies define common and simple principles for the action of SSRIs, SNRIs and TCAs on BATs.

AKAP79-mediated Targeting of the Cyclic AMP-dependent Protein Kinase to the β1-Adrenergic Receptor Promotes Recycling and Functional Resensitization of the Receptor

Resensitization of G protein-coupled receptors (GPCR) following prolonged agonist exposure is critical for restoring the responsiveness of the receptor to subsequent challenges by agonist. The 3′-5′ cyclic AMP-dependent protein kinase (PKA) and serine 312 in the third intracellular loop of the human β1-adrenergic receptor (β1-AR) were both necessary for efficient recycling and resensitization of the agonist-internalized β1-AR (Gardner, L. A., Delos Santos, N. M., Matta, S. G., Whitt, M. A., and Bahouth, S. W. (2004) J. Biol. Chem. 279, 21135-21143). Because PKA is compartmentalized near target substrates by interacting with protein kinase A anchoring proteins (AKAPs), the present study was undertaken to identify the AKAP involved in PKA-mediated phosphorylation of the β1-AR and in its recycling and resensitization. Here, we report that Ht-31 peptide-mediated disruption of PKA/AKAP interactions prevented the recycling and functional resensitization of heterologously expressed β1-AR in HEK-293 cells and endogenously expressed β1-AR in SK-N-MC cells and neonatal rat cortical neurons. Whereas several endogenous AKAPs were identified in HEK-293 cells, small interfering RNA-mediated down-regulation of AKAP79 prevented the recycling of the β1-AR in this cell line. Co-immunoprecipitations and fluorescence resonance energy transfer (FRET) microscopy experiments in HEK-293 cells revealed that the β1-AR, AKAP79, and PKA form a ternary complex at the carboxyl terminus of the β1-AR. This complex was involved in PKA-mediated phosphorylation of the third intracellular loop of the β1-AR because disruption of PKA/AKAP interactions or small interfering RNA-mediated down-regulation of AKAP79 both inhibited this response. Thus, AKAP79 provides PKA to phosphorylate the β1-AR and thereby dictate the recycling and resensitization itineraries of the β1-AR.

Orchestration of synaptic plasticity through AKAP signaling complexes.

Significant progress has been made toward understanding the mechanisms by which organisms learn from experiences and how those experiences are translated into memories. Advances in molecular, electrophysiological and genetic technologies have permitted great strides in identifying biochemical and structural changes that occur at synapses during processes that are thought to underlie learning and memory. Cellular events that generate the second messenger cyclic AMP (cAMP) and activate protein kinase A (PKA) have been linked to synaptic plasticity and long-term memory. In this review we will focus on the role of PKA in synaptic plasticity and discuss how the compartmentalization of PKA through its association with A-Kinase Anchoring Proteins (AKAPs) affect PKA function in this process.

mAKAP Compartmentalizes Oxygen-Dependent Control of HIF-1α

The scaffold protein muscle A kinase–anchoring protein (mAKAP) regulates the stability and localization of the transcription factor HIF-1α. Anchor’s a Way to Regulate HIF-1α Under conditions in which oxygen concentrations are normal (normoxia), the transcription factor hypoxia-inducible factor 1α (HIF-1α) is ubiquitinated and undergoes proteasome-dependent degradation. When oxygen becomes scarce (hypoxia), such as occurs in tumors and during myocardial infarction, HIF-1α is no longer degraded; it forms a heterodimer with the constitutively expressed HIF-1β subunit and induces the expression of hypoxia-associated genes. The products of these genes, including proteins such as vascular endothelial growth factor and glucose transporter 1, help the cell to adapt to conditions of low oxygen concentration. Wong et al. now provide evidence that the scaffolding protein muscle A kinase–anchoring protein (mAKAP), best known for its role in organizing protein kinase A and other signaling molecules, binds to HIF-1α and components of the ubiquitin machinery to regulate the stability of HIF-1α in a bidirectional fashion. Under normoxia, components of the mAKAP complex target HIF-1α for degradation; under hypoxia, however, mAKAP organizes factors that stabilize HIF-1α. In addition, the perinuclear localization of mAKAP is required to position HIF-1α close to its target genes. Together, these results suggest that mAKAP functions as an important regulatory component of the hypoxic response in cardiomyocytes. The activity of the transcription factor hypoxia-inducible factor 1α (HIF-1α) is increased in response to reduced intracellular oxygen. Enzymes of the protein ubiquitin machinery that signal the destruction or stabilization of HIF-1α tightly control this transcriptional response. Here, we show that muscle A kinase–anchoring protein (mAKAP) organized ubiquitin E3 ligases that managed the stability of HIF-1α and optimally positioned it close to its site of action inside the nucleus. Functional experiments in cardiomyocytes showed that depletion of mAKAP or disruption of its targeting to the perinuclear region altered the stability of HIF-1α and transcriptional activation of genes associated with hypoxia. Thus, we propose that compartmentalization of oxygen-sensitive signaling components may influence the fidelity and magnitude of the hypoxic response.

MyRIP Anchors Protein Kinase A to the Exocyst Complex

The movement of signal transduction enzymes in and out of multi-protein complexes coordinates the spatial and temporal resolution of cellular events. Anchoring and scaffolding proteins are key to this process because they sequester protein kinases and phosphatases with a subset of their preferred substrates. The protein kinase A-anchoring family of proteins (AKAPs), which target the cAMP-dependent protein kinase (PKA) and other enzymes to defined subcellular microenvironments, represent a well studied group of these signal-organizing molecules. In this report we demonstrate that the Rab27a GTPase effector protein MyRIP is a member of the AKAP family. The zebrafish homolog of MyRIP (Ze-AKAP2) was initially detected in a two-hybrid screen for AKAPs. A combination of biochemical, cell-based, and immunofluorescence approaches demonstrate that the mouse MyRIP ortholog targets the type II PKA holoenzyme via an atypical mechanism to a specific perinuclear region of insulin-secreting cells. Similar approaches show that MyRIP interacts with the Sec6 and Sec8 components of the exocyst complex, an evolutionarily conserved protein unit that controls protein trafficking and exocytosis. These data indicate that MyRIP functions as a scaffolding protein that links PKA to components of the exocytosis machinery.

Cryo-EM structures of the triheteromeric NMDA receptor and its allosteric modulation.

Added complexity in an asymmetric receptor N-methyl-d-aspartate receptors (NMDARs) are heterotetrameric ion channels that initiate chemical and electrical signals in postsynaptic cells. They play key roles in brain development and function and are the targets of drugs for treating neurological disorders such as schizophrenia, depression, and epilepsy. For the channel to open, it must bind glutamate and glycine and release a blocking magnesium ion. Most NMDARs have three different subunits that bind glycine and glutamine, but structural studies have focused on tetramers of only two subunits. Lü et al. determined the structure of triheteromeric NMDAR. The structural studies show how having three different subunits modifies receptor symmetry and subunit interactions and increases the complexity of receptor regulation. Science, this issue p. eaal3729 Having three different subunits allows complex regulation of the neuronal NMDA ionotropic glutamate receptor involved in synaptic plasticity. INTRODUCTION Chemical neurotransmission is fundamental to communication between neurons and to the alternation of the “strength” of neuron-to-neuron connections in an experience-dependent manner. N-methyl-d-aspartate receptors (NMDARs) are neurotransmitter-activated ion channels that act as Hebbian-like coincidence detectors, requiring the binding of glutamate and glycine together with the voltage-dependent relief of magnesium block from the ion channel pore. Because the open NMDAR ion channel pore conducts both monovalent ions and Ca2+, not only does the activation of NMDARs elicit an electrical signal but also the entry of Ca2+ provides a chemical signal, initiating intracellular calcium-dependent signaling processes. NMDARs are ubiquitously dispersed throughout the central nervous system, play crucial roles in brain development and function, and are the targets of clinically relevant drugs for treatment of mild cognitive impairment, schizophrenia, depression, and epilepsy. Diversity in NMDAR function is the consequence of receptor assembly as heterotetramers with different receptor subunit combinations found in distinct brain regions. The palette of NMDAR building blocks includes the extensively studied glycine-binding and glutamate-binding GluN1 and GluN2A to -D subunits, respectively, together with the rather enigmatic glycine-binding GluN3A and -B subunits. The canonical NMDAR is composed of two GluN1 subunits and two GluN2 subunits, where the two GluN2 subunits can be either identical or different, thus giving rise to diheteromeric or triheteromeric NMDARs, respectively. Despite the prevalence of triheteromeric receptors throughout the brain, such as the GluN1/GluN2A/GluN2B receptor, the dominant NMDAR in the hippocampus and cortex, physiological and structural studies on NMDARs have been almost exclusively restricted to diheteromeric receptors. However, triheteromeric NMDARs are endowed with channel gating kinetics and receptor pharmacology distinct from the GluN2A- and GluN2B-containing diheteromeric receptors. Furthermore, the triheteromeric receptor is uniquely modulated by GluN2A- and GluN2B-specific allosteric antagonists. RATIONALE To determine how incorporation of two different GluN2 subunits alters receptor symmetry and subunit-subunit interactions, we resolved the structure of the GluN1/GluN2A/GluN2B receptor by single-particle cryogenic electron microscopy. Because the GluN2A and GluN2B subunits are structurally related, we used a GluN2B-specific Fab to unambiguously distinguish the two GluN2 subunits. To understand the molecular basis for the action of GluN2B-specific allosteric modulator in the context of a GluN2A subunit, we carried out structural studies in the presence or absence of the GluN2B-specific allosteric antagonist Ro 25-6981 (Ro). RESULTS The triheteromeric NMDAR adopts a bouquet-like shape assembled as a GluN1/GluN2A/GluN1/GluN2B heterotetramer, with each subunit at the canonical A/B/C/D positions, respectively. The amino-terminal domains (ATDs) and ligand-binding domains (LBDs) define a large, synaptically localized extracellular structure, and the transmembrane domains (TMDs) form the ion-conducting channel. Throughout the extracellular regions, the receptor displays a “dimer-of-dimers” arrangement, with a swapping of domains between the ATD and LBD layers. The presence of GluN2A and GluN2B subunits in the triheteromeric receptor disrupts the 2-fold symmetry in the ATD and LBD layers and the pseudo–4-fold symmetry in the TMD layer. Within the ATD layer, the GluN2A and GluN2B ATDs adopt “closed” and “open” clefts, respectively. Upon binding Ro, the GluN2B ATD clamshell transitions from an open to a closed conformation. Compared with the GluN2B subunit, the GluN2A ATD interacts more extensively with the GluN1 subunit within the ATD heterodimer and thus is poised to modify the conformational properties of its GluN1 ATD partner to a greater extent than the GluN2B. At the ATD-LBD interface, the GluN2A ATD caps the LBD layer, participating in extensive interactions with the LBD layer. By contrast, the GluN2B ATD is located farther away from the LBD layer. In the LBD layer, the GluN2A LBD interacts extensively with both GluN1 subunits, whereas the GluN2B LBD is primarily coupled to the GluN1 subunit within the LBD heterodimer. Therefore, the GluN2A subunit interacts more extensively with GluN1 subunits throughout the receptor, in comparison with the GluN2B subunit, consistent with the predominant role of the GluN2A subunit in sculpting the ion channel kinetics of the triheteromeric receptor. CONCLUSION The structural studies reveal the architecture of the triheteromeric receptor, define the molecular action of GluN2B-specific modulator Ro, and show how the GluN2A and GluN2B subunits participate in distinct interactions throughout the receptor assembly. Schematic representation of the triheteromeric NMDAR. (A) NMDARs are localized in the postsynapse. (B) Binding of glycine to the GluN1 subunits and glutamate to the GluN2 subunits promotes closure of the LBD “clamshells” and opening of the ion channel