Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 29
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Biol Chem ; 296: 100316, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33516724

RESUMO

A central role for the influenza matrix protein 1 (M1) is to form a polymeric coat on the inner leaflet of the host membrane that ultimately provides shape and stability to the virion. M1 polymerizes upon binding membranes, but triggers for conversion of M1 from a water-soluble component of the nucleus and cytosol into an oligomer at the membrane surface are unknown. While full-length M1 is required for virus viability, the N-terminal domain (M1NT) retains membrane binding and pH-dependent oligomerization. We studied the structural plasticity and oligomerization of M1NT in solution using NMR spectroscopy. We show that the isolated domain can be induced by sterol-containing compounds to undergo a conformational change and self-associate in a pH-dependent manner consistent with the stacked dimer oligomeric interface. Surface-exposed residues at one of the stacked dimer interfaces are most sensitive to sterols. Several perturbed residues are at the interface between the N-terminal subdomains and are also perturbed by changes in pH. The effects of sterols appear to be indirect and most likely mediated by reduction in water activity. The local changes are centered on strictly conserved residues and consistent with a priming of the N-terminal domain for polymerization. We hypothesize that M1NT is sensitive to changes in the aqueous environment and that this sensitivity is part of a mechanism for restricting polymerization to the membrane surface. Structural models combined with information from chemical shift perturbations indicate mechanisms by which conformational changes can be transmitted from one polymerization interface to the other.


Assuntos
Vírus da Influenza A/genética , Influenza Humana/genética , Conformação Proteica , Proteínas da Matriz Viral/genética , Humanos , Vírus da Influenza A/patogenicidade , Vírus da Influenza A/ultraestrutura , Influenza Humana/virologia , Multimerização Proteica/genética , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/ultraestrutura
2.
PLoS Pathog ; 16(8): e1008716, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32780760

RESUMO

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.


Assuntos
Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Influenza Humana/virologia , Rimantadina/farmacologia , Proteínas da Matriz Viral/antagonistas & inibidores , Zanamivir/farmacologia , Antivirais/farmacologia , Farmacorresistência Viral , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/tratamento farmacológico , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
3.
Biomol NMR Assign ; 14(1): 157-161, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32157574

RESUMO

The cytoplasmic C-terminal tail of the matrix protein 2 (M2) from influenza A virus has a well conserved sequence and is involved in interactions with several host proteins as well as the influenza matrix protein 1 (M1). Whereas the transmembrane domain of M2 has been well characterised structurally and functionally, high resolution information about the distal cytoplasmic tail is lacking. Here we report the chemical shifts of the cytoplasmic tail of M2 and the chemical shift perturbations at low pH and in the presence of membrane mimetics. The cytoplasmic tail residues are mostly disordered but an extended backbone conformation is adopted by the LC3 binding motif and the putative M1 interaction site has partial helical content with a small pH-dependence. The chemical shift assignments provide a basis for further investigations into interactions of the M2 cytoplasmic tail with viral and host cell factors.


Assuntos
Citoplasma/química , Vírus da Influenza A/metabolismo , Proteínas da Matriz Viral/química , Concentração de Íons de Hidrogênio , Estrutura Secundária de Proteína
4.
Nat Commun ; 10(1): 4619, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601809

RESUMO

Lipid availability within transmembrane nano-pockets of ion channels is linked with mechanosensation. However, the effect of hindering lipid-chain penetration into nano-pockets on channel structure has not been demonstrated. Here we identify nano-pockets on the large conductance mechanosensitive channel MscL, the high-pressure threshold channel. We restrict lipid-chain access to the nano-pockets by mutagenesis and sulfhydryl modification, and monitor channel conformation by PELDOR/DEER spectroscopy. For a single site located at the entrance of the nano-pockets and distal to the channel pore we generate an allosteric response in the absence of tension. Single-channel recordings reveal a significant decrease in the pressure activation threshold of the modified channel and a sub-conducting state in the absence of applied tension. Threshold is restored to wild-type levels upon reduction of the sulfhydryl modification. The modification associated with the conformational change restricts lipid access to the nano-pocket, interrupting the contact between the membrane and the channel that mediates mechanosensitivity.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Regulação Alostérica , Proteínas de Bactérias/genética , Cisteína/genética , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Canais Iônicos/genética , Bicamadas Lipídicas/química , Lipídeos/química , Mutação , Conformação Proteica , Domínios Proteicos
5.
Sci Rep ; 9(1): 8383, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182749

RESUMO

The hepatitis C virus (HCV) viroporin p7 oligomerizes to form ion channels, which are required for the assembly and secretion of infectious viruses. The 63-amino acid p7 monomer has two putative transmembrane domains connected by a cytosolic loop, and has both N- and C- termini exposed to the endoplasmic reticulum (ER) lumen. NMR studies have indicated differences between p7 structures of distantly related HCV genotypes. A critical question is whether these differences arise from the high sequence variation between the different isolates and if so, how the divergent structures can support similar biological functions. Here, we present a side-by-side characterization of p7 derived from genotype 1b (isolate J4) in the detergent 6-cyclohexyl-1-hexylphosphocholine (Cyclofos-6) and p7 derived from genotype 5a (isolate EUH1480) in n-dodecylphosphocholine (DPC). The 5a isolate p7 in conditions previously associated with a disputed oligomeric form exhibits secondary structure, dynamics, and solvent accessibility broadly like those of the monomeric 1b isolate p7. The largest differences occur at the start of the second transmembrane domain, which is destabilized in the 5a isolate. The results show a broad consensus among the p7 variants that have been studied under a range of different conditions and indicate that distantly related HCVs preserve key features of structure and dynamics.


Assuntos
Hepacivirus/ultraestrutura , Hepatite C/genética , Canais Iônicos/ultraestrutura , Proteínas não Estruturais Virais/ultraestrutura , Proteínas Virais/ultraestrutura , Sequência de Aminoácidos/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/ultraestrutura , Genótipo , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepatite C/virologia , Humanos , Canais Iônicos/química , Canais Iônicos/genética , Estrutura Secundária de Proteína , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Proteínas Viroporinas
7.
Chem Rev ; 118(7): 3559-3607, 2018 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-29488756

RESUMO

Membrane proteins perform a host of vital cellular functions. Deciphering the molecular mechanisms whereby they fulfill these functions requires detailed biophysical and structural investigations. Detergents have proven pivotal to extract the protein from its native surroundings. Yet, they provide a milieu that departs significantly from that of the biological membrane, to the extent that the structure, the dynamics, and the interactions of membrane proteins in detergents may considerably vary, as compared to the native environment. Understanding the impact of detergents on membrane proteins is, therefore, crucial to assess the biological relevance of results obtained in detergents. Here, we review the strengths and weaknesses of alkyl phosphocholines (or foscholines), the most widely used detergent in solution-NMR studies of membrane proteins. While this class of detergents is often successful for membrane protein solubilization, a growing list of examples points to destabilizing and denaturing properties, in particular for α-helical membrane proteins. Our comprehensive analysis stresses the importance of stringent controls when working with this class of detergents and when analyzing the structure and dynamics of membrane proteins in alkyl phosphocholine detergents.


Assuntos
Membrana Celular/ultraestrutura , Detergentes/química , Proteínas de Membrana/química , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Animais , Fenômenos Biofísicos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Espectroscopia de Ressonância Magnética/métodos , Micelas , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Solubilidade
8.
Adv Exp Med Biol ; 964: 15-29, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28315262

RESUMO

The Sigma-1 Receptor (S1R) is a small, ligand-regulated integral membrane protein involved in cell homeostasis and the cellular stress response. The receptor has a multitude of protein and small molecule interaction partners with therapeutic potential. Newly reported structures of the human S1R in ligand-bound states provides essential insights into small molecule binding in the context of the overall protein structure. The structure also raises many interesting questions and provides an excellent starting point for understanding the molecular tricks employed by this small membrane receptor to modulate a large number of signaling events. Here, we review insights from the structures of ligand-bound S1R in the context of previous biochemical studies and propose, from a structural viewpoint, a set of important future directions.


Assuntos
Receptores sigma/química , Receptores sigma/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Ligantes , Alinhamento de Sequência , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Receptor Sigma-1
9.
Proc Natl Acad Sci U S A ; 113(39): 10902-7, 2016 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-27621477

RESUMO

Reticulons (RTNs) are a class of endoplasmic reticulum (ER) membrane proteins that are capable of maintaining high membrane curvature, thus helping shape the ER membrane into tubules. The mechanism of action of RTNs is hypothesized to be a combination of wedging, resulting from the transmembrane topology of their conserved reticulon homology domain, and scaffolding, arising from the ability of RTNs to form low-mobility homo-oligomers within the membrane. We studied the plant RTN isoform RTN13, which has previously been shown to locate to ER tubules and the edges of ER cisternae and to induce constrictions in ER tubules when overexpressed, and identified a region in the C terminus containing a putative amphipathic helix (APH). Here we show that deletion of this region or disruption of the hydrophobic face of the predicted helix abolishes the ability of RTN13 to induce constrictions of ER tubules in vivo. These mutants, however, still retain their ability to interact and form low-mobility oligomers in the ER membrane. Hence, our evidence indicates that the conserved APH is a key structural feature for RTN13 function in vivo, and we propose that RTN, like other membrane morphogens, rely on APHs for their function.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Transferência Ressonante de Energia de Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/metabolismo , Mutação/genética , Epiderme Vegetal/citologia , Estrutura Secundária de Proteína , Deleção de Sequência , Relação Estrutura-Atividade , Nicotiana/citologia
10.
Phys Chem Chem Phys ; 17(30): 19766-76, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26153345

RESUMO

Permeation of drugs across lipid bilayers is a key factor in dictating how effective they will be. In vivo, the issue is compounded by the presence of drug-exporter proteins such as P-glycoprotein. However, despite intense effort, exactly what controls permeation and susceptibility to export is still poorly understood. In this work we examine two well-studied drugs for which interaction with P-glycoprotein has been studied before: amitriptyline, a known substrate and clozapine, which is not a substrate. Extensive MD simulations, including potential of mean force (PMF) profiles of the compounds in all possible protonation states, reveal that the preferred location of the compounds in different bilayers in different protonation states is remarkably similar. For both molecules in charged states, there is a substantial barrier to crossing the bilayer. Clozapine however, shows an energetic barrier to movement across the bilayer even in a protonation state that results in an uncharged molecule. For amitriptyline there is only a very small barrier of approximately 1.3 kcal mol(-1). Further analysis revealed that the conformational and orientational behavior of the two compounds was also similar, with the sidechain interacting with the lipid headgroups. This effect was much stronger if the sidechain was charged (protonated). These interactions with lipid bilayers were confirmed by NMR ROESY experiments. The results are discussed in terms of their potential interactions with export proteins like P-glycoprotein.


Assuntos
Clozapina/química , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Conformação Molecular , Simulação de Dinâmica Molecular , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Prótons , Termodinâmica
11.
FEBS Lett ; 589(5): 659-65, 2015 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-25647032

RESUMO

The sigma-1 receptor (S1R) is a ligand-regulated membrane chaperone protein associated with endoplasmic reticulum stress response, and modulation of ion channel activities at the plasma membrane. We report here a solution NMR study of a S1R construct (S1R(Δ35)) in which only the first transmembrane domain and the eight-residue N-terminus have been removed. The second transmembrane helix is found to be composed of residues 91-107, which corresponds to the first steroid binding domain-like region. The cytosolic domain is found to contain three helices, and the secondary structure and backbone dynamics of the chaperone domain are consistent with that determined previously for the chaperone domain alone. The position of TM2 provides a framework for ongoing studies of S1R ligand binding and oligomerisation.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Receptores sigma/química , Dicroísmo Circular , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor Sigma-1
12.
Proc Natl Acad Sci U S A ; 112(7): E639-48, 2015 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-25646439

RESUMO

The integral membrane proteins of the DP1 (deleted in polyposis) and reticulon families are responsible for maintaining the high membrane curvature required for both smooth endoplasmic reticulum (ER) tubules and the edges of ER sheets, and mutations in these proteins lead to motor neuron diseases, such as hereditary spastic paraplegia. Reticulon/DP1 proteins contain reticulon homology domains (RHDs) that have unusually long hydrophobic segments and are proposed to adopt intramembrane helical hairpins that stabilize membrane curvature. We have characterized the secondary structure and dynamics of the DP1 family protein produced from the YOP1 gene (Yop1p) and identified a C-terminal conserved amphipathic helix (APH) that, on its own, interacts strongly with negatively charged membranes and is necessary for membrane tubule formation. Analyses of DP1 and reticulon family members indicate that most, if not all, contain C-terminal sequences capable of forming APHs. Together, these results indicate that APHs play a previously unrecognized role in RHD membrane curvature stabilization.


Assuntos
Proteínas de Escherichia coli/fisiologia , Biopolímeros , Membrana Celular/fisiologia , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Micelas , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína
13.
J Biol Chem ; 289(20): 13876-89, 2014 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-24668806

RESUMO

Endoglycosidase S (EndoS) is a glycoside-hydrolase secreted by the bacterium Streptococcus pyogenes. EndoS preferentially hydrolyzes the N-linked glycans from the Fc region of IgG during infection. This hydrolysis impedes Fc functionality and contributes to the immune evasion strategy of S. pyogenes. Here, we investigate the mechanism of human serum IgG deactivation by EndoS. We expressed fragments of IgG1 and demonstrated that EndoS was catalytically active against all of them including the isolated CH2 domain of the Fc domain. Similarly, we sought to investigate which domains within EndoS could contribute to activity. Bioinformatics analysis of the domain organization of EndoS confirmed the previous predictions of a chitinase domain and leucine-rich repeat but also revealed a putative carbohydrate binding module (CBM) followed by a C-terminal region. Using expressed fragments of EndoS, circular dichroism of the isolated CBM, and a CBM-C-terminal region fusion revealed folded domains dominated by ß sheet and α helical structure, respectively. Nuclear magnetic resonance analysis of the CBM with monosaccharides was suggestive of carbohydrate binding functionality. Functional analysis of truncations of EndoS revealed that, whereas the C-terminal of EndoS is dispensable for activity, its deletion impedes the hydrolysis of IgG glycans.


Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Streptococcus pyogenes/enzimologia , Glicosídeo Hidrolases/genética , Glicosilação , Células HEK293 , Humanos , Fragmentos de Imunoglobulinas/química , Modelos Moleculares , Polissacarídeos/metabolismo , Estrutura Terciária de Proteína , Deleção de Sequência
14.
Biochemistry ; 52(47): 8420-9, 2013 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-24168642

RESUMO

The protein M2 from influenza is a tetrameric membrane protein with several roles in the viral life cycle. The transmembrane helix (TMH) of M2 has proton channel activity that is required for unpackaging the viral genome. Additionally a C-terminal juxtamembrane region includes an amphipathic helix (APH) important for virus budding and scission. The APH interacts with membranes and is required for M2 localization to the site of viral budding. As a step toward obtaining high resolution information on the structure and lipid interactions of the M2 APH, we sought to develop a fast tumbling bicelle system, which would make studies of M2 in a membrane-like environment by solution NMR possible. Since M2 is highly sensitive to the solubilizing environment, an M2 construct containing the APH was studied under micelle and bicelle conditions while maintaining the same detergent and lipid headgroup chemistry to facilitate interpretation of the spectroscopic results. The sequence from a human H1N1 "swine flu" isolate was used to design an M2 construct (swM2) similar in amino acid sequence to currently circulating viruses. Comparison of swM2 solubilized in either the diacyl detergent 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) or a mixture of DHPC and the lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (q = 0.4) indicated that the largest changes were a decrease in helicity at the N-terminus of the TMH and a decrease in dynamics for the juxtamembrane linker residues connecting the TMH and the APH. Whereas the linker region is very dynamic and the amide protons are rapidly exchanged with water protons in micelles, the dynamics and water exchange are largely suppressed in the presence of lipid. Chemical shift changes and relaxation measurements were consistent with an overall stabilization of the linker region, with only modest changes in conformation or environment of the APH itself. Such changes are consistent with differences observed in structures of M2 in lipid bilayers and detergent micelles, indicating that the bicelle system provides a more membrane-like environment.


Assuntos
Canais Iônicos/química , Bicamadas Lipídicas/química , Modelos Moleculares , Proteínas da Matriz Viral/química , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Reagentes de Ligações Cruzadas/química , Emulsões , Vírus da Influenza A Subtipo H1N1/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , Bicamadas Lipídicas/metabolismo , Micelas , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Conformação Proteica , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tensoativos/química , Tensoativos/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
15.
J Biol Chem ; 288(29): 21448-21457, 2013 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-23760505

RESUMO

The sigma-1 receptor (S1R) is a ligand-regulated membrane protein chaperone involved in the ER stress response. S1R activity is implicated in diseases of the central nervous system including amnesia, schizophrenia, depression, Alzheimer disease, and addiction. S1R has been shown previously to regulate the Hsp70 binding immunoglobulin protein (BiP) and the inositol triphosphate receptor calcium channel through a C-terminal domain. We have developed methods for bacterial expression and reconstitution of the chaperone domain of human S1R into detergent micelles that enable its study by solution NMR spectroscopy. The chaperone domain is found to contain a helix at the N terminus followed by a largely dynamic region and a structured, helical C-terminal region that encompasses a membrane associated domain containing four helices. The helical region at residues ∼198-206 is strongly amphipathic and proposed to anchor the chaperone domain to micelles and membranes. Three of the helices in the C-terminal region closely correspond to previously identified cholesterol and drug recognition sites. In addition, it is shown that the chaperone domain interacts with full-length BiP or the isolated nucleotide binding domain of BiP, but not the substrate binding domain, suggesting that the nucleotide binding domain is sufficient for S1R interactions.


Assuntos
Proteínas de Choque Térmico/metabolismo , Receptores sigma/química , Receptores sigma/metabolismo , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Chaperona BiP do Retículo Endoplasmático , Humanos , Espectroscopia de Ressonância Magnética , Micelas , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Prótons , Água/química , Receptor Sigma-1
16.
Biochemistry ; 52(27): 4605-19, 2013 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-23758161

RESUMO

Correlated networks of amino acids have been proposed to play a fundamental role in allostery and enzyme catalysis. These networks of amino acids can be traced from surface-exposed residues all the way into the active site, and disruption of these networks can decrease enzyme activity. Substitution of the distal Gly121 residue in Escherichia coli dihydrofolate reductase results in an up to 200-fold decrease in the hydride transfer rate despite the fact that the residue is located 15 Å from the active-site center. In this study, nuclear magnetic resonance relaxation experiments are used to demonstrate that dynamics on the picosecond to nanosecond and microsecond to millisecond time scales are changed significantly in the G121V mutant of dihydrofolate reductase. In particular, picosecond to nanosecond time scale dynamics are decreased in the FG loop (containing the mutated residue at position 121) and the neighboring active-site loop (the Met20 loop) in the mutant compared to those of the wild-type enzyme, suggesting that these loops are dynamically coupled. Changes in methyl order parameters reveal a pathway by which dynamic perturbations can be propagated more than 25 Å across the protein from the site of mutation. All of the enzyme complexes, including the model Michaelis complex with folate and nicotinamide adenine dinucleotide phosphate bound, assume an occluded ground-state conformation, and we do not observe sampling of a higher-energy closed conformation by (15)N R2 relaxation dispersion experiments. This is highly significant, because it is only in the closed conformation that the cofactor and substrate reactive centers are positioned for reaction. The mutation also impairs microsecond to millisecond time scale fluctuations that have been implicated in the release of product from the wild-type enzyme. Our results are consistent with an important role for Gly121 in controlling protein dynamics critical for enzyme function and further validate the dynamic energy landscape hypothesis of enzyme catalysis.


Assuntos
Aminoácidos/química , Mutação , Tetra-Hidrofolato Desidrogenase/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Tetra-Hidrofolato Desidrogenase/genética
17.
Proc Natl Acad Sci U S A ; 110(12): E1092-101, 2013 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-23471988

RESUMO

The twin-arginine translocase (Tat) carries out the remarkable process of translocating fully folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Tat is required for bacterial pathogenesis and for photosynthesis in plants. TatA, the protein-translocating element of the Tat system, is a small transmembrane protein that assembles into ring-like oligomers of variable size. We have determined a structural model of the Escherichia coli TatA complex in detergent solution by NMR. TatA assembly is mediated entirely by the transmembrane helix. The amphipathic helix extends outwards from the ring of transmembrane helices, permitting assembly of complexes with variable subunit numbers. Transmembrane residue Gln8 points inward, resulting in a short hydrophobic pore in the center of the complex. Simulations of the TatA complex in lipid bilayers indicate that the short transmembrane domain distorts the membrane. This finding suggests that TatA facilitates protein transport by sensitizing the membrane to transient rupture.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Bicamadas Lipídicas/química , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Multimerização Proteica , Transporte Biológico Ativo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Plantas/química , Plantas/genética , Plantas/metabolismo , Estrutura Quaternária de Proteína , Tilacoides/química , Tilacoides/genética , Tilacoides/metabolismo
18.
Methods Mol Biol ; 831: 165-79, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22167674

RESUMO

Advances in solution nuclear magnetic resonance (NMR) methodology that enable studies of very large proteins have also paved the way for studies of membrane proteins that behave like large proteins due to the added weight of surfactants. Solution NMR has been used to determine the high-resolution structures of several small, membrane proteins dissolved in detergent micelles and small bicelles. However, the usual difficulties with membrane proteins in producing, purifying, and stabilizing the proteins away from native membranes remain, requiring intensive screening efforts. Low levels of heterologous expression can be the most detrimental aspect to studying membrane proteins. This is exacerbated for NMR studies because of the costs of isotopically enriched media. Thus, solution NMR studies have tended to focus on relatively small, membrane proteins that can be expressed into inclusion bodies and refolded. Here, we describe the methods used to produce, purify, and refold the proton channel M2 into detergent micelles, and the procedures used to determine chemical shift assignments and the atomic level structure of the closed form of the homotetrameric channel.


Assuntos
Reatores Biológicos , Escherichia coli/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes de Fusão/química , Proteínas da Matriz Viral/química , Isótopos de Carbono/metabolismo , Isótopos de Nitrogênio/metabolismo , Conformação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Triptofano/química , Triptofano/metabolismo , Proteínas da Matriz Viral/biossíntese , Proteínas da Matriz Viral/isolamento & purificação , Proteínas da Matriz Viral/metabolismo
19.
Proc Natl Acad Sci U S A ; 106(18): 7379-84, 2009 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-19383794

RESUMO

The influenza A virus M2 proton channel equilibrates pH across the viral membrane during entry and across the trans-Golgi membrane of infected cells during viral maturation. It is an important target of adamantane-family antiviral drugs, but drug resistance has become a critical problem. Two different sites for drug interaction have been proposed. One is a lipid-facing pocket between 2 adjacent transmembrane helices (around Asp-44), at which the drug binds and inhibits proton conductance allosterically. The other is inside the pore (around Ser-31), at which the drug directly blocks proton passage. Here, we describe structural and functional experiments on the mechanism of drug inhibition and resistance. The solution structure of the S31N drug-resistant mutant of M2, a mutant of the highly pathogenic avian influenza subtype H5N1, shows that replacing Ser-31 with Asn has little effect on the structure of the channel pore, but dramatically reduces drug binding to the allosteric site. Mutagenesis and liposomal proton flux assays show that replacing the key residue (Asp-44) in the lipid-facing binding pocket with Ala has a dramatic effect on drug sensitivity, but that the channel remains fully drug sensitive when replacing Ser-31 with Ala. Chemical cross-linking studies indicate an inverse correlation between channel stability and drug resistance. The lipid-facing pocket contains residues from 2 adjacent channel-forming helices. Therefore, it is present only when the helices are tightly packed in the closed conformation. Thus, drug-resistant mutants impair drug binding by destabilizing helix-helix assembly.


Assuntos
Adamantano/farmacologia , Antivirais/farmacologia , Farmacorresistência Viral , Vírus da Influenza A/efeitos dos fármacos , Proteínas da Matriz Viral/antagonistas & inibidores , Adamantano/química , Antivirais/química , Sítios de Ligação , Farmacorresistência Viral/genética , Humanos , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína/efeitos dos fármacos , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética
20.
Cell ; 135(4): 702-13, 2008 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-19013279

RESUMO

Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live-cell imaging studies showed a close interaction of the CD3epsilon cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3epsilon residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3epsilon ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation.


Assuntos
Complexo CD3/metabolismo , Membrana Celular/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Tirosina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Citoplasma/metabolismo , Transferência Ressonante de Energia de Fluorescência , Bicamadas Lipídicas/química , Lipídeos/química , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Quinases da Família src/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...