Cytoplasmic End Of Transmembrane Helix Research Articles

Understanding the Role of Polar Residues on the c‐Ring of E. coli F1Fo ATP Synthase

F1Fo ATP synthases catalyze the synthesis of adenosine triphosphate (ATP), the universal biological energy carrier, and are thus integral to life. The membrane‐embedded Fo sector utilizes the proton electrochemical gradient to generate rotation in the rotor subunits, which drives ATP synthesis in the F1 sector. In addition to ATP synthesis, F1Fo can also hydrolyze ATP to pump protons to create an electrochemical gradient. Recent structural models of ATP synthase from cryo‐electron microscopy have revealed the architecture of the rotor‐stator interface; however, the intricacies of the rotation mechanism are not fully understood. Previous research has identified specific residues of the c subunit of E. coli F1Fo, including Arg50 and Thr51 on the cytoplasmic end of transmembrane helix 2, that may be necessary for the H+ translocation mechanism. Mutation of Arg50 or Thr51 to Cys abolished ATP‐driven H+ pumping and blocked H+‐permeability. Interestingly, these mutants were able to grow in succinate minimal medium, suggesting that ATP synthesis activity was unaffected. Arg50 is part of a proposed charge interaction between subunits a and c, and the polar Thr51 may also participate in this interaction. Therefore, we studied the importance of positive charge and polarity at positions 50 and 51 using chemical and genetic modifications. Chemically appending positive charge onto Cys50 with methanethiosulfonate did not restore ATP‐driven H+ pumping activity. However, mutation of Arg50 to Lys or His supported activity. Mutation of Thr51 to Ser or Asp also supported H+ pumping activity. These studies provide insight into the role of polar residues on the surface of the c‐ring and their contribution to the H+‐driven rotational mechanism of F1Fo ATP synthase.Support or Funding InformationSupported by the North Carolina GlaxoSmithKline Foundation and the University of North Carolina Asheville Undergraduate Research ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Residues contributing to drug transport by ABCG2 are localised to multiple drug-binding pockets.

Multidrug binding and transport by the ATP-binding cassette transporter ABCG2 is a factor in the clinical resistance to chemotherapy in leukaemia, and a contributory factor to the pharmacokinetic profiles of many other prescribed drugs. Despite its importance, the structural basis of multidrug transport, i.e. the ability to transport multiple distinct chemicals, has remained elusive. Previous research has shown that at least two residues positioned towards the cytoplasmic end of transmembrane helix 3 (TM3) of the transporter play a role in drug transport. We hypothesised that other residues, either in the longitudinal span of TM3, or a perpendicular slice through the intracellular end of other TM helices would also contribute to drug binding and transport by ABCG2. Single-point mutant isoforms of ABCG2 were made at ∼30 positions and were analysed for effects on protein expression, localisation (western blotting, confocal microscopy) and function (flow cytometry) in a mammalian stable cell line expression system. Our data were interpreted in terms of recent structural data on the ABCG protein subfamily and enabled us to propose a surface-binding site for the drug mitoxantrone (MX) as well as a second, buried site for the same drug. Further mutational analysis of residues that spatially separate these two sites prompts us to suggest a molecular and structural pathway for MX transport by ABCG2.

Structure of the adenosine A(2A) receptor bound to an engineered G protein.

G-protein-coupled receptors (GPCRs) are essential components of the signalling network throughout the body. To understand the molecular mechanism of G-protein-mediated signalling, solved structures of receptors in inactive conformations and in the active conformation coupled to a G protein are necessary. Here we present the structure of the adenosine A(2A) receptor (A(2A)R) bound to an engineered G protein, mini-Gs, at 3.4 Å resolution. Mini-Gs binds to A(2A)R through an extensive interface (1,048 Å2) that is similar, but not identical, to the interface between Gs and the β2-adrenergic receptor. The transition of the receptor from an agonist-bound active-intermediate state to an active G-protein-bound state is characterized by a 14 Å shift of the cytoplasmic end of transmembrane helix 6 (H6) away from the receptor core, slight changes in the positions of the cytoplasmic ends of H5 and H7 and rotamer changes of the amino acid side chains Arg3.50, Tyr5.58 and Tyr7.53. There are no substantial differences in the extracellular half of the receptor around the ligand binding pocket. The A(2A)R-mini-Gs structure highlights both the diversity and similarity in G-protein coupling to GPCRs and hints at the potential complexity of the molecular basis for G-protein specificity.

Control of serotonin transporter phosphorylation by conformational state

Serotonin transporter (SERT) is responsible for reuptake and recycling of 5-hydroxytryptamine (5-HT; serotonin) after its exocytotic release during neurotransmission. Mutations in human SERT are associated with psychiatric disorders and autism. Some of these mutations affect the regulation of SERT activity by cGMP-dependent phosphorylation. Here we provide direct evidence that this phosphorylation occurs at Thr276, predicted to lie near the cytoplasmic end of transmembrane helix 5 (TM5). Using membranes from HeLa cells expressing SERT and intact rat basophilic leukemia cells, we show that agents such as Na(+) and cocaine that stabilize outward-open conformations of SERT decreased phosphorylation and agents that stabilize inward-open conformations (e.g., 5-HT, ibogaine) increased phosphorylation. The opposing effects of the inhibitors cocaine and ibogaine were each reversed by an excess of the other inhibitor. Inhibition of phosphorylation by Na(+) and stimulation by ibogaine occurred at concentrations that induced outward opening and inward opening, respectively, as measured by the accessibility of cysteine residues in the extracellular and cytoplasmic permeation pathways, respectively. The results are consistent with a mechanism of SERT regulation that is activated by the transport of 5-HT, which increases the level of inward-open SERT and may lead to unwinding of the TM5 helix to allow phosphorylation.

Mutations of charged amino acids at the cytoplasmic end of transmembrane helix 2 affect transport activity of the budding yeast multidrug resistance protein Pdr5p.

Pdr5p is a major ATP-binding cassette (ABC) transporter in Saccharomyces cerevisiae. It displays a sequence and functional homologyto the pathogenic Candida albicans multidrug resistance protein Cdr1p. The transmembrane helices of Pdr5p act in substrate recognition, binding, translocation and eventual removal of toxic substances out of the plasma membrane via the formation of a binding pocket. In this study, we identify two novel Pdr5 mutants (E574K and E580K), which exhibit impaired substrate efflux functions. Both mutants remained hypersensitive to all tested Pdr5p substrates without affecting their protein expression levels, localization or ATPase activities. As E574 and E580 are both located adjacent to the predicted cytoplasmic end of transmembrane helix 2, this implies that such charged residues are functionally essential for Pdr5p. Molecular docking studies suggest the possibility that oppositely charged substitution at residue E574 may disturb the interaction between the substrates and Pdr5p, resulting in impaired transport activity. Our results present new evidence, suggesting that transmembrane helix 2 plays an important role for the efflux function of Pdr5p.

The hydrophobic amino acid cluster at the cytoplasmic end of transmembrane helix III modulates the coupling of the ß1-adrenergic receptor to Gs

A cluster of hydrophobic amino acids at the cytoplasmic end of trans-membranal helix III (TM-III) is a common feature among class-A of G protein-coupled receptors (GPCR). We mutagenized alanine 1593.53 to glutamic acid and isoleucine1603.54 to arginine (A159E/I160R) in TM-III of the human ß1-adrenergic receptor (ß1-AR) to disrupt the function of the hydrophobic cluster. Structurally, the combined mutations of A159E/I160R caused an almost 90° tilt in the rotation of Arg1563.50 in the E/DRY motif of TM-III and displaced Tyr1663.60 in intracellular loop 2. The A159E/I160R ß1-AR was uncoupled from Gs as determined by cyclic AMP/adenylyl cyclase assays and by FRET-based proximity measurements between the ß1-AR and Gsα. Isoproterenol induced ß-arrestin trafficking in cells expressing both the wild-type ß1-AR and the A159E/I160R ß1-AR. Isoproterenol markedly increased the phosphorylation of ERK1/2 in cells expressing the WT ß1-AR and this effect was dependent on the activation of the Gs-cyclic AMP-dependent protein kinase → Rap → B-raf axis. However, in cells bearing the A159E/I160R ß1-AR, isoproterenol failed to increase the phosphorylation of ERK1/2. These results indicate that mutations in the Gsα-binding pocket of the GPCR interfered with receptor coupling to Gs and with its downstream signaling cascades.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR): THREE-DIMENSIONAL STRUCTURE AND LOCALIZATION OF A CHANNEL GATE

Cystic fibrosis affects about 1 in 2500 live births and involves loss of transmembrane chloride flux due to a lack of a membrane protein channel termed the cystic fibrosis transmembrane conductance regulator (CFTR). We have studied CFTR structure by electron crystallography. The data were compared with existing structures of other ATP-binding cassette transporters. The protein was crystallized in the outward facing state and resembled the well characterized Sav1866 transporter. We identified regions in the CFTR map, not accounted for by Sav1866, which were potential locations for the regulatory region as well as the channel gate. In this analysis, we were aided by the fact that the unit cell was composed of two molecules not related by crystallographic symmetry. We also identified regions in the fitted Sav1866 model that were missing from the map, hence regions that were either disordered in CFTR or differently organized compared with Sav1866. Apart from the N and C termini, this indicated that in CFTR, the cytoplasmic end of transmembrane helix 5/11 and its associated loop could be partly disordered (or alternatively located).

Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation

Adenosine receptors and β-adrenoceptors are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins on binding the agonists adenosine or noradrenaline, respectively. GPCRs have similar structures consisting of seven transmembrane helices that contain well-conserved sequence motifs, indicating that they are probably activated by a common mechanism. Recent structures of β-adrenoceptors highlight residues in transmembrane region 5 that initially bind specifically to agonists rather than to antagonists, indicating that these residues have an important role in agonist-induced activation of receptors. Here we present two crystal structures of the thermostabilized human adenosine A(2A) receptor (A(2A)R-GL31) bound to its endogenous agonist adenosine and the synthetic agonist NECA. The structures represent an intermediate conformation between the inactive and active states, because they share all the features of GPCRs that are thought to be in a fully activated state, except that the cytoplasmic end of transmembrane helix 6 partially occludes the G-protein-binding site. The adenine substituent of the agonists binds in a similar fashion to the chemically related region of the inverse agonist ZM241385 (ref. 8). Both agonists contain a ribose group, not found in ZM241385, which extends deep into the ligand-binding pocket where it makes polar interactions with conserved residues in H7 (Ser 277(7.42) and His 278(7.43); superscripts refer to Ballesteros-Weinstein numbering) and non-polar interactions with residues in H3. In contrast, the inverse agonist ZM241385 does not interact with any of these residues and comparison with the agonist-bound structures indicates that ZM241385 sterically prevents the conformational change in H5 and therefore it acts as an inverse agonist. Comparison of the agonist-bound structures of A(2A)R with the agonist-bound structures of β-adrenoceptors indicates that the contraction of the ligand-binding pocket caused by the inward motion of helices 3, 5 and 7 may be a common feature in the activation of all GPCRs.

Two distinct conformations of helix 6 observed in antagonist-bound structures of a β 1 -adrenergic receptor

The β(1)-adrenergic receptor (β(1)AR) is a G-protein-coupled receptor whose inactive state structure was determined using a thermostabilized mutant (β(1)AR-M23). However, it was not thought to be in a fully inactivated state because there was no salt bridge between Arg139 and Glu285 linking the cytoplasmic ends of transmembrane helices 3 and 6 (the R(3.50) - D/E(6.30) "ionic lock"). Here we compare eight new structures of β(1)AR-M23, determined from crystallographically independent molecules in four different crystals with three different antagonists bound. These structures are all in the inactive R state and show clear electron density for cytoplasmic loop 3 linking transmembrane helices 5 and 6 that had not been seen previously. Despite significantly different crystal packing interactions, there are only two distinct conformations of the cytoplasmic end of helix 6, bent and straight. In the bent conformation, the Arg139-Glu285 salt bridge is present, as in the crystal structure of dark-state rhodopsin. The straight conformation, observed in previously solved structures of β-receptors, results in the ends of helices 3 and 6 being too far apart for the ionic lock to form. In the bent conformation, the R(3.50)-E(6.30) distance is significantly longer than in rhodopsin, suggesting that the interaction is also weaker, which could explain the high basal activity in β(1)AR compared to rhodopsin. Many mutations that increase the constitutive activity of G-protein-coupled receptors are found in the bent region at the cytoplasmic end of helix 6, supporting the idea that this region plays an important role in receptor activation.

Hydrophobic amino acids at the cytoplasmic ends of helices 3 and 6 of rhodopsin conjointly modulate transducin activation

Rhodopsin is the visual photoreceptor responsible for dim light vision. This receptor is located in the rod cell of the retina and is a prototypical member of the G-protein-coupled receptor superfamily. The structural details underlying the molecular recognition event in transducin activation by photoactivated rhodopsin are of key interest to unravel the molecular mechanism of signal transduction in the retina. We constructed and expressed rhodopsin mutants in the second and third cytoplasmic domains of rhodopsin – where the natural amino acids were substituted by the human M3 acetylcholine muscarinic receptor homologous residues – in order to determine their potential involvement in G-protein recognition. These mutants showed normal chromophore formation and a similar photobleaching behavior than WT rhodopsin, but decreased thermal stability in the dark state. The single mutant V138 3.53 and the multiple mutant containing V227 5.62 and a combination of mutations at the cytoplasmic end of transmembrane helix 6 caused a reduction in transducin activation upon rhodopsin photoactivation. Furthermore, combination of mutants at the second and third cytoplasmic domains revealed a cooperative role, and partially restored transducin activation. The results indicate that hydrophobic interactions by V138 3.53, V227 5.62, V250 6.33, V254 6.37 and I255 6.38 are critical for receptor activation and/or efficient rhodopsin–transducin interaction.

The Magnitude of the Light-induced Conformational Change in Different Rhodopsins Correlates with Their Ability to Activate G Proteins

Light converts rhodopsin, the prototypical G protein-coupled receptor, into a form capable of activating G proteins. Recent work has shown that the light-activated state of different rhodopsins can possess different molecular properties, especially different abilities to activate G protein. For example, bovine rhodopsin is approximately 20-fold more effective at activating G protein than parapinopsin, a non-visual rhodopsin, although these rhodopsins share relatively high sequence similarity. Here we have investigated possible structural aspects that might underlie this difference. Using a site-directed fluorescence labeling approach, we attached the fluorescent probe bimane to cysteine residues introduced in the cytoplasmic ends of transmembrane helices V and VI in both rhodopsins. The fluorescence spectra of these probes as well as their accessibility to aqueous quenching agents changed dramatically upon photoactivation in bovine rhodopsin but only moderately so in parapinopsin. We also compared the relative movement of helices V and VI upon photoactivation of both rhodopsins by introducing a bimane label and the bimane-quenching residue tryptophan into helices VI and V, respectively. Both receptors showed movement in this region upon activation, although the movement appears much greater in bovine rhodopsin than in parapinopsin. Together, these data suggest that a larger conformational change in helices V and VI of bovine rhodopsin explains why it has greater G protein activation ability than other rhodopsins. The different amplitude of the helix movement may also be responsible for functional diversity of G protein-coupled receptors.

Repulsive Separation of the Cytoplasmic Ends of Transmembrane Helices 3 and 6 Is Linked to Receptor Activation in a Novel Thyrotropin Receptor Mutant (M626I)

Ligand-dependent activation of G protein-coupled receptors (GPCRs) involves repositioning of the juxtacytoplasmic ends of transmembrane helices TM3 and TM6. This concept, inferred from site-directed spin labeling studies, is supported by chemical cross-linking of the cytoplasmic ends of TM3 and TM6 blocking GPCR activation. Here we report a novel constitutive active mutation (M626I) in TM6 of the TSH receptor (TSHR), identified in affected members of a family with nonautoimmune hyperthyroidism. The specific constitutive activity of M626I, measured by its basal cAMP generation corrected for cell surface expression, was 13-fold higher than that of wild-type TSHR. Homology modeling of the TSHR serpentine domain based on the rhodopsin crystal structure suggests that M626 faces the side chain of I515 of TM3 near the membrane-cytoplasmic junction. Steric hindrance of the introduced isoleucine by I515 is consistent with the fact that shorter or more flexible side chains at position 626 did not increase constitutivity. Furthermore, a reciprocal mutation at position 515 (I515M), when introduced into the M626I background, acts as revertant mutation by allowing accommodation of the isoleucine sidechain at position 626 and fully restoring the constitutive activity to the level of wild-type TSHR. Thus, repulsive separation of the juxtacytoplasmic TM6 and TM3 in the M626I model conclusively demonstrates a direct link between the opening of this cytoplasmic face of the receptor structure and G protein coupling.

Agonist-Induced Conformational Changes in Thyrotropin-Releasing Hormone Receptor Type I: Disulfide Cross-Linking and Molecular Modeling Approaches

The conformational changes at the cytoplasmic ends of transmembrane helices 5 and 6 (TMH5 and TMH6) of thyrotropin-releasing hormone (TRH) receptor type I (TRH-R1) during activation were analyzed by cysteine-scanning mutagenesis followed by disulfide cross-linking and molecular modeling. Sixteen double cysteine mutants were constructed by substitution of one residue at the cytoplasmic end of TMH5 and the other at that of TMH6. The cross-linking experiments indicate that four mutants, Q263C/G212C, Q263C/Y211C, T265C/G212C, and T265C/Y211C, exhibited disulfide bond formation that was sensitive to TRH occupancy. We refined our previous TRH-R1 models by embedding them into a hydrated explicit lipid bilayer. Molecular dynamics simulations of the models, as well as in silico double cysteine models, generated trajectories that were in agreement with experimental results. Our findings suggest that TRH binding induces a separation of the cytoplasmic ends of TMH5 and TMH6 and a rotation of TMH6. These changes likely increase the surface accessible area at the juxtamembrane region of intracellular loop 3 that could promote interactions between G proteins and key residues within the receptor.

Surface-exposed positions in the transmembrane helices of the lactose permease of Escherichia coli determined by intermolecular thiol cross-linking.

Intermolecular thiol cross-linking was used to determine surface-exposed positions in 250 lactose permease mutants containing single-Cys replacements in each transmembrane helix. Significant cross-linking of monomers to produce homodimers is observed in nine mutants with a 5-A-long cross-linking agent containing bis-methane thiosulfonate reactive groups [position 78 (helix III); positions 185, 186, and 187 (helix VI); positions 263, 275, and 278 (helix VIII); and positions 308 (helix IX) and 398 (helix XII)]. The results are consistent with a current helix-packing model of the permease. Seven of the nine mutants that exhibit intermolecular cross-linking are located at or near the cytoplasmic ends of transmembrane helices; two are near periplasmic ends. The results suggest that only those Cys replacements accessible from the aqueous phase and not from the hydrophobic core of the membrane are susceptible to cross-linking because of the much higher reactivity of the thiolate anion relative to the thiol. Single-Cys mutants at positions 278 (helix VIII) and 398 (helix XII), which are located in opposite sides of the 12-helix bundle, exhibit similar rates of cross-linking with sigmoid kinetics. Furthermore, cross-linking is markedly decreased at 0 degrees C, suggesting that lateral diffusion of the permease within the plane of the membrane is important for intermolecular cross-linking. The findings confirm previous observations indicating that intermolecular cross-linking is a stochastic process resulting from random collisions and support a number of other lines of evidence that lactose permease is a monomer.

Structural motifs as functional microdomains in G‐protein‐coupled receptors: Energetic considerations in the mechanism of activation of the serotonin 5‐HT2A receptor by disruption of the ionic lock of the arginine cage*

AbstractStudies of the structural changes associated with the activation of several G‐protein coupled receptors (GPCRs) have suggested that the cytoplasmic ends of transmembrane helices 6 and 3 move apart, but the molecular mechanisms involved in the helix rearrangements are not known. The cytoplasmic end of helix 3 contains the structural motif DRY that is universally conserved in the sequences of GPCRs in the rhodopsin family. Using computational modeling of the 5‐HT2A receptor, we find that an interaction of the central arginine in this motif, R3.50, with the conserved acidic side chain of the neighboring Asp (D3.49) is supplemented by a concomitant interaction with the conserved glutamate in helix 6, E6.30. Thus, R3.50 is caged in the inactive state of the receptor by its interaction with D3.49 and other conserved residues including E6.30. In the model, these interactions support the close packing of the ends of helices 3 and 6 that contributes to the stabilization of the inactive receptor state. To understand the energetic determinants for the stabilization of the inactive form of the receptor, and the mode in which activation can free the caged R3.50, we compared the electrostatic properties of the conserved residues in the conformations corresponding to the active and inactive forms of the receptor. These comparisons were analyzed with respect to a hypothesis about the role of protonation of D3.49 and E6.30 in the activation mechanism. The inferences from the computational analysis were used to design mutations to probe the mechanistic hypothesis. The experiments with mutant receptor constructs show that the removal of the acidic side‐chain at either position 3.49 or 6.30, or both, increases the level of ligand‐independent (constitutive) activity of the receptor. These results support the hypothesis regarding the role of these acidic residues in stabilizing the inactive state, because mutations that weaken the interactions should facilitate the receptor's transition to the active conformation. Hence we find a constitutively active state for receptor constructs in which E6.30 is mutated to N, Q, or L. The weakening of the helix 3–helix 6 link that results from these substitutions allows the helices to move apart in the activated state of the receptor. From the results of the computational analysis combined with related experimental probing of receptor function we thus propose a general molecular model for the role of the structural motif that include D3.49, R3.50, and E6.30, as a functional microdomain in the receptor activation mechanism. © 2002 Wiley Periodicals, Inc. Int J Quantum Chem 88: 65–75, 2002

Light-induced exposure of the cytoplasmic end of transmembrane helix seven in rhodopsin.

A key step in signal transduction in the visual cell is the light-induced conformational change of rhodopsin that triggers the binding and activation of the guanine nucleotide-binding protein. Site-directed mAbs against bovine rhodopsin were produced and used to detect and characterize these conformational changes upon light activation. Among several antibodies that bound exclusively to the light-activated state, an antibody (IgG subclass) with the highest affinity (Ka approximately 6 x 10(-9) M) was further purified and characterized. The epitope of this antibody was mapped to the amino acid sequence 304-311. This epitope extends from the central region to the cytoplasmic end of the seventh transmembrane helix and incorporates a part of a highly conserved NPXXY motif, a critical region for signaling and agonist-induced internalization of several biogenic amine and peptide receptors. In the dark state, no binding of the antibody to rhodopsin was detected. Accessibility of the epitope to the antibody correlated with formation of the metarhodopsin II photointermediate and was reduced significantly at the metarhodopsin III intermediate. Further, incubation of the antigen-antibody complex with 11-cis-retinal failed to regenerate the native rhodopsin chromophore. These results suggest significant and reversible conformational changes in close proximity to the cytoplasmic end of the seventh transmembrane helix of rhodopsin that might be important for folding and signaling.

280. - Effects of amphetamine on cognitive impairment in schizotypal personality disorder

280. - Effects of amphetamine on cognitive impairment in schizotypal personality disorder