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PubMed2003

Structure and activation of muscarinic acetylcholine receptors.

A homology model of the M(1) muscarinic acetylcholine receptor, based on the X-ray structure of bovine rhodopsin, has been used to interpret the results of scanning and point mutagenesis studies on the receptor's transmembrane (TM) domain. Potential intramolecular interactions that are important for the stability of the protein fold have been identified. The residues contributing to the binding site for the antagonist, N -methyl scopolamine, and the agonist, acetylcholine, have been mapped. The positively charged headgroups of these ligands probably bind in a charge-stabilized aromatic cage formed by amino acid side chains in TM helices TM3, TM6 and TM7, while residues in TM4 may participate as part of a peripheral docking site. Closure of the cage around the headgroup of acetylcholine may be part of the mechanism for transducing binding energy into receptor activation, probably by disrupting a set of Van der Waals interactions between residues lying beneath the binding site that help to constrain the receptor to the inactive state, in the absence of agonist. This may trigger the reorganization of a hydrogen-bonding network between highly conserved residues in the core of the receptor, whose integrity is crucial for achievement of the activated state.

Acetylcholine↗
PubMed1997

A variant of the EPB3 gene of the anti-Lepore type in hereditary spherocytosis.

The EPB3 gene encodes band 3 (anion exchanger 1) of the red cell membrane. A subset of hereditary spherocytosis (HS) is associated with EPB3 gene mutations and band 3 deficiency. We report a large Italian family in which 10 of the 27 members investigated displayed an autosomal dominant HS. SDS-PAGE revealed a reduction in band 3 in the patients. Screening of the Pst I polymorphic site confirmed the linkage of HS with the EPB3 gene. Analysis of complementary and genomic DNA showed a large additional segment. Nucleotide sequencing disclosed an in-frame duplication of 69 nucleotides (nt) including a triplet of intronic origin and a genuine exonic duplication of 66 nt. Two CCTGC sequences occurred close to one another, one near the intron 12 acceptor splice site (nt -7 to -3), and the other within exon 13 (nt 1494-1498). We assumed that the abnormal allele arose from an unequal recombination event of the anti-Lepore type between the two CCTGC sequences. At the level of the mutated protein, termed band 3 Milano, the additional segment (Gln plus duplication of residues 478-499) corresponded to the last part of the third transmembrane domain (TM3), the entire second outer loop and part of TM4 as it is currently defined in hydropathy analysis. After deglycosylation of band 3, only the normal band was detected, supporting the view that band 3 Milano is probably not incorporated into the membrane.

Adolescent↗
PubMed1997

Key residues defining the mu-opioid receptor binding pocket: a site-directed mutagenesis study.

Structural elements of the rat mu-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the mu, delta, and kappa receptors (Asn150 to Ala, His297 to Ala, and Tyr326 to Phe) and two designed to test for mu/delta selectivity (Ile196 to Val and Val202 to Ile). Mutation of His297 in transmembrane domain 6 (TM6) resulted in no detectable binding with [3H]DAMGO (3H-labeled D-Ala2, N-Me-Phe4, Gly-ol5-enkephalin), [3H]bremazocine, or [3H]ethylketocyclazocine. Mutation of Asn150 in TM3 produces a three- to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, beta-endorphin1-31, JOM-13, deltorphin II, dynorphin1-13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor-binaltorphamine. In contrast, the Tyr326 mutation in TM7 resulted in a decreased affinity for a wide spectrum of mu, delta, and kappa agonists and antagonists. Altering Val202 to Ile in TM4 produced no change on ligand affinity, but Ile196 to Val resulted in a four- to fivefold decreased affinity for the mu agonists morphine and DAMGO, with no change in the binding affinities of kappa and delta ligands.

Animals↗
PubMed2002

Hydroxylated residues influence desensitization behaviour of recombinant alpha3 glycine receptor channels.

The human glycine receptor subunit alpha3 exists in two splice variants (alpha3K/L), with alpha3L bearing an additional segment of 15 amino acids within the cytoplasmic TM3-4 loop. Homomeric alpha3K glycine receptors show faster desensitization than alpha3L receptors. Ion channel properties were compared of alpha3L, alpha3K, and of the triple mutant alpha3LDeltaOH = alpha3L(T358A/Y367F/S370A), where hydroxyl functions of the spliced insert had been removed by site-directed mutagenesis. Upon recombinant expression in HEK 293 cells, patch-clamp recording experiments revealed that removal of hydroxyl functions primarily affected receptor desensitization. The fraction of non-desensitizing current was 68 +/- 13% for alpha3L, 21 +/- 13% for alpha3K, and 48 +/- 16% for alpha3LDeltaOH. Desensitization time constants at saturating glycine concentration were 8.4 +/- 2.8 s, 1.9 +/- 2.3 s, and 2.8 +/- 0.4 s, for alpha3L, alpha3K, and the triple mutant alpha3LDeltaOH, respectively. In contrast, single-channel and whole-cell properties were similar for all three constructs. Thus, ion channel activation, desensitization, and conductance properties are independently controlled by distinct structural elements. Hydroxyl functions within the M3-4 loop of the glycine receptor alpha3 subunit are crucial, but not exclusive, determinants of receptor desensitization.

Alternative Splicing↗
PubMed2003

A new Chrna4 mutation with low penetrance in nocturnal frontal lobe epilepsy.

PURPOSE: To identify and characterize the mutation(s) causing nocturnal frontal lobe epilepsy in a German extended family. METHODS: Neuronal nicotinic acetylcholine receptor (nAChR) subunit genes were screened by direct sequencing. Once a CHRNA4 mutation was identified, its biophysical and pharmacologic properties were characterized by expression experiments in Xenopus oocytes. RESULTS: We report a new CHRNA4 mutation, causing a alpha4-T265I amino acid exchange at the extracellular end of the second transmembrane domain (TM). Functional studies of alpha4-T265I revealed an increased ACh sensitivity of the mutated receptors. alpha4-T265I is associated with an unusual low penetrance of the epilepsy phenotype. Sequencing of the TM1-TM3 parts of the 1 known nAChR subunits did not support a two-locus model involving a second nAChR sequence variation. CONCLUSIONS: nAChR mutations found in familial epilepsy are not always associated with an autosomal dominant mode of inheritance. alpha4-T265I is the first nAChR allele showing a markedly reduced penetrance consistent with a major gene effect. The low penetrance of the mutation is probably caused by unknown genetic or environmental factors or both.

Adolescent↗
PubMed1994

Identification of a mammary transforming gene (MAT1) associated with mouse mammary carcinogenesis.

We have developed an efficient in vitro transformation system using N-methyl-N-nitrosourea that allows us to study the role of hormones and growth factors in mouse mammary tumorigenesis. Utilizing this system, we reported earlier that mammary tumors induced in vitro with N-methyl-N-nitrosourea in the presence of mammogenic hormones (progesterone and prolactin) contain predominately an activated c-Ki-ras protooncogene with a G35 --> A35 transitional mutation in the 12th codon. Mammary tumors induced in the presence of another mitogen, lithium (Li), do not have a mutation in the c-Ki-ras protooncogene. By using an expression cloning system, a plasmid clone containing a 1.75-kb cDNA insert has been isolated from this group of tumors. Nucleic acid sequence analysis of the insert reveals that it has a short open reading frame of 61 amino acids and that it does not have sequence homology with any known gene. The gene, designated MAT1, can neoplastically transform NIH 3T3 cells and also the mammary epithelial cell line TM3. Expression of this gene occurs in normal mouse tissues including mammary gland and is overexpressed in the original mammary tumors as indicated by Northern blot analysis. In vitro transcription and translation of the clone shows a protein product of 6000 Da, which agrees with the predicted open reading frame.

3T3 Cells↗
PubMed1999

Five-transmembrane domains appear sufficient for a G protein-coupled receptor: functional five-transmembrane domain chemokine receptors.

The putative seven-transmembrane (TM) domains have been the structural hallmark for the superfamily of heterotrimeric G protein-coupled receptors (GPCRs) that regulate a variety of cellular functions by mediating a large number of extracellular signals. Five-TM GPCR mutants of chemokine receptor CCR5 and CXCR4, the N-terminal segment of which connected directly to TM3 as a result of a deletion of TM1-2 and the first intracellular and extracellular loops, have been obtained in this study. Laser confocal microscopy and flow cytometry analysis revealed that these five-TM mutant GPCRs were expressed stably on the cell surface after transfection into human embryonic kidney 293 cells. The five-TM CCR5 and CXCR4 functioned as normal chemokine receptors in mediating chemokine-stimulated chemotaxis, Ca2+ influx, and activation of pertussis toxin-sensitive G proteins. Like the wild-type GPCRs, the five-TM mutant receptors also underwent agonist-dependent internalization and desensitization and were subjected to regulation by GPCR kinases and arrestins. Our study indicates that five-TM domains, at least in the case of CCR5 and CXCR4, appear to meet the minimum structural requirements for a functional GPCR and suggests possible existence of functional five-TM GPCRs in nature during evolution.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Membrane topology of a cysteine-less mutant of human P-glycoprotein.

A human P-glycoprotein devoid of cysteine residues was constructed by site-directed mutagenesis for studying its topology. The cDNA for human P-glycoprotein-A52 in which codons for cysteines 137, 431, 717, 956, 1074, 1125, 1227, 1288, and 1304 were changed to Ala, was transfected into NIH 3T3 cells and analyzed with respect to its ability to confer resistance to various drugs. The cysteine-less P-glycoprotein-A52 retained the ability to confer resistance to vinblastine, colchicine, doxorubicin, and actinomycin D with only a small decrease in efficiency relative to wild-type enzyme. Cysteine residues were then reintroduced into predicted extracellular or cytoplasmic loops of the cysteine-less P-glycoprotein-A52, and the topology of the protein was determined using membrane-permeant and impermeant thiol-specific reagents. It was found that 8 of 15 cysteine residues introduced into P-glycoprotein-A52 could be biotinylated, when cells expressing the mutant P-glycoprotein were incubated with membrane-permeant biotin maleimide. Biotinylation of a cysteine residue placed in predicted extracellular loops between transmembrane segment (TM) 5 and TM6, TM7 and TM8, or TM11 and TM12 was blocked by pretreatment of the cells with a membrane-impermeant maleimide, suggesting that these residues have an extracellular location. By contrast, biotinylation of cysteine residues located in the predicted cytoplasmic loops between TM2 and TM3, TM4 and TM5, TM8 and TM9, or TM10 and TM11 were not blocked by pretreatment with membrane impermeant maleimide, suggesting that these residues were in the cytoplasm. These results are consistent with the model of P-glycoprotein, which predicts six transmembrane segments in each of the two homologous halves of the molecule.

3T3 Cells↗
PubMed1996

Transmembrane organization of mouse P-glycoprotein determined by epitope insertion and immunofluorescence.

P-glycoprotein (P-gp) is an integral membrane protein that causes multidrug resistance when overexpressed in tumor cells. Efforts to identify the position and polarity of its 12 putative transmembrane (TM) domains have so far failed to yield a consistent topological model. Recently, we have described a method for topology mapping based on the insertion of a small antigenic peptide epitope (YPYDVPDYA) in predicted intra- or extracellular loops of the protein. The tagged proteins are then functionally expressed in Chinese hamster ovary cells, and the polarity of the inserted tag with respect to plasma membrane is deduced by immunofluorescence in intact or permeabilized cells. We previously localized segments between TM1 and TM2, and TM5 and TM6 as extracellular and segments between TM2 and TM3 and downstream of TM6 as intracellular (Kast, C., Canfield, V., Levenson, R., and Gros, P. (1995) Biochemistry 34, 4402-4411). We have now inserted single epitope tags at positions 207, 235, 276, 741, 782, 797, 815, 849, 887, 961, and 1024; double epitope tags at positions 736, 849, and 961; and a triple epitope tag at position 849. Insertions of epitopes at positions 235, 736, 741, 849, 887, 961, and 1024 resulted in functional proteins, whereas insertions at positions 207, 276, 782, 797, and 815 abrogated the capacity of P-gp to confer multidrug resistance. The epitope tags inserted at positions 736, 849, and 961 were localized extracellularly, whereas tags at positions 235, 887, and 1024 mapped intracellularly. These results indicate that the intervening segments separated by TM4-TM5, TM10-TM11, and downstream of TM12 are cytoplasmic; segments delineated by TM7-TM8, TM9-TM10, and TM11-TM12 are extracellular. Our combined analysis of the amino- and carboxyl-terminal halves of P-gp supports a 12-TM domain topology with intracellular amino and carboxyl termini and ATP binding sites and an extracellular glycosylated loop (TM1-TM2) in agreement with hydropathy prediction. These results are clearly distinct from those obtained by the analysis of truncated P-gps in vitro and in heterologous expression systems.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Molecular basis for the interaction of [Nle4,D-Phe7]melanocyte stimulating hormone with the human melanocortin-1 receptor.

The melanocortin-1 receptor (MC1R) is a seven-transmembrane (TM) G-protein-coupled receptor whose natural ligands are the melanocortin peptides, adrenocorticotropic hormone, and alpha-, beta-, and gamma- melanocyte stimulating hormone (MSH). To test a previously constructed three-dimensional model of the molecular interaction between the long-acting, superpotent alpha-MSH analog [Nle4,D-Phe7]MSH (NDP-MSH) and the human MC1R we examined the effects of site-directed receptor mutagenesis on the binding affinity and potency of NDP-MSH. In addition, we also examined the effects of these same mutations on the binding affinity and potency of the structurally related agonists alpha-MSH, gamma-MSH, and Ac-Nle4-cyclic-[Asp5,His6,D-Phe7,Arg8,Trp9,Lys10]NH2 (MT-II). Mutagenesis of acidic receptor residues Glu94 in TM2 and Asp117 or Asp121 in TM3 significantly altered the binding affinity and potency of all four agonists suggesting that these receptor residues are important to the ligand-receptor interactions of all. A disproportionate change in agonist potency versus affinity observed with simultaneous mutation of these acidic residues (mutant constructs D117A/D121A or E94A/D117A/D121A) or introduction of a single positive charge (mutant construct D121K) also implicates these residues in receptor activation. In addition, results from the individual mutation of aromatic receptor residues Phe175, Phe196, and Phe257, and simultaneous mutation of multiple TM4, -5, and -6 tyrosine and phenylalanine residues suggests that aromatic-aromatic ligand-receptor interactions also participate in binding these melanocortins to the MC1R. These experiments appear to have identified some of the critical receptor residues involved in the ligand-receptor interactions between these melanocortins and the hMC1R.

Binding Sites↗
PubMed1998

The human glycine receptor subunit alpha3. Glra3 gene structure, chromosomal localization, and functional characterization of alternative transcripts.

The neuronal glycine receptor is a ligand-gated chloride channel composed of ligand binding alpha and structural beta polypeptides. Homology screening of a human fetal brain cDNA library resulted in the identification of two alternative splice variants of the glycine receptor alpha3 subunit. The amino acid sequence predicted for the alpha3L variant was largely identical to the corresponding rat subunit. In contrast, the novel splice variant alpha3K lacked the coding sequence for 15 amino acids located within the cytoplasmic loop connecting transmembrane spanning region 3 (TM3) and TM4. Using P1 artificial chromosome (PAC) clones, the structure of the GLRA3 gene was elucidated and its locus assigned to human chromosomal bands 4q33-q34 by fluorescence in situ hybridization. Two transcripts of 2.4 and 9 kilobases, corresponding to alpha3L and alpha3K, respectively, were identified and found to be widely distributed throughout the human central nervous system. Structural analysis of the GLRA3 gene revealed that the alpha3K transcript resulted from a complex splice event where excision of the novel exon 8A comprising the alternative sequence of 45 base pairs coincides with the persistence of a large intronic sequence in the 3'-untranslated region. Functional expression in HEK 293 cells of alpha3L and alpha3K subunits resulted in the formation of glycine-gated chloride channels that differed significantly in desensitization behavior, thus defining the cytoplasmic loop as an important determinant of channel inactivation kinetics.

Alternative Splicing↗
PubMed2001

Determinants of the trans-dominant negative effect of truncated forms of the CCR5 chemokine receptor.

The human immunodeficiency virus, type 1 (HIV-1) entry process is triggered by interaction between the viral envelope and a seven membrane-spanning domain receptor at the cell surface, usually the CCR5 chemokine receptor. Different naturally occurring mutations in the CCR5 gene abolish receptor function, the most frequent being a 32-nucleotide deletion resulting in a truncated protein (Delta32) lacking the last three transmembrane domains (TM5-7). This mutant is retained in the endoplasmic reticulum and exerts a trans-dominant negative (TDN) effect on the wild type, preventing its exit from this compartment. This TDN effect is often considered as evidence for the oligomerization of CCR5 during transport to the cell surface. Here we use a genetic approach to define the structural determinants of the TDN effect of the Delta32 mutant. It was abolished by certain deletions and by mutations of cysteine residues preventing formation of a disulfide link between the first and second extracellular loops, suggesting that conformation of Delta32 is important for its interaction with CCR5. To circumvent this problem, we used chimeric forms of the Delta32 and wild type CCR5, consisting in substitutions with homologous domains from the mouse CCR5. All chimeric full-length receptors were expressed at the cell surface and were functional for interaction with HIV-1 or with a chemokine ligand, when assayed. The TDN effect was only observed if both the TM3 domain in CCR5 and the TM4 domain in Delta32 were from human origin, whereas the rest of the proteins could be from either origin. This suggests that the TDN effect involves some form of interaction between these transmembrane domains. Alternatively, but less likely to us, substitutions in TM4 could affect the conformation of CCR5 in the endoplasmic reticulum but not at the cell surface. However that may be, it seems that the TDN effect of the Delta32 mutant has no bearing to the issue of CCR5 dimerization and to its possible role in the processing of the receptor to the cell surface.

Amino Acid Sequence↗
PubMed2002

Beta2 adrenergic receptor activation. Modulation of the proline kink in transmembrane 6 by a rotamer toggle switch.

In many rhodopsin-like G-protein-coupled receptors, agonist binding to a cluster of aromatic residues in TM6 may promote receptor activation by altering the configuration of the TM6 Pro-kink and by the subsequent movement of the cytoplasmic end of TM6 away from TM3. We hypothesized that the highly conserved Cys(6.47), in the vicinity of the conserved Pro(6.50), modulates the configuration of the aromatic cluster and the TM6 Pro-kink through specific interactions in its different rotamer configurations. In the beta(2) adrenergic receptor, mutation of Cys(6.47) to Thr, which in an alpha-helix has a different rotamer distribution from Cys and Ser, produced a constitutively active receptor, whereas the Ser mutant was similar to wild-type receptor. Use of the biased Monte Carlo technique of Conformational Memories showed that the rotamer changes among Cys/Ser/Thr(6.47), Trp(6.48), and Phe(6.52) are highly correlated, representing a rotamer "toggle switch" that may modulate the TM6 Pro-kink. Differential modulation of the accessibility of Cys(6.47) and an engineered Cys(6.52) in wild type and a constitutively active background provides experimental support for the association of this rotamer switch with receptor activation.

Adrenergic beta-Antagonists↗
PubMed2003

Residues in internal repeats of the rice cation/H+ exchanger are involved in the transport and selection of cations.

In plants, the cation/H+ exchanger (CAX) translocates Ca2+ and other metal ions into vacuoles using the H+ gradient formed by H+-ATPase and H+-pyrophosphatase. Such exchangers carrying 11 transmembrane domains (TMs) have been isolated from plants, yeast, and bacteria. In this study, multiple sequence alignment of several CAXs revealed the presence of highly conserved 36-residue regions between TM3 and TM4 and between TM8 and TM9. These two repetitive motifs are designated repeats c-1 and c-2. Using site-directed mutagenesis, we generated 31 mutations in the repeats of the Oryza sativa CAX, which translocates Ca2+ and Mn2+. Mutant exchangers were expressed in a Saccharomyces cerevisiae strain that is sensitive to Ca2+ and Mn2+ because of the absence of vacuolar Ca2+-ATPase and the Ca2+/H+ exchanger. Mutant exchangers were classified into six classes according to their tolerance for Ca2+ and Mn2+. For example, the class III mutants had no tolerance for either ion, and the class IV mutants had tolerance only for Ca2+. The biochemical function of each residue was estimated. We investigated the membrane topology of the repeats using a method combining cysteine mutagenesis and sulfhydryl reagents. Our results suggest that repeat c-1 re-enters the membrane from the vacuolar luminal side and forms a solution-accessible region. Furthermore, several residues in repeats c-1 and c-2 were found to be conserved in animal Na+/Ca2+ exchangers. Finally, we suggest that these re-entrant repeats may form a vestibule or filter for cation selection.

Amino Acid Sequence↗
PubMed2004

Subunits of a yeast oligomeric G protein-coupled receptor are activated independently by agonist but function in concert to activate G protein heterotrimers.

G protein-coupled receptors (GPCRs) form dimeric or oligomeric complexes in vivo. However, the function of oligomerization in receptor-mediated G protein activation is unclear. Previous studies of the yeast alpha-factor receptor (STE2 gene product) have indicated that oligomerization promotes signaling. Here we have addressed the mechanism by which oligomerization facilitates G protein signaling by examining the ability of ligand binding- and G protein coupling-defective alpha-factor receptors to form complexes in vivo and to correct their signaling defects when co-expressed (trans complementation). Newly and previously identified receptor mutants indicated that ligand binding involves the exofacial end of transmembrane domain (TM) 4, whereas G protein coupling involves ic1, ic3, the C-terminal tail, and the intracellular ends of TM2 and TM3. Mutant receptors bearing substitutions in these domains formed homo-oligomeric or hetero-oligomeric complexes in vivo, as indicated by results of fluorescence resonance energy transfer experiments. Co-expression of ligand binding- and G protein coupling-defective mutant receptors did not significantly improve signaling. In contrast, co-expression of ic1 and ic3 mutations in trans but not in cis significantly increased signaling efficiency. Therefore, we suggest that subunits of the alpha-factor receptor: 1) are activated independently rather than cooperatively by agonist, and 2) function in a concerted fashion to promote G protein activation, possibly by contacting different subunits or regions of the G protein heterotrimer.

Cell Division↗
PubMed2004

Analysis of transmembrane domain 2 of rat serotonin transporter by cysteine scanning mutagenesis.

The second transmembrane domain (TM2) of neurotransmitter transporters has been invoked to control oligomerization and surface expression. This transmembrane domain lies between TM1 and TM3, which have both been proposed to contain residues that contribute to the substrate binding site. Rat serotonin transporter (SERT) TM2 was investigated by cysteine scanning mutagenesis. Six mutants in which cysteine replaced an endogenous TM2 residue had low transport activity, and two were inactive. Most of the reduction in transport activity was due to decreased surface expression. In contrast, M124C and G128C showed increased activity and surface expression. Random mutagenesis at positions 124 and 128 revealed that hydrophobic residues at these positions also increased activity. When modeled as an alpha-helix, positions where mutation to cysteine strongly affects expression levels clustered on the face of TM2 surrounding the leucine heptad repeat conserved within this transporter family. 2-(Aminoethyl)-methanethiosulfonate hydrobromide (MTSEA)-biotin labeled A116C and Y136C but not F117C, M135C, or Y134C, suggesting that these residues may delimit the transmembrane domain. None of the cysteine substitution mutants from 117 through 135 were sensitive to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) or MTSEA. However, treatment with MTSEA increased 5-hydroxytryptamine transport by A116C. Activation of A116C by MTSEA was observed only in mutants containing Cys to Ile mutation at position 357, suggesting that modification of Cys-116 activated transport by compensating for a disruption in transport in response to Cys-357 replacement. The reactivity of A116C toward MTSEA was substantially increased in the presence of substrates but not inhibitors. This increase required Na+ and Cl-, and was likely to result from conformational changes during the transport process.

Animals↗
PubMed2003

Mechanism of proton gating of a urea channel.

The size and complexity of many pH-gated channels have frustrated the development of specific structural models. The small acid-activated six-membrane segment urea channel of Helicobacter hepaticus (HhUreI), homologous to the essential UreI of the gastric pathogen Helicobacter pylori, enables identification of all the periplasmic sites of proton gating by site-directed mutagenesis. Exposure to external acidity enhances [(14)C]urea uptake by Xenopus oocytes expressing HhUreI, with half-maximal activity (pH(0.5)) at pH 6.8. A downward shift of pH(0.5) in single site mutants identified four of six protonatable periplasmic residues (His-50 at the boundary of the second transmembrane segment TM2, Glu-56 in the first periplasmic loop, Asp-59 at the boundary of TM3, and His-170 at the boundary of TM6) that affect proton gating. Asp-59 was the only site at which a protonatable residue appeared to be essential for pH gating. Mutation of Glu-110 or Glu-114 in PL2 did not affect the pH(0.5) of gating. A chimera, where the entire periplasmic domain of HhUreI was fused to the membrane domain of Streptococcus salivarius UreI (SsUreI), retained the pH-independent properties of SsUreI. Hence, proton gating of HhUreI likely depends upon the formation of hydrogen bonds by periplasmic residues that in turn produce conformational changes of the transmembrane domain. Further studies on HhUreI may facilitate understanding of other physiologically important pH-responsive channels.

Amino Acid Sequence↗
PubMed2004

Evidence of an intramolecular interaction between the two domains of the BlaR1 penicillin receptor during the signal transduction.

The BlaR1 protein is a penicillin-sensory transducer involved in the induction of the Bacillus licheniformis beta-lactamase. The amino-terminal domain of the protein exhibits four transmembrane segments (TM1-TM4) that form a four-alpha-helix bundle embedded in the plasma bilayer. The carboxyl-terminal domain of 250 amino acids (BlaR-CTD) fused at the carboxyl end of TM4 possesses the amino acid sequence signature of penicillin-binding proteins. This membrane topology suggests that BlaR-CTD and the BlaR-amino-terminal domain are responsible for signal reception and signal transduction, respectively. With the use of phage display experiments, we highlight herein an interaction between BlaR-CTD and the extracellular, 63-amino acid L2 loop connecting TM2 and TM3. This interaction does not occur in the presence of penicillin. This result suggests that binding of the antibiotic to BlaR1 might entail the release of the interaction between L2 and BlaR-CTD, causing a motion of the alpha-helix bundle and transfer of the information to the cytoplasm of the cell. In addition, fluorescence spectroscopy, CD, and Fourier transform IR spectroscopy experiments indicate that in contrast to the behavior of the corresponding Staphylococcus aureus protein, the beta-lactam antibiotic does not induce a drastic conformational change in B. licheniformis BlaR-CTD.

Acylation↗