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ABSTRACT: Coronavirus envelope (E) proteins are short (~100 residues) polypeptides that contain at least one transmembrane (TM) domain and a cluster of 2-3 juxtamembrane cysteines. These proteins are involved in viral morphogenesis and tropism, and their absence leads in some cases to aberrant virions, or to viral attenuation. In common to other viroporins, coronavirus envelope proteins increase membrane permeability to ions. Although an NMR-based model for the TM domain of the E protein in the severe acute respiratory syndrome virus (SARS-CoV E) has been reported, structural data and biophysical studies of full length E proteins are not available because efficient expression and purification methods for these proteins are lacking. Herein we have used a novel fusion protein consisting of a modified β-barrel to purify both wild type and cysteine-less mutants of two representatives of coronavirus E proteins: the shortest (76 residues), from SARS-CoV E, and one of the longest (109 residues), from the infectious bronchitis virus (IBV E). The fusion construct was subsequently cleaved with cyanogen bromide and all polypeptides were obtained with high purity. This is an approach that can be used in other difficult hydrophobic peptides.
Protein Expression and Purification 07/2012; 85(1):133-41. · 1.59 Impact Factor
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Siok-Wan Gan,
Edward Tan,
Xin Lin,
Dejie Yu,
Juejin Wang,
Gregory Ming-Yeong Tan, Ardcharaporn Vararattanavech,
Chiew Ying Yeo,
Cin Huang Soon,
Tuck Wah Soong,
Konstantin Pervushin,
Jaume Torres
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ABSTRACT: The small hydrophobic (SH) protein is encoded by the human respiratory syncytial virus. Its absence leads to viral attenuation in the context of whole organisms, and it prevents apoptosis in infected cells. Herein, we have examined the structure of SH protein in detergent micelles and in lipid bilayers, by solution NMR and attenuated total reflection-Fourier transform infrared spectroscopy, respectively. We found that SH protein has a single α-helical transmembrane domain and forms homopentamers in several detergents. In detergent micelles, the transmembrane domain is flanked N-terminally by an α-helix that forms a ring around the lumen of the pore and C-terminally by an extended β-turn. SH protein was found in the plasma membrane of transiently expressing HEK 293 cells, which showed pH-dependent (acid-activated) channel activity. Channel activity was abolished in mutants lacking both native His residues, His(22) and His(51), but not when either His was present. Herein, we propose that the pentameric model of SH protein presented is a physiologically relevant conformation, albeit probably not the only one, in which SH contributes to RSV infection and replication. Viroporins are short (∼100 amino acids) viral membrane proteins that form oligomers of a defined size, act as proton or ion channels, and in general enhance membrane permeability in the host. However, with some exceptions, their precise biological role of their channel activity is not understood. In general, viroporins resemble poorly specialized proteins but are nevertheless critical for viral fitness. In vivo, viruses lacking viroporins usually exhibit an attenuated or weakened phenotype, altered tropism, and diminished pathological effects. We have chosen to study the SH protein, 64 amino acids long, found in the human respiratory syncytial virus because of the effect of RSV on human health and the lack of adequate antivirals. We show that SH protein forms oligomers that behave as ion channels when activated at low pH. This study adds SH protein to a growing group of viroporins that have been structurally characterized. Although the precise biological role of this pentameric channel is still unknown, this report is nevertheless essential to fill some of the many gaps that exist in the understanding of SH protein function.
Journal of Biological Chemistry 05/2012; 287(29):24671-89. · 4.77 Impact Factor
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ABSTRACT: This paper describes a method to create giant protein vesicles (GPVs) of ≥10 μm by solvent-driven fusion of large vesicles (0.1-0.2 μm) with reconstituted membrane proteins. We found that formation of GPVs proceeded from rotational mixing of protein-reconstituted large unilamellar vesicles (LUVs) with a lipid-containing solvent phase. We made GPVs by using n-decane and squalene as solvents, and applied generalized polarization (GP) imaging to monitor the polarity around the protein transmembrane region of aquaporins labeled with the polarity-sensitive probe Badan. Specifically, we created GPVs of spinach SoPIP2;1 and E. coli AqpZ aquaporins. Our findings show that hydrophobic interactions within the bilayer of formed GPVs are influenced not only by the solvent partitioning propensity, but also by lipid composition and membrane protein isoform.
ChemBioChem 11/2011; 12(18):2856-62. · 3.94 Impact Factor
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ABSTRACT: This study describes the interaction between sodium dodecyl sulfate (SDS) and membrane proteins reconstituted into large unilamellar lipid vesicles and detergent micelles studied by circular dichroism (CD) and polarity sensitive probe labeling. Specifically, we carried out a comparative study of two aquaporins with high structural homology SoPIP2;1 and AqpZ using identical reconstitution conditions. Our CD results indicate that SDS, when added to membrane-reconstituted aquaporins in concentrations below the SDS critical micelle concentration (CMC, ~8mM), causes helical rearrangements of both aquaporins. However, we do not find compelling evidence for unfolding. In contrast when SDS is added to detergent stabilized aquaporins, SoPIP2;1 partly unfolds, while AqpZ secondary structure is unaffected. Using a fluorescent polarity sensitive probe (Badan) we show that SDS action on membrane reconstituted SoPIP2;1 as well as AqpZ is associated with initial increased hydrophobic interactions in protein transmembrane (TM) spanning regions up to a concentration of 0.1× CMC. At higher SDS concentrations TM hydrophobic interactions, as reported by Badan, decrease and reach a plateau from SDS CMC up to 12.5× CMC. Combined, our results show that SDS does not unfold neither SoPIP2;1 nor AqpZ during transition from a membrane reconstituted form to a detergent stabilized state albeit the native folds are changed.
Biochimica et Biophysica Acta 06/2011; 1808(10):2600-7. · 4.66 Impact Factor
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ABSTRACT: The use of blue native polyacrylamide gel electrophoresis (BN-PAGE) has been reported in the literature to retain both water-soluble and membrane protein complexes in their native hetero-oligomeric state and to determine the molecular weight of membrane proteins. However, membrane proteins show abnormal mobility when compared with water-soluble markers. Although one could use membrane proteins as markers or apply a conversion factor to the observed molecular weight to account for the bound Coomassie blue dye, when one just wants to assess homo-oligomeric size, these methods appear to be too time-consuming or might not be generally applicable. Here, during detergent screening studies to identify the best detergent for achieving a monodisperse sample, we observed that under certain conditions membrane proteins tend to form ladders of increasing oligomeric size. Although the ladders themselves contain no indication of which band represents the correct oligomeric size, they provide a scale that can be compared with a single band, representing the native homo-oligomeric size, obtained in other conditions of the screen. We show that this approach works for three membrane proteins: CorA (42 kDa), aquaporin Z (25 kDa), and small hydrophobic (SH) protein from respiratory syncytial virus (8 kDa). In addition, polydispersity results and identification of the most suitable detergent correlate optimally not only with size exclusion chromatography (SEC) but also with results from sedimentation velocity and equilibrium experiments. Because it involves minute quantities of sample and detergent, this method can be used in high-throughput approaches as a low-cost technique.
Analytical Biochemistry 05/2011; 416(1):100-6. · 3.00 Impact Factor
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ABSTRACT: Novel cancer cellular therapy approaches involving long-term ex vivo IL-2 stimulated highly cytotoxic natural killer (NK) cells are emerging. However, adhesion properties of such NK cells are not very well understood. Herein, we describe the novel observation of permanently activated alphaLbeta2 integrin leukocyte function-associated antigen (LFA)-1 adhesion receptor in long-term IL-2 activated NK cells and the permanent NK cell lines KHYG-1 and NK-92. We show that such cytokine activated NK effectors constitutively adhered to the LFA-1-ligand ICAM-1, whereas binding to the lower affinity ligand ICAM-3 required additional exogenous activating conditions. The results demonstrate an extended conformation and an intermediate affinity state for the LFA-1 population expressed by the NK cells. Interestingly, adhesion to ICAM-1 or K562 induced pronounced cell spreading in KHYG-1, but not in NK-92, and partially in long-term IL-2 stimulated primary NK cells. It is conceivable that such differential adhesion characteristics may impact motility potential of such NK effectors with relevance to clinical tumor targeting. KHYG-1 could be a useful model in planning future targeted therapeutic approaches involving NK effectors with augmented functions.
Frontiers in bioscience (Elite edition) 01/2011; 3:166-78.
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ABSTRACT: Integrins are heterodimeric transmembrane (TM) receptors formed by noncovalent associations of alpha and beta subunits. Each subunit contains a single alpha-helical TM domain. Inside-out activation of an integrin involves the separation of its cytoplasmic tails, leading to disruption of alphabeta TM packing. The leukocyte integrin alpha L beta 2 is required for leukocyte adhesion, migration, proliferation, cytotoxic function, and antigen presentation. In this study, we show by mutagenesis experiments that the packing of alpha L beta 2 TMs is consistent with that of the integrin alpha IIb beta 3 TMs. However, molecular dynamics simulations of alpha L beta 2 TMs in lipids predicted a polar interaction involving the side chains of alpha L Ser1071 and beta2 Thr686 in the outer-membrane association clasp (OMC). This is supported by carbonyl vibrational shifts observed in isotope-labeled alpha L beta 2 TM peptides that were incorporated into lipid bilayers. Molecular dynamics studies simulating the separation of alpha L beta 2 tails showed the presence of polar interaction during the initial perturbation of the inner-membrane association clasp. When the TMs underwent further separation, the polar interaction was disrupted. OMC polar interaction is important in regulating the functions of beta2 integrins because mutations that disrupt the OMC polar interaction generated constitutively activated alpha L beta 2, alpha M beta 2, and alpha X beta 2 in 293T transfectants. We also show that the expression of mutant beta2 Thr686Gly in beta2-deficient T cells rescued cell adhesion to intercellular adhesion molecule 1, but the cells showed overt elongated morphologies in response to chemokine stromal-cell-derived factor 1 alpha treatment as compared to wild-type beta2-expressing cells. These two TM polar residues are totally conserved in other members of the beta2 integrins in humans and across different species. Our results provide an example of the stabilizing effect of polar interactions within the low dielectric environment of the membrane interior and demonstrate its importance in the regulation of alpha L beta 2 function.
Journal of Molecular Biology 03/2010; 398(4):569-83. · 4.00 Impact Factor
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ABSTRACT: The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one alpha-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric alpha-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular alpha-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.
PLoS Pathogens 08/2009; 5(7):e1000511. · 9.13 Impact Factor
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ABSTRACT: Glutathione transferases, GSTs, are detoxification proteins that are found in most organisms. The acGSTE3-3 had the ability to conjugate 4-hydroxynonenal, a cytotoxic lipid peroxidation product. Although other Epsilon GSTs showed roles in insecticide metabolism, the acGSTE3-3 appeared to have a major role in detoxifying lipid peroxidation products conferring protection against oxidative damage.
Protein and Peptide Letters 02/2009; 16(1):75-81. · 1.94 Impact Factor
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ABSTRACT: Integrins are type I heterodimeric cell adhesion molecules that mediate a wide array of biological processes. Integrin bidirectional
signaling allows communication between the cell interior with its microenvironment. The integrin transmembrane domains (TMs)
are the transducers of activation signal that is relayed from the cytoplasmic domains to the distal ligand binding site located
in the ectodomain of the integrin and vice versa. In this study, we showed that the disruption of the αLβ2 TMs by mutation of a key interface residue Thr-686 in the β2 TM
promoted αLβ2 activation with ICAMs binding properties that are reminiscent of an intermediate affinity receptor. The activated
αLβ2 TM mutants, however, showed minimal reactivity with the reporter mAb KIM127 that recognizes a highly extended αLβ2. Two
models of αLβ2 TM interaction were proposed previously. One with GXXXG-type interaction, and another that is based on TM cysteine-scanning analyses. Our data are consistent with a GXXXG-type interaction of the αLβ2 TMs. Finally, we observed by FRET analyses that perturbation of the αLβ2 TMs by β2 Thr-686
mutation facilitated αL micro-cluster formation. This was diminished by linking the αLβ2 TMs with a disulfide bond, which
served to clasp the TMs. These data suggest that disruption of the TM interface changes αLβ2 ligand binding affinity, and
it may contribute to αL micro-cluster formation.
Journal of Biological Chemistry 01/2009; 284(5):3239-3249. · 4.77 Impact Factor
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ABSTRACT: Integrins are type I heterodimeric cell adhesion molecules that mediate a wide array of biological processes. Integrin bidirectional signaling allows communication between the cell interior with its microenvironment. The integrin transmembrane domains (TMs) are the transducers of activation signal that is relayed from the cytoplasmic domains to the distal ligand binding site located in the ectodomain of the integrin and vice versa. In this study, we showed that the disruption of the alphaLbeta2 TMs by mutation of a key interface residue Thr-686 in the beta2 TM promoted alphaLbeta2 activation with ICAMs binding properties that are reminiscent of an intermediate affinity receptor. The activated alphaLbeta2 TM mutants, however, showed minimal reactivity with the reporter mAb KIM127 that recognizes a highly extended alphaLbeta2. Two models of alphaLbeta2 TM interaction were proposed previously. One with GXXXG-type interaction, and another that is based on TM cysteine-scanning analyses. Our data are consistent with a GXXXG-type interaction of the alphaLbeta2 TMs. Finally, we observed by FRET analyses that perturbation of the alphaLbeta2 TMs by beta2 Thr-686 mutation facilitated alphaL micro-cluster formation. This was diminished by linking the alphaLbeta2 TMs with a disulfide bond, which served to clasp the TMs. These data suggest that disruption of the TM interface changes alphaLbeta2 ligand binding affinity, and it may contribute to alphaL micro-cluster formation.
Journal of Biological Chemistry 12/2008; 284(5):3239-49. · 4.77 Impact Factor
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ABSTRACT: The glycosylphosphatidylinositol-linked urokinase-type plasminogen activator receptor (uPAR) interacts with the heterodimer cell adhesion molecules integrins to modulate cell adhesion and migration. Devoid of a cytoplasmic domain, uPAR triggers intracellular signaling via its associated molecules that contain cytoplasmic domains. Interestingly, uPAR changes the ectodomain conformation of one of its partner molecules, integrin alpha(5)beta(1), and elicits cytoplasmic signaling. The separation or reorientation of integrin transmembrane domains and cytoplasmic tails are required for integrin outside-in signaling. However, there is a lack of direct evidence showing these conformational changes of an integrin that interacts with uPAR. In this investigation we used reporter monoclonal antibodies and fluorescence resonance energy transfer analyses to show conformational changes in the alpha(M)beta(2) headpiece and reorientation of its transmembrane domains when alpha(M)beta(2) interacts with uPAR.
Journal of Biological Chemistry 09/2008; 283(37):25392-403. · 4.77 Impact Factor
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ABSTRACT: The current paradigm is that integrin is activated via inside-out signalling when its cytoplasmic tails and TMs (transmembrane helices) are separated by specific cytosolic protein(s). Perturbations of the helical interface between the alpha- and beta-TMs of an integrin, as a result of mutations, affect its function. Previous studies have shown the requirement for specific pairing between integrin subunits by ectodomain-exchange analyses. It remains unknown whether permissive alpha/beta-TM pairing of an integrin is also required for pairing specificity and the expression of a functionally regulated receptor. We performed scanning replacement of integrin beta2-TM with a TM of other integrin beta-subunits. With the exception of beta4 substitution, others presented beta2-integrins with modified phenotypes, either in their expression or ligand-binding properties. Subsequently, we adopted alphaLbeta2 for follow-on experiments because its conformation and affinity-state transitions have been well defined as compared with other members of the beta2-integrins. Replacement of beta2- with beta3-TM generated a chimaeric alphaLbeta2 of an intermediate affinity that adhered to ICAM-1 (intercellular adhesion molecule 1) but not to ICAM-3 constitutively. Replacing alphaL-TM with alphaIIb-TM, forming a natural alphaIIb/beta3-TM pair, reversed the phenotype of the chimaera to that of wild-type alphaLbeta2. Interestingly, the replacement of alphaLbeta2- with beta3-TM showed neither an extended conformation nor the separation of its cytoplasmic tails, which are well-reported hallmarks of an activated alphaLbeta2, as determined by reporter mAb (monoclonal antibody) KIM127 reactivity and FRET (fluorescence resonance energy transfer) measurements respectively. Collectively, our results suggest that TM pairing specificity is required for the expression of a functionally regulated integrin.
Biochemical Journal 04/2008; 410(3):495-502. · 4.90 Impact Factor
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ABSTRACT: The present study characterized conserved residues in a GST (glutathione transferase) in the active-site region that interacts with glutathione. This region of the active site is near the glycine moiety of glutathione and consists of a hydrogen bond network. In the GSTD (Delta class GST) studied, adGSTD4-4, the network consisted of His(38), Met(39), Asn(47), Gln(49), His(50) and Cys(51). In addition to contributing to glutathione binding, this region also had major effects on enzyme catalysis, as shown by changes in kinetic parameters and substrate-specific activity. The results also suggest that the electron distribution of this network plays a role in stabilization of the ionized thiol of glutathione as well as impacting on the catalytic rate-limiting step. This area constitutes a second glutathione active-site network involved in glutathione ionization distinct from a network previously observed interacting with the glutamyl end of glutathione. This second network also appears to be functionally conserved in GSTs. In the present study, His(50) is the key basic residue stabilized by this network, as shown by up to a 300-fold decrease in k(cat) and 5200-fold decrease in k(cat)/K(m) for glutathione. Although these network residues have a minor role in structural integrity, the replaced residues induced changes in active-site topography as well as generating positive co-operativity towards glutathione. Moreover, this network at the glycine moiety of GSH (glutathione) also contributed to the 'base-assisted deprotonation model' for GSH ionization. Taken together, the results indicate a critical role for the functionally conserved basic residue His(50) and this hydrogen bond network in the active site.
Biochemical Journal 10/2007; 406(2):247-56. · 4.90 Impact Factor
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ABSTRACT: GSTs (glutathione S-transferases; E.C.2.5.1.18) are a supergene family of dimeric multifunctional enzymes that have a major role in detoxification pathways. Using a GST from the mosquito Anopheles dirus (adGSTD4-4), we have characterized the enzymatic and physical properties of Leu-6, Thr-31, Leu-33, Ala-35, Glu-37, Lys-40 and Glu-42. These residues generate two motifs located in the N-terminal domain (domain I) that are functionally conserved across GST classes. The aim of this study was to understand the function of these two motifs. The first motif is a small hydrophobic core in the G-site (glutathione-binding site) wall, and the second motif contains an ionic bridge at the N-terminus of the alpha2 helix and is also part of the G-site. The mutations in the small hydrophobic core appear to have structural effects, as shown by the thermal stability, refolding rate and intrinsic fluorescence differences. In the Delta class GST, interactions form an ionic bridge motif located at the beginning of the alpha2 helix. The data suggest that electrostatic interactions in the alpha2 helix are involved in alpha-helix stabilization, and disruption of this ionic bridge interaction changes the movement of the alpha2-helix region, thereby modulating the interaction of the enzyme with substrates. These results show that the small hydrophobic core and ionic bridge have a major impact on structural stabilization, as well as being required to maintain structural conformation of the enzyme. These structural effects are also transmitted to the active site to influence substrate binding and specificity. Therefore changes in the conformation of the G-site wall in the active site appear to be capable of exerting influences on the tertiary structural organization of the whole GST protein.
Biochemical Journal 02/2006; 393(Pt 1):89-95. · 4.90 Impact Factor
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ABSTRACT: This study was designed to characterize residues in the glutathione binding site of AdGSTD4-4 from the mosquito malaria vector Anopheles dirus. The data revealed that Leu33, His38 and His50 each play a role in enzyme catalysis and glutathione binding. The mutants of these three residues also displayed differences in hydrophobic substrate specificity, suggesting that changes in the active site conformation occurred. Differences in conformations was also suggested by protein stability changes. These results indicate that residues in the glutathione binding site are not only important in the catalytic function but also play a role in the structural integrity of the enzyme.
Protein and Peptide Letters 11/2003; 10(5):441-8. · 1.94 Impact Factor
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ABSTRACT: Integrins are heterodimeric transmembrane (TM) receptors formed by noncovalent associations of α and β subunits. Each subunit contains a single α-helical TM domain. Inside-out activation of an integrin involves the separation of its cytoplasmic tails, leading to disruption of αβ TM packing. The leukocyte integrin αLβ2 is required for leukocyte adhesion, migration, proliferation, cytotoxic function, and antigen presentation. In this study, we show by mutagenesis experiments that the packing of αLβ2 TMs is consistent with that of the integrin αIIbβ3 TMs. However, molecular dynamics simulations of αLβ2 TMs in lipids predicted a polar interaction involving the side chains of αL Ser1071 and β2 Thr686 in the outer-membrane association clasp (OMC). This is supported by carbonyl vibrational shifts observed in isotope-labeled αLβ2 TM peptides that were incorporated into lipid bilayers. Molecular dynamics studies simulating the separation of αLβ2 tails showed the presence of polar interaction during the initial perturbation of the inner-membrane association clasp. When the TMs underwent further separation, the polar interaction was disrupted. OMC polar interaction is important in regulating the functions of β2 integrins because mutations that disrupt the OMC polar interaction generated constitutively activated αLβ2, αMβ2, and αXβ2 in 293T transfectants. We also show that the expression of mutant β2 Thr686Gly in β2-deficient T cells rescued cell adhesion to intercellular adhesion molecule 1, but the cells showed overt elongated morphologies in response to chemokine stromal-cell-derived factor 1α treatment as compared to wild-type β2-expressing cells. These two TM polar residues are totally conserved in other members of the β2 integrins in humans and across different species. Our results provide an example of the stabilizing effect of polar interactions within the low dielectric environment of the membrane interior and demonstrate its importance in the regulation of αLβ2 function.
Journal of Molecular Biology.
