RESUMO
Infection by HIV-1 requires protein-protein interactions involving gp120, CD4 and CCR5. We have previously demonstrated that the transferred nuclear Overhauser effect (TRNOE), in combination with asymmetric deuteration of a protein and a peptide ligand can be used to detect intermolecular interactions in large protein complexes with molecular weights up to ~ 100 kDa. Here, using this approach, we reveal interactions between tyrosine residues of a 27-residue peptide corresponding to the N-terminal segment of the CCR5 chemokine receptor, and a dimeric extended core YU2 gp120 envelope protein of HIV-1 complexed with a CD4-mimic miniprotein. The TRNOE crosspeaks in the ternary complex were assigned to the specific Tyr protons in the CCR5 peptide and to methyl protons of isoleucine, leucine and/or valine residues of gp120. Site directed mutagenesis combined with selective deuteration and TRNOE resulted in the first discernment by a biophysical method of specific pairwise interactions between gp120 residues in the bridging sheet of gp120 and the N-terminus of CCR5.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , Mutação , Ressonância Magnética Nuclear Biomolecular/métodos , Receptores CCR5/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Receptores CCR5/química , Receptores CCR5/genéticaRESUMO
The effects of various lipid bound paramagnetic metal ions on liposomes prepared in the presence of trehalose and chelator lipids are evaluated to observe site-specific signal changes on liposome samples with optimal resolution in solid-state NMR spectroscopy. We found that Mn2+, Gd3+ and Dy3+ have different influences on the lipid 13C sites depending on their penetration depths into the bilayer, which can be extracted as distance information. The trehalose-liposome mixture is efficiently packed into solid-state NMR rotors and provides optimal resolution at reasonable instrument temperatures (10-50⯰C). The effectiveness and convenience of the trehalose preparation for studying a membrane protein in liposomes are demonstrated by a membrane sample with a model membrane peptide to show that trehalose is useful to prepare consistent and stable membrane protein liposome samples for solid-state NMR.
Assuntos
Quelantes/química , Lipossomos/química , Espectroscopia de Ressonância Magnética , Lipídeos de Membrana/química , Metais/química , Trealose/química , Bicamadas Lipídicas/químicaRESUMO
The inflammatory chemokine CCL5, which binds the chemokine receptor CCR5 in a two-step mechanism so as to activate signaling pathways in hematopoetic cells, plays an important role in immune surveillance, inflammation, and development as well as in several immune system pathologies. The recently published crystal structure of CCR5 bound to a high-affinity variant of CCL5 lacks the N-terminal segment of the receptor that is post-translationally sulfated and is known to be important for high-affinity binding. Here, we report the NMR solution structure of monomeric CCL5 bound to a synthetic doubly sulfated peptide corresponding to the missing first 27 residues of CCR5. Our structures show that two sulfated tyrosine residues, sY10 and sY14, as well as the unsulfated Y15 form a network of strong interactions with a groove on a surface of CCL5 that is formed from evolutionarily conserved basic and hydrophobic amino acids. We then use our NMR structures, in combination with available crystal data, to create an atomic model of full-length wild-type CCR5:CCL5. Our findings reveal the structural determinants involved in the recognition of CCL5 by the CCR5 N terminus. These findings, together with existing structural data, provide a complete structural framework with which to understand the specificity of receptor:chemokine interactions. DATABASE: Structural data are available in the PDB under the accession number 6FGP.
Assuntos
Quimiocina CCL5/química , Conformação Proteica , Receptores CCR5/química , Sequência de Aminoácidos/genética , Sítios de Ligação , Quimiocina CCL5/genética , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica/genética , Receptores CCR5/genéticaRESUMO
NMR is a powerful tool for studying structural details of protein/peptide complexes exhibiting weak to medium binding (KD > 10 µm). However, it has been assumed that intermolecular nuclear Overhauser effect (NOE) interactions are difficult to observe in such complexes. We demonstrate that intermolecular NOEs can be revealed by combining the 13 C-edited/13 C-filtered experiment with the transferred NOE effect (TRNOE). Due to the TRNOE phenomenon, intermolecular NOE cross peaks are characterized by both the chemical shifts (CSs) of the protein protons and the average CSs of the peptide protons, which are dominated by the CSs of the protons of the free peptide. Previously, the TRNOE phenomenon was used almost exclusively to investigate the conformation of small ligands bound to large biomolecules. Here, we demonstrate that TRNOE can be extended to enable the study of intermolecular interactions in small- and medium-sized protein complexes. We used the 13 C-edited/13 C-filtered TRNOE experiment to study the interactions of the chemokine regulated upon activation, normal T cell, expressed and secreted (RANTES) with a 27-residue peptide, containing two sulfotyrosine residues, representing the N-terminal segment of the CCR5 receptor ((Nt-CCR5(1-27). The TRNOE phenomenon led to more than doubling of the signal-to-noise ratios (SNRs) for the intermolecular NOEs observed in the 13 C-edited/13 C-filtered experiment for the 11.5-kDa monomeric RANTES/Nt-CCR5(1-27) complex. An even better improvement in the SNR was achieved with dimeric Nt-CCR5(1-27)/RANTES (23 kDa), especially in comparison with the spectra measured with a 1 : 1 protein to peptide ratio. In principle, the isotope-edited/isotope-filtered TRNOE spectrum can discern all intermolecular interactions involving nonexchangeable protons in the complex.
Assuntos
Quimiocina CCL5/química , Ressonância Magnética Nuclear Biomolecular/métodos , Receptores CCR5/química , Sequência de Aminoácidos , Sítios de Ligação , Isótopos de Carbono , Quimiocina CCL5/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Cinética , Peso Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores CCR5/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem/métodosRESUMO
V3-directed antibodies are present in practically all HIV-1 infected patients and in individuals vaccinated with gp120. The levels of maternal V3-directed antibodies were recently shown to correlate with reduced mother to child transmission, and V3 IgGs were found to be a negative correlate of risk in the RV-144 human trial. mAb directed to the tip of the V3 are capable of broad neutralization of Tier-1 and some Tier-2 viruses. Here we report an immunofocusing approach using conformationally constrained V3 peptides of different lengths. Immunofocusing with short constrained V3 peptides following immunizations with long constrained V3 peptides resulted in sera with improved neutralization of Tier-1B viruses in comparison with immunizations with the long constrained peptide alone. Immunizations only with the short constrained peptide were ineffective. Our results demonstrate that immunofocusing with constrained V3 peptides of different lengths could improve the induction of HIV-1 neutralizing antibodies.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Animais , Antígenos HIV/imunologia , Humanos , CoelhosRESUMO
Weak protein-protein and protein-ligand interactions play important roles in biological recognition. In many cases, simplification of structural studies of large protein complexes is achieved by investigation of the interaction between the protein and a weakly binding segment of its protein ligand. Detection of pairwise interactions in such complexes is a major challenge for both X-ray crystallography and nuclear magnetic resonance. We demonstrate that transferred nuclear Overhauser effect (TRNOE), in combination with asymmetric deuteration of a protein and a peptide ligand can be used to detect intermolecular interactions in large protein complexes with molecular weights up to ~ 100 kDa. Using this approach, we revealed interactions between tyrosine residues of a 27-residue peptide (deuterated at Ile and Val residues) corresponding to the N-terminal segment of the human C-C chemokine receptor 5 (CCR5) chemokine receptor, and a 43 kDa construct of gp120 envelope protein of human immunodeficiency virus type 1 (deuterated on all aromatics) complexed with a cluster of differentiation 4-mimic miniprotein. The complex was present mostly as a dimer as determined by T2 relaxation measurements. The TRNOE crosspeaks in the ternary complex were assigned to the specific Tyr protons in the CCR5 peptide and to methyl protons, predominantly of isoleucine residues, and also of leucine and/or valine residues of gp120. The TRNOE/asymmetric deuteration method benefits from the sensitivity of the homonuclear NOESY experiment and does not suffer the sensitivity losses associated with isotope-edited/isotope-filtered approaches that rely on magnetization transfer between protons and heteronuclei that are bonded to them. The technique can be widely applied for studying large protein complexes that exhibit fast off-rates.
Assuntos
Proteína gp120 do Envelope de HIV/química , Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeos/química , Receptores CCR5/química , Sítios de Ligação , Cristalografia por Raios X , Deutério , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Isoleucina/química , Isoleucina/metabolismo , Leucina/química , Leucina/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Prótons , Receptores CCR5/metabolismo , Tirosina/química , Tirosina/metabolismo , Valina/química , Valina/metabolismoRESUMO
The peptide T20, which corresponds to a sequence in the C-terminal segment of the HIV-1 transmembrane glycoprotein gp41, is a strong entry inhibitor of HIV-1. It has been assumed that T20 inhibits HIV-1 infection by binding to the trimer formed by the N-terminal helical region (HR1) of gp41, preventing the formation of a six helix bundle by the N- and C-terminal helical regions of gp41. In addition to binding to gp41, T20 was found to bind to gp120 of X4 viruses and this binding was suggested to be responsible for an alternative mechanism of HIV-1 inhibition by this peptide. In the present study, T20 also was found to bind R5 gp120. Using NMR spectroscopy, the segments of T20 that interact with both gp120 and a gp120/CD4M33 complex were mapped. A peptide corresponding to the fourth constant region of gp120, sC4, was found to partially recapitulate gp120 binding to T20 and the segment of this peptide interacting with T20 was mapped. The present study concludes that an amphiphilic helix on the T20 C-terminus binds through mostly hydrophobic interactions to a nonpolar gp120 surface formed primarily by the C4 region. The ten- to thousand-fold difference between the EC50 of T20 against viral fusion and the affinity of T20 to gp120 implies that binding to gp120 is not a major factor in T20 inhibition of HIV-1 fusion. Nevertheless, this hydrophobic gp120 surface could be a target for anti-HIV therapeutics.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Inibidores da Fusão de HIV/metabolismo , HIV-1/efeitos dos fármacos , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , Internalização do Vírus , Substituição de Aminoácidos , Antígenos CD4/química , Antígenos CD4/metabolismo , Enfuvirtida , Células HEK293 , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/farmacologia , Inibidores da Fusão de HIV/química , Inibidores da Fusão de HIV/farmacologia , HIV-1/fisiologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Peptídeos/química , Peptídeos/genética , Peptídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Solubilidade , Ressonância de Plasmônio de Superfície , Internalização do Vírus/efeitos dos fármacosRESUMO
The F-recruitment site (FRS) of active ERK2 binds F-site (Phe-x-Phe-Pro) sequences found downstream of the Ser/Thr phospho-acceptor on cellular substrates. Here we apply NMR methods to analyze the interaction between active ERK2 (ppERK2), and a 13-residue F-site-bearing peptide substrate derived from its cellular target, the transcription factor Elk-1. Our results provide detailed insight into previously elusive structural and dynamic features of FRS/F-site interactions and FRS-driven substrate phosphorylation. We show that substrate F-site engagement significantly quenches slow dynamics involving the ppERK2 activation-loop and the FRS. We also demonstrate that the F-site phenylalanines make critical contacts with ppERK2, in contrast to the proline whose cis-trans isomerization has no significant effect on F-site recognition by the kinase FRS. Our results support a mechanism where phosphorylation of the disordered N-terminal phospho-acceptor is facilitated by its increased productive encounters with the ppERK2 active site due to docking of the proximal F-site at the kinase FRS.
Assuntos
Domínio Catalítico/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismo , Sítios de Ligação/fisiologia , DNA/metabolismo , Humanos , Modelos Moleculares , Fosforilação , Conformação ProteicaRESUMO
UNLABELLED: C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV-1. The CCR5 N-terminal segment, the second extracellular loop (ECL2) and the transmembrane helices have been implicated in binding the envelope glycoprotein gp120. Peptides corresponding to the sequence of the putative ECL2 as well as peptides containing extracellular loops 1 and 3 (ECL1 and ECL3) were found to inhibit HIV-1 infection. The aromatic residues in the C-terminal half of an ECL2 peptide were shown to interact with gp120. In the present study, we found that, in aqueous buffer, the segment Q188-Q194 in an elongated ECL2 peptide (R168-K197) forms an amphiphilic helix, which corresponds to the beginning of the fifth transmembrane helix in the crystal structure of CCR5. Two-dimensional saturation transfer difference NMR spectroscopy and dynamic filtering studies revealed involvement of Y187, F189, W190 and F193 of the helical segment in the interaction with gp120. The crystal structure of CCR5 shows that the aromatic side chains of F189, W190 and F193 point away from the binding pocket and interact with the membrane or with an adjacent CCR5 molecule, and therefore could not interact with gp120 in the intact CCR5 receptor. We conclude that these three aromatic residues of ECL2 peptides interact with gp120 through hydrophobic interactions that are not representative of the interactions of the intact CCR5 receptor. The HIV-1 inhibition by ECL2 peptides, as well as by ECL1 and ECL3 peptides and peptides corresponding to ECL2 of CXCR4, which serves as an alternative HIV-1 co-receptor, suggests that there is a hydrophobic surface in the envelope spike that could be a target for HIV-1 entry inhibitors. DATABASE: The structures and NMR data of ECL2S (Q186-T195) were deposited under Protein Data Bank ID 2mzx and BioMagResBank ID 25505.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Receptores CCR5/química , Receptores CCR5/metabolismo , Animais , Bovinos , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Ligação Proteica , Estrutura Secundária de Proteína , Soroalbumina Bovina/metabolismoRESUMO
This report summarizes recent biophysical and protein expression experiments on polypeptides containing the N-terminus, the first, second, and third transmembrane (TM) domains and the contiguous loops of the α-factor receptor Ste2p, a G protein-coupled receptor. The 131-residue polypeptide Ste2p(G31-R161), TM1-TM3, was investigated by solution NMR in trifluoroethanol/water. TM1-TM3 contains helical TM domains at the predicted locations, supported by continuous sets of medium-range NOEs. In addition, a short helix N-terminal to TM1 was detected, as well as a short helical stretch in the first extracellular loop. Two 161-residue polypeptides, [Ste2p(M1-R161), NT-TM1-TM3], that contain the entire N-terminal sequence, one with a single mutation, were directly expressed and isolated from Escherichia coli in yields as high as 30 mg/L. Based on its increased stability, the L11P mutant will be used in future experiments to determine long-range interactions. The study demonstrated that 3-TM domains of a yeast G protein-coupled receptor can be produced in isotopically labeled form suitable for solution NMR studies. The quality of spectra is superior to data recorded in micelles and allows more rapid data analysis. No tertiary contacts have been determined, and if present, they are likely transient. This observation supports earlier studies by us that secondary structure was retained in smaller fragments, both in organic solvents and in detergent micelles, but that stable tertiary contacts may only be present when the protein is imbedded in lipids.
Assuntos
Receptores de Fator de Acasalamento/química , Proteínas de Saccharomyces cerevisiae/química , Trifluoretanol/química , Água/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Marcação por Isótopo , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Fator de Acasalamento/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , TermodinâmicaRESUMO
Structural analysis by NMR of G protein-coupled receptors (GPCRs) has proven to be extremely challenging. To reduce the number of peaks in the NMR spectra by segmentally labeling a GPCR, we have developed a Guided Reconstitution method that includes the use of charged residues and Cys activation to drive heterodimeric disulfide bond formation. Three different cysteine-activating reagents: 5-5'-dithiobis(2-nitrobenzoic acid) [DTNB], 2,2'-dithiobis(5-nitropyridine) [DTNP], and 4,4'-dipyridyl disulfide [4-PDS] were analyzed to determine their efficiency in heterodimer formation at different pHs. Short peptides representing the N-terminal (NT) and C-terminal (CT) regions of the first extracellular loop (EL1) of Ste2p, the Saccharomyces cerevisiae alpha-factor mating receptor, were activated using these reagents and the efficiencies of activation and rates of heterodimerization were analyzed. Activation of NT peptides with DTNP and 4-PDS resulted in about 60% yield, but heterodimerization was rapid and nearly quantitative. Double transmembrane domain protein fragments were biosynthesized and used in Guided Reconstitution reactions. A 102-residue fragment, 2TM-tail [Ste2p(G31-I120C)], was heterodimerized with CT-EL1-tail(DTNP) at pH 4.6 with a yield of â¼75%. A 132-residue fragment, 2TMlong-tail [Ste2p(M1-I120C)], was expressed in both unlabeled and (15)N-labeled forms and used with a peptide comprising the third transmembrane domain, to generate a 180-residue segmentally labeled 3TM protein that was found to be segmentally labeled using [(15)N,(1)H]-HSQC analysis. Our data indicate that the Guided Reconstitution method would be applicable to the segmental labeling of a membrane protein with 3 transmembrane domains and may prove useful in the preparation of an intact reconstituted GPCR for use in biophysical analysis and structure determination.
Assuntos
Bioquímica/métodos , Proteínas de Membrana/química , Sequência de Aminoácidos , Brometo de Cianogênio/química , Cisteína/química , Dissulfetos/metabolismo , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Mutação/genética , Peptídeos/química , Multimerização Proteica , Receptores de Fator de Acasalamento/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de TempoRESUMO
Chemokines constitute a large family of small proteins that regulate leukocyte trafficking to the site of inflammation by binding to specific cell-surface receptors belonging to the G-protein-coupled receptor (GPCR) superfamily. The interactions between N-terminal (Nt-) peptides of these GPCRs and chemokines have been studied extensively using NMR spectroscopy. However, because of the lower affinities of peptides representing the three extracellular loops (ECLs) of chemokine receptors to their respective chemokine ligands, information concerning these interactions is scarce. To overcome the low affinity of ECL peptides to chemokines, we linked two or three CC chemokine receptor 5 (CCR5) extracellular domains using either biosynthesis in Escherichia coli or chemical synthesis. Using such chimeras, CCR5 binding to RANTES was followed using (1)H-(15)N-HSQC spectra to monitor titration of the chemokine with peptides corresponding to the extracellular surface of the receptor. Nt-CCR5 and ECL2 were found to be the major contributors to CCR5 binding to RANTES, creating an almost closed ring around this protein by interacting with opposing faces of the chemokine. A RANTES positively charged surface involved in Nt-CCR5 binding resembles the positively charged surface in HIV-1 gp120 formed by the C4 and the base of the third variable loop of gp120 (V3). The opposing surface on RANTES, composed primarily of ß2-ß3 hairpin residues, binds ECL2 and was found to be analogous to a surface in the crown of the gp120 V3. The chemical and biosynthetic approaches for linking GPCR surface regions discussed herein should be widely applicable to the investigation of interactions of extracellular segments of chemokine receptors with their respective ligands.
Assuntos
Quimiocina CCL5/química , Receptores CCR5/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Cistina/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Propriedades de SuperfícieRESUMO
To conduct biophysical analyses on large domains of GPCRs, multimilligram quantities of highly homogeneous proteins are necessary. This communication discusses the biosynthesis of four transmembrane and five transmembrane-containing fragments of Ste2p, a GPCR recognizing the Saccharomyces cerevisiae tridecapeptide pheromone α-factor. The target fragments contained the predicted four N-terminal Ste2p[G(31) -A(198) ] (4TMN), four C-terminal Ste2p[T(155) -L(340) ] (4TMC), or five C-terminal Ste2p[I(120) -L(340) ] (5TMC) transmembrane segments of Ste2p. 4TMN was expressed as a fusion protein using a modified pMMHa vector in L-arabinose-induced Escherichia coli BL21-AI, and cleaved with cyanogen bromide. 4TMC and 5TMC were obtained by direct expression using a pET21a vector in IPTG-induced E. coli BL21(DE3) cells. 4TMC and 5TMC were biosynthesized on a preparative scale, isolated in multimilligram amounts, characterized by MS and investigated by biophysical methods. CD spectroscopy indicated the expected highly α-helical content for 4TMC and 5TMC in membrane mimetic environments. Tryptophan fluorescence showed that 5TMC integrated into the nonpolar region of 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) micelles. HSQC-TROSY investigations revealed that [(15) N]-labeled 5TMC in 50% trifluoroethanol-d(2) /H(2) O/0.05%-trifluoroacetic acid was stable enough to conduct long multidimensional NMR measurements. The entire Ste2p GPCR was not readily reconstituted from the first two and last five or first three and last four transmembrane domains.
Assuntos
Fragmentos de Peptídeos/biossíntese , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/isolamento & purificação , Receptores de Fator de Acasalamento/química , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Biomimética , Fenômenos Biofísicos , Membrana Celular/química , Brometo de Cianogênio/química , Eletroforese em Gel de Poliacrilamida , Escherichia coli/química , Vetores Genéticos/química , Corpos de Inclusão/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Plasmídeos/química , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas G/química , Proteínas Recombinantes de Fusão/química , Saccharomyces cerevisiae/química , Triptofano/químicaRESUMO
Interaction of CC chemokine receptor 5 (CCR5) with the human immunodeficiency virus type 1 (HIV-1) gp120/CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of two to four tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied both in its free form and in a ternary complex with deglycosylated gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27), which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by 2-fold the length of Nt-CCR5's known structure. Two-dimensional saturation transfer experiments and measurement of relaxation times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4, while the C-terminal segment of Nt-CCR5(1-27) points toward the target cell membrane, reflecting an Nt-CCR5 orientation that differs by 180° from that of a previous model. A gp120 site that could accommodate (CCR5)Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated (CCR5)Tyr14 with (gp120)Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion, furthering our understanding of CCR5 recognition by HIV-1.
Assuntos
Aminoácidos/metabolismo , Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Peptídeos/metabolismo , Receptores CCR5/química , Receptores CCR5/metabolismo , Glicosilação , HIV-1/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Peptídeos/química , Ligação Proteica , Estrutura Secundária de Proteína , Eletricidade Estática , TermodinâmicaRESUMO
Structural characterization of G protein-coupled receptors (GPCRs) is hindered by the inherent hydrophobicity, flexibility, and large size of these signaling proteins. Insights into conformational preferences and the three-dimensional (3D) structure of domains of these receptors can be obtained using polypeptide fragments of these proteins. Herein, we report the expression, purification, and biophysical characterization of a three-transmembrane domain-containing 131-residue fragment of the yeast α-factor receptor, Ste2p. Ste2p TM1TM3 (G31R161) was expressed as a TrpΔLE fusion protein in Escherichia coli. The expressed protein was subject to CNBr cleavage to remove the fusion tag and TM1TM3 was purified by reverse-phased HPLC. The cleavage product was isolated in yields of up to 20 mg per liter of culture in both unlabeled and uniformly [15N]-labeled and [15N, 13C, 2H]-labeled forms. The secondary structure of TM1TM3 was determined to be helical in a number of membrane mimetic environments, including 2,2,2-trifluoroethanol (TFE):water and lysomyristoylphosphatidylglycerol (LMPG) detergent micelles by circular dichroism. Preliminary HSQC analysis in 50% TFE:water and LMPG micelles prepared in sodium phosphate and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffers revealed that this fragment is suitable for structural analysis by nuclear magnetic resonance (NMR). Complete backbone assignments and a detailed localization of the secondary structural elements of TM1TM3 in 50% TFE:water have been achieved.
Assuntos
Fragmentos de Peptídeos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Biofísica , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas G/químicaRESUMO
Fundamental knowledge about how G protein-coupled receptors and their ligands interact is important for understanding receptor-ligand binding and the development of new drug discovery strategies. We have used cross-linking and tandem mass spectrometry analyses to investigate the interaction of the N terminus of the Saccharomyces cerevisiae tridecapeptide pheromone, α-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of α-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys(7) for detection purposes to create the peptide [DOPA(1),Lys(7)(BioACA),Nle(12)]α-factor, called Bio-DOPA(1)-α-factor. This ligand analog was a potent agonist and bound to Ste2p with â¼65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA(1)-α-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native α-factor or an α-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-α-factor cross-linked to a fragment of Ste2p encompassing residues Ser(251)-Met(294). Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA(1) of the α-factor analog to the Ste2p Lys(269) side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA(1)-α-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of α-factor binding to Ste2p is proposed. The model for bound α-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway.
Assuntos
Di-Hidroxifenilalanina/química , Ácido Periódico/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Ligação Competitiva , Reagentes de Ligações Cruzadas/química , Cinética , Ligantes , Espectrometria de Massas/métodos , Mitógenos/química , Mutagênese Sítio-Dirigida , Peptídeos/química , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Synthetic peptides offer an attractive option for development of a V3-directed vaccine. However, immunization with flexible linear peptides may result in an immune response to multiple conformations, many of which differ from the native conformation of the corresponding region in the protein. Here we show that optimization of the location of a disulfide bond in peptides constrained to mimic the beta-hairpin conformation of the V3, yields an immunogen that elicits a 30-fold stronger HIV-1 neutralizing response in rabbits compared with the homologous linear V3 peptide. The HIV-1 neutralizing response elicited by the optimally constrained peptide is also significantly stronger than that elicited by a gp120 construct in which the V3 is exposed. Neutralization of an HIV-1 strain that shares only 72% identity with the immunizing peptide was demonstrated. The most effective immunogen was also able to neutralize primary isolates that are more resistant to neutralization such as SS1196 and 6535.
Assuntos
Vacinas contra a AIDS/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/química , Animais , Anticorpos Neutralizantes/sangue , Reações Cruzadas , Feminino , Anticorpos Anti-HIV/sangue , Humanos , Coelhos , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
The V3 region of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1) is a potential target for an anti-HIV-1 vaccine. Peptides corresponding to V3 form three variations of a beta-hairpin conformation when bound to anti-V3 HIV-1 neutralizing antibodies. The conformation of a V3(IIIB) peptide bound to the 0.5beta antibody, generated against an X4 gp120, has been postulated to represent the V3 conformation of X4 viruses while the conformations of a V3(MN) and a V3(CONSENSUS) peptide bound to the 447-52D human monoclonal antibody were postulated to represent the R5A and R5B V3 conformations of R5 viruses, respectively. To constrain the conformation of synthetic V3 peptides to these X4, R5A, and R5B conformations, we formed disulfide bonds between Cys residues whose location in a peptide template representing the entire V3(CONSENSUS) epitope recognized by the broadly neutralizing 447-52D antibody was changed systematically. In a previous study [Mor, A., et al. (2009) Biochemistry 48, 3288-3303] we showed that these constrained peptides adopted conformations resembling the three antibody-bound V3 conformations according to the location of the disulfide bonds. Here we show that these constrained peptides, with the exception of peptides in which the disulfide bond flanks the GPGR segment, retain high-affinity binding to the 447-52D antibody. Compared with peptides designed to mimic the X4 conformation, peptides designed to mimic either the R5A or R5B conformation had higher affinity to 447-52D. It is possible that constrained peptides which mimic the R5A and R5B conformations of the V3 and retain high-affinity binding to 447-52D are good candidates for eliciting a broad neutralizing antibody response similar to that of 447-52D.
Assuntos
Vacinas contra a AIDS/metabolismo , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Região Variável de Imunoglobulina/metabolismo , Fragmentos de Peptídeos/metabolismo , Vacinas contra a AIDS/síntese química , Vacinas contra a AIDS/imunologia , Sítios de Ligação de Anticorpos , Proteína gp120 do Envelope de HIV/síntese química , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/imunologia , HIV-1/isolamento & purificação , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Testes de Neutralização , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Ligação Proteica/imunologia , Conformação Proteica , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/metabolismoRESUMO
The structure and dynamic properties of an 80-residue fragment of Ste2p, the G-protein-coupled receptor for alpha-factor of Saccharomyces cerevisiae, was studied in LPPG micelles with the use of solution NMR spectroscopy. The fragment Ste2p(G31-T110) (TM1-TM2) consisted of 19 residues from the N-terminal domain, the first TM helix (TM1), the first cytoplasmic loop, the second TM helix (TM2), and seven residues from the first extracellular loop. Multidimensional NMR experiments on [(15)N], [(15)N, (13)C], [(15)N, (13)C, (2)H]-labeled TM1-TM2 and on protein fragments selectively labeled at specific amino acid residues or protonated at selected methyl groups resulted in >95% assignment of backbone and side-chain nuclei. The NMR investigation revealed the secondary structure of specific residues of TM1-TM2. TALOS constraints and NOE connectivities were used to calculate a structure for TM1-TM2 that was highlighted by the presence of three alpha-helices encompassing residues 39-47, 49-72, and 80-103, with higher flexibility around the internal Arg(58) site of TM1. RMSD values of individually superimposed helical segments 39-47, 49-72, and 80-103 were 0.25 +/- 0.10 A, 0.40 +/- 0.13 A, and 0.57 +/- 0.19 A, respectively. Several long-range interhelical connectivities supported the folding of TM1-TM2 into a tertiary structure typified by a crossed helix that splays apart toward the extracellular regions and contains considerable flexibility in the G(56)VRSG(60) region. (15)N-relaxation and hydrogen-deuterium exchange data support a stable fold for the TM parts of TM1-TM2, whereas the solvent-exposed segments are more flexible. The NMR structure is consistent with the results of biochemical experiments that identified the ligand-binding site within this region of the receptor.