Stephen B. Long

Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment.

Voltage-dependent K+ (Kv) channels repolarize the action potential in neurons and muscle. This type of channel is gated directly by membrane voltage through protein domains known as voltage sensors, which are molecular voltmeters that read the membrane voltage and regulate the pore. Here we describe the structure of a chimaeric voltage-dependent K+ channel, which we call the ‘paddle-chimaera channel’, in which the voltage-sensor paddle has been transferred from Kv2.1 to Kv1.2. Crystallized in complex with lipids, the complete structure at 2.4 ångström resolution reveals the pore and voltage sensors embedded in a membrane-like arrangement of lipid molecules. The detailed structure, which can be compared directly to a large body of functional data, explains charge stabilization within the membrane and suggests a mechanism for voltage-sensor movements and pore gating.

Crystal Structure of the Calcium Release–Activated Calcium Channel Orai

Architecture of a CRAC The calcium release–activated calcium (CRAC) channel generates intracellular calcium signals in response to depletion of calcium from the endoplasmic reticulum. Hou et al. (p. 1308, published online 22 November) report a high-resolution crystal structure of Orai, the CRAC channel pore from Drosophila melanogaster. Six Orai subunits surround a central pore that extends into the cytosol. The pore is in a closed conformation that is stabilized by anions binding to a basic region near the intracellular side. A ring of glutamates on the extracellular side form a selectivity filter. The channel architecture allows calcium permeation while regulating the flow to prevent overloading the cell with calcium. The unusual architecture of this ion-channel pore regulates the flow of calcium into cells. The plasma membrane protein Orai forms the pore of the calcium release–activated calcium (CRAC) channel and generates sustained cytosolic calcium signals when triggered by depletion of calcium from the endoplasmic reticulum. The crystal structure of Orai from Drosophila melanogaster, determined at 3.35 angstrom resolution, reveals that the calcium channel is composed of a hexameric assembly of Orai subunits arranged around a central ion pore. The pore traverses the membrane and extends into the cytosol. A ring of glutamate residues on its extracellular side forms the selectivity filter. A basic region near the intracellular side can bind anions that may stabilize the closed state. The architecture of the channel differs markedly from other ion channels and gives insight into the principles of selective calcium permeation and gating.

Merlin/NF2 Suppresses Tumorigenesis by Inhibiting the E3 Ubiquitin Ligase CRL4DCAF1 in the Nucleus

Current models imply that the FERM domain protein Merlin, encoded by the tumor suppressor NF2, inhibits mitogenic signaling at or near the plasma membrane. Here, we show that the closed, growth-inhibitory form of Merlin accumulates in the nucleus, binds to the E3 ubiquitin ligase CRL4(DCAF1), and suppresses its activity. Depletion of DCAF1 blocks the promitogenic effect of inactivation of Merlin. Conversely, enforced expression of a Merlin-insensitive mutant of DCAF1 counteracts the antimitogenic effect of Merlin. Re-expression of Merlin and silencing of DCAF1 implement a similar, tumor-suppressive program of gene expression. Tumor-derived mutations invariably disrupt Merlins ability to interact with or inhibit CRL4(DCAF1). Finally, depletion of DCAF1 inhibits the hyperproliferation of Schwannoma cells from NF2 patients and suppresses the oncogenic potential of Merlin-deficient tumor cell lines. We propose that Merlin suppresses tumorigenesis by translocating to the nucleus to inhibit CRL4(DCAF1).

Crystal structure of the human two-pore domain potassium channel K2P1.

Potassium Permeation Two–pore domain potassium (K2P) channels conduct K+ ions across the plasma membrane of eukaryotic cells. They help to maintain the cellular resting potential and their modulation can tune cellular excitability (see the Perspective by Poulsen and Nissen). Miller and Long (p. 432) describe a high-resolution crystal structure of the human K2P channel K2P1 (TWIK-1) and Brohawn et al. (p. 436) present a high-resolution structure of the lipid and mechanosensitive human channel TRAAK. In both structures an extracellular domain constricts the channel entrance so that K+ ions reach the selectivity filter through side portals. Openings in the transmembrane region expose the central cavity to the lipid bilayer and a helix is kinked so that its C-terminal part lies in the cytosol-membrane interface. The structural features explain K2P conductance and gating and give insight into how the channels are regulated by diverse stimuli. Structural features provide a basis for understanding gating and ion conduction of these channels. Two–pore domain potassium (K+) channels (K2P channels) control the negative resting potential of eukaryotic cells and regulate cell excitability by conducting K+ ions across the plasma membrane. Here, we present the 3.4 angstrom resolution crystal structure of a human K2P channel, K2P1 (TWIK-1). Unlike other K+ channel structures, K2P1 is dimeric. An extracellular cap domain located above the selectivity filter forms an ion pathway in which K+ ions flow through side portals. Openings within the transmembrane region expose the pore to the lipid bilayer and are filled with electron density attributable to alkyl chains. An interfacial helix appears structurally poised to affect gating. The structure lays a foundation to further investigate how K2P channels are regulated by diverse stimuli.

Reaction path of protein farnesyltransferase at atomic resolution.

Protein farnesyltransferase (FTase) catalyses the attachment of a farnesyl lipid group to numerous essential signal transduction proteins, including members of the Ras superfamily. The farnesylation of Ras oncoproteins, which are associated with 30% of human cancers, is essential for their transforming activity. FTase inhibitors are currently in clinical trials for the treatment of cancer. Here we present a complete series of structures representing the major steps along the reaction coordinate of this enzyme. From these observations can be deduced the determinants of substrate specificity and an unusual mechanism in which product release requires binding of substrate, analogous to classically processive enzymes. A structural model for the transition state consistent with previous mechanistic studies was also constructed. The processive nature of the reaction suggests the structural basis for the successive addition of two prenyl groups to Rab proteins by the homologous enzyme geranylgeranyltransferase type-II. Finally, known FTase inhibitors seem to differ in their mechanism of inhibiting the enzyme.

The crystal structure of human protein farnesyltransferase reveals the basis for inhibition by CaaX tetrapeptides and their mimetics

Protein farnesyltransferase (FTase) catalyzes the attachment of a farnesyl lipid group to the cysteine residue located in the C-terminal tetrapeptide of many essential signal transduction proteins, including members of the Ras superfamily. Farnesylation is essential both for normal functioning of these proteins, and for the transforming activity of oncogenic mutants. Consequently FTase is an important target for anti-cancer therapeutics. Several FTase inhibitors are currently undergoing clinical trials for cancer treatment. Here, we present the crystal structure of human FTase, as well as ternary complexes with the TKCVFM hexapeptide substrate, CVFM non-substrate tetrapeptide, and L-739,750 peptidomimetic with either farnesyl diphosphate (FPP), or a nonreactive analogue. These structures reveal the structural mechanism of FTase inhibition. Some CaaX tetrapeptide inhibitors are not farnesylated, and are more effective inhibitors than farnesylated CaaX tetrapeptides. CVFM and L-739,750 are not farnesylated, because these inhibitors bind in a conformation that is distinct from the TKCVFM hexapeptide substrate. This non-substrate binding mode is stabilized by an ion pair between the peptide N terminus and the α-phosphate of the FPP substrate. Conformational mapping calculations reveal the basis for the sequence specificity in the third position of the CaaX motif that determines whether a tetrapeptide is a substrate or non-substrate. The presence of β-branched amino acids in this position prevents formation of the non-substrate conformation; all other aliphatic amino acids in this position are predicted to form the non-substrate conformation, provided their N terminus is available to bind to the FPP α-phosphate. These results may facilitate further development of FTase inhibitors.

Merlin/NF2 Functions Upstream of the Nuclear E3 Ubiquitin Ligase CRL4DCAF1 to Suppress Oncogenic Gene Expression

The closed conformer of Merlin inhibits tumorigenesis by inhibiting a ubiquitin ligase implicated in promoting cell cycle progression and inhibiting growth arrest, apoptosis, and adhesion. Integrin-mediated activation of PAK (p21-activated kinase) causes phosphorylation and inactivation of the FERM (4.1, ezrin, radixin, moesin) domain–containing protein Merlin, which is encoded by the NF2 (neurofibromatosis type 2) tumor suppressor gene. Conversely, cadherin engagement inactivates PAK, thus leading to accumulation of unphosphorylated Merlin. Current models imply that Merlin inhibits cell proliferation by inhibiting mitogenic signaling at or near the plasma membrane. We have recently shown that the unphosphorylated, growth-inhibiting form of Merlin accumulates in the nucleus and binds to the E3 ubiquitin ligase CRL4DCAF1 to suppress its activity. Depletion of DCAF1 blocks the hyperproliferation caused by inactivation of Merlin. Conversely, expression of a Merlin-insensitive DCAF1 mutant counteracts the antimitogenic effect of Merlin. Expression of Merlin or silencing of DCAF1 in Nf2-deficient cells induce an overlapping, tumor-suppressive program of gene expression. Mutations present in some tumors from NF2 patients disrupt Merlin’s ability to interact with or inhibit CRL4DCAF1. Lastly, depletion of DCAF1 inhibits the hyperproliferation of Schwannoma cells isolated from NF2 patients and suppresses the oncogenic potential of Merlin-deficient tumor cell lines. Current studies are aimed at identifying the substrates and mechanism of action of CRL4DCAF1 and examining its role in NF2-dependent tumorigenesis in mouse models. We propose that Merlin mediates contact inhibition and suppresses tumorigenesis by translocating to the nucleus to inhibit CRL4DCAF1.

Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel

Significance BEST1 is a Ca2+-activated chloride channel found in a variety of cell types that allows chloride to traverse the plasma membrane. Mutations in BEST1 can cause macular degeneration. The mechanisms for anion selectivity and Ca2+-dependent activation of BEST1 are unknown. Here, we show that a hydrophobic “neck” region of the channel’s pore does not play a major role in ion selectivity but acts as an effective gate, responding to Ca2+ binding at a cytosolic sensor. Mutation of a cytosolic “aperture” dramatically affects relative permeabilities among anions. These insights help rationalize how disease-causing mutations in BEST1 affect channel behavior and contribute to a broader understanding of ion channel gating and selectivity mechanisms. Cytoplasmic calcium (Ca2+) activates the bestrophin anion channel, allowing chloride ions to flow down their electrochemical gradient. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Previously, we determined an X-ray structure of chicken BEST1 that revealed the architecture of the channel. Here, we present electrophysiological studies of purified wild-type and mutant BEST1 channels and an X-ray structure of a Ca2+-independent mutant. From these experiments, we identify regions of BEST1 responsible for Ca2+ activation and ion selectivity. A “Ca2+ clasp” within the channel’s intracellular region acts as a sensor of cytoplasmic Ca2+. Alanine substitutions within a hydrophobic “neck” of the pore, which widen it, cause the channel to be constitutively active, irrespective of Ca2+. We conclude that the primary function of the neck is as a “gate” that controls chloride permeation in a Ca2+-dependent manner. In contrast to what others have proposed, we find that the neck is not a major contributor to the channel’s ion selectivity. We find that mutation of a cytosolic “aperture” of the pore does not perturb the Ca2+ dependence of the channel or its preference for anions over cations, but its mutation dramatically alters relative permeabilities among anions. The data suggest that the aperture functions as a size-selective filter that permits the passage of small entities such as partially dehydrated chloride ions while excluding larger molecules such as amino acids. Thus, unlike ion channels that have a single “selectivity filter,” in bestrophin, distinct regions of the pore govern anion-vs.-cation selectivity and the relative permeabilities among anions.

An allosteric mechanism of inactivation in the calcium-dependent chloride channel BEST1

Bestrophin proteins are calcium (Ca2+)-activated chloride channels. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Whole-cell recordings show that ionic currents through BEST1 run down over time, but it is unclear whether this behavior is intrinsic to the channel or the result of cellular factors. Here, using planar lipid bilayer recordings of purified BEST1, we show that current rundown is an inherent property of the channel that can now be characterized as inactivation. Inactivation depends on the cytosolic concentration of Ca2+, such that higher concentrations stimulate inactivation. We identify a C-terminal inactivation peptide that is necessary for inactivation and dynamically interacts with a receptor site on the channel. Alterations of the peptide or its receptor dramatically reduce inactivation. Unlike inactivation peptides of voltage-gated channels that bind within the ion pore, the receptor for the inactivation peptide is on the cytosolic surface of the channel and separated from the pore. Biochemical, structural, and electrophysiological analyses indicate that binding of the peptide to its receptor promotes inactivation, whereas dissociation prevents it. Using additional mutational studies we find that the “neck” constriction of the pore, which we have previously shown to act as the Ca2+-dependent activation gate, also functions as the inactivation gate. Our results indicate that unlike a ball-and-chain inactivation mechanism involving physical occlusion of the pore, inactivation in BEST1 occurs through an allosteric mechanism wherein binding of a peptide to a surface-exposed receptor controls a structurally distant gate.

Molecular mechanisms of gating in the calcium-activated chloride channel bestrophin

Bestrophin (BEST1–4 in humans) channels are ligand gated chloride (Cl−) channels that are activated by calcium (Ca2+). Mutations in BEST1 cause retinal degenerative diseases. Partly because these channels have no sequence or structural similarity to other ion channels, the molecular mechanisms underlying gating are unknown. Here, we present a series of cryo-electron microscopy (cryo-EM) structures of chicken BEST1, determined at 3.1 Å resolution or better, that represent the principal gating states of the channel. Unlike other channels, opening of the pore is due to the repositioning of tethered pore-lining helices within a surrounding protein shell that dramatically widens a “neck” of the pore through a concertina of amino acid rearrangements within the protein core. The neck serves as both the activation and the inactivation gate. The binding of Ca2+ to a cytosolic domain instigates pore opening and the structures reveal that, unlike voltage-gated Na+ and K+ channels, similar molecular rearrangements are responsible for inactivation and deactivation. A single aperture within the 95 Å-long opened pore separates the cytosol from the extracellular milieu and controls anion permeability. The studies define the basis for Ca2+-activated Cl− channel function and reveal a new molecular paradigm for gating in ligand-gated ion channels.