RESUMEN
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.
Asunto(s)
Canales Iónicos/química , Membrana Dobles de Lípidos/química , Modelos Moleculares , Proteínas de la Matriz Viral/química , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Reactivos de Enlaces Cruzados/química , Emulsiones , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Canales Iónicos/genética , Canales Iónicos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Micelas , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Conformación Proteica , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tensoactivos/química , Tensoactivos/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismoRESUMEN
To perform their physiological functions, amino methyl propionic acid receptors (AMPARs) cycle through active, resting, and desensitized states, and dysfunction in AMPAR activity is associated with various neurological disorders. Transitions among AMPAR functional states, however, are largely uncharacterized at atomic resolution and are difficult to examine experimentally. Here, we report long-timescale molecular dynamics simulations of dimerized AMPAR ligand-binding domains (LBDs), whose conformational changes are tightly coupled to changes in AMPAR functional states, in which we observed LBD dimer activation and deactivation upon ligand binding and unbinding at atomic resolution. Importantly, we observed the ligand-bound LBD dimer transition from the active conformation to several other conformations, which may correspond with distinct desensitized conformations. We also identified a linker region whose structural rearrangements heavily affected the transitions to and among these putative desensitized conformations, and confirmed, using electrophysiology experiments, the importance of the linker region in these functional transitions.
Asunto(s)
Simulación de Dinámica Molecular , Receptores AMPA , Receptores AMPA/química , Ligandos , Dominios Proteicos , DimerizaciónRESUMEN
The sulphonylurea receptor (SUR1) subunit of the ATP-sensitive potassium (KATP) channel is a member of the ATP-binding cassette (ABC) protein family. Binding of MgADP to nucleotide-binding domain 2 (NBD2) is critical for channel activation.We identified a residue in NBD2 (G1401) that is fully conserved among ABC proteins and whose functional importance is unknown. Homology modelling places G1401 on the outer surface of the protein, distant from the nucleotide-binding site. The ATPase activity of purified SUR1-NBD2-G1410R (bound to maltose-binding protein) was slightly inhibited when compared to the wild-type protein, but its inhibition by MgADP was unchanged, indicating that MgADP binding is not altered. However, MgADP activation of channel activity was abolished. This implies that the G1401R mutation impairs the mechanism by which MgADP binding to NBD2 is translated into opening of the KATP channel pore. The location of G1401 would be consistent with interaction of this residue with the pore-forming Kir6.2 subunit. Channel activity in the presence of MgATP reflects the balance between the stimulatory (at SUR1) and inhibitory (at Kir6.2) effects of nucleotides. Mutant channels were 2.5-fold less sensitive to MgATP inhibition and not activated by MgATP. This suggests that ATP block of the channel is reduced by the SUR1 mutation. Interestingly, this effect was dependent on the functional integrity of the NBDs. These results therefore suggest that SUR1 modulates both nucleotide inhibition and activation of the KATP channel.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/fisiología , Activación del Canal Iónico/fisiología , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/fisiología , Receptores de Droga/química , Receptores de Droga/fisiología , Adenosina Trifosfatasas/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Secuencia Conservada , Humanos , Técnicas In Vitro , Proteínas de Unión a Maltosa/química , Datos de Secuencia Molecular , Mutación , Nucleótidos/fisiología , Oocitos/fisiología , Ratas , Alineación de Secuencia , Receptores de Sulfonilureas , Xenopus laevisRESUMEN
Infection with the hepatitis C virus (HCV) has a huge impact on global health putting more than 170 million people at risk of developing severe liver disease. The HCV encoded p7 ion channel is essential for the production of infectious viruses. Despite a growing body of functional data, little is known about the 3-dimensional (3D) structure of the channel. Here, we present the 3D structure of a full-length viroporin, the detergent-solubilized hexameric 42 kDa form of the HCV p7 ion channel, as determined by single-particle electron microscopy using the random conical tilting approach. The reconstruction of such a small protein complex was made possible by a combination of high-contrast staining, the symmetry, and the distinct structural features of the channel. The orientation of the p7 monomers within the density was established using immunolabeling with N and C termini specific F(ab) fragments. The density map at a resolution of approximately 16 A reveals a flower-shaped protein architecture with protruding petals oriented toward the ER lumen. This broadest part of the channel presents a comparatively large surface area providing potential interaction sites for cellular and virally encoded ER resident proteins.
Asunto(s)
Proteínas Virales/química , Imagenología Tridimensional , Microscopía Electrónica , Microscopía Inmunoelectrónica , Modelos MolecularesRESUMEN
ABC transporters are a large family of membrane proteins involved in a variety of cellular processes, including multidrug and tumor resistance and ion channel regulation. Advances in the structural and functional understanding of ABC transporters have revealed that hydrolysis at the two canonical nucleotide-binding sites (NBSs) is co-operative and non-simultaneous. A conserved core architecture of bacterial and eukaryotic ABC exporters has been established, as exemplified by the crystal structure of the homodimeric multidrug exporter Sav1866. Currently, it is unclear how sequential ATP hydrolysis arises in a symmetric homodimeric transporter, since it implies at least transient asymmetry at the NBSs. We show by molecular dynamics simulation that the initially symmetric structure of Sav1866 readily undergoes asymmetric transitions at its NBSs in a pre-hydrolytic nucleotide configuration. MgATP-binding residues and a network of charged residues at the dimer interface are shown to form a sequence of putative molecular switches that allow ATP hydrolysis only at one NBS. We extend our findings to eukaryotic ABC exporters which often consist of two non-identical half-transporters, frequently with degeneracy substitutions at one of their two NBSs. Interestingly, many residues involved in asymmetric conformational switching in Sav1866 are substituted in degenerate eukaryotic NBS. This finding strengthens recent suggestions that the interplay of a consensus and a degenerate NBS in eukaroytic ABC proteins pre-determines the sequence of hydrolysis at the two NBSs.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Simulación de Dinámica Molecular , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Simulación por Computador , Farmacorresistencia Bacteriana Múltiple , Metabolismo de los Lípidos , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Activating mutations in the pore-forming Kir6.2 (KCNJ11) and regulatory sulphonylurea receptor SUR1 (ABCC8) subunits of the K(ATP) channel are a common cause of transient neonatal diabetes mellitus (TNDM). We identified a new TNDM mutation (R826W) in the first nucleotide-binding domain (NBD1) of SUR1. The mutation was found in a region that heterodimerizes with NBD2 to form catalytic site 2. Functional analysis showed that this mutation decreases MgATP hydrolysis by purified maltose-binding protein MBP-NBD1 fusion proteins. Inhibition of ATP hydrolysis by MgADP or BeF was not changed. The results indicate that the ATPase cycle lingers in the post-hydrolytic MgADP.P(i)-bound state, which is associated with channel activation. The extent of MgADP-dependent activation of K(ATP) channel activity was unaffected by the R826W mutation, but the time course of deactivation was slowed. Channel inhibition by MgATP was reduced, leading to an increase in resting whole-cell currents. In pancreatic beta cells, this would lead to less insulin secretion and thereby diabetes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/metabolismo , Diabetes Mellitus/enzimología , Diabetes Mellitus/genética , Enfermedades del Recién Nacido/genética , Mutación/genética , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/genética , Receptores de Droga/química , Receptores de Droga/genética , Adenosina Difosfato/farmacología , Adenosina Trifosfatasas/química , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Arginina/genética , Niño , Humanos , Recién Nacido , Enfermedades del Recién Nacido/enzimología , Activación del Canal Iónico/efectos de los fármacos , Cinética , Masculino , Datos de Secuencia Molecular , Proteínas Mutantes/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Sulfonilureas , Triptófano/genéticaRESUMEN
Gain-of-function mutations in the genes encoding the ATP-sensitive potassium (K(ATP)) channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) are a common cause of neonatal diabetes mellitus. Here we investigate the molecular mechanism by which two heterozygous mutations in the second nucleotide-binding domain (NBD2) of SUR1 (R1380L and R1380C) separately cause neonatal diabetes. SUR1 is a channel regulator that modulates the gating of the pore formed by Kir6.2. K(ATP) channel activity is inhibited by ATP binding to Kir6.2 but is stimulated by MgADP binding, or by MgATP binding and hydrolysis, at the NBDs of SUR1. Functional analysis of purified NBD2 showed that each mutation enhances MgATP hydrolysis by purified isolated fusion proteins of maltose-binding protein and NBD2. Inhibition of ATP hydrolysis by MgADP was unaffected by mutation of R1380, but inhibition by beryllium fluoride (which traps the ATPase cycle in the prehydrolytic state) was reduced. MgADP-dependent activation of K(ATP) channel activity was unaffected. These data suggest that the R1380L and R1380C mutations enhance the off-rate of P(i), thereby enhancing the hydrolytic rate. Molecular modeling studies supported this idea. Because mutant channels were inhibited less strongly by MgATP, this would increase K(ATP) currents in pancreatic beta cells, thus reducing insulin secretion and producing diabetes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Diabetes Mellitus Tipo 1/genética , Mutación Missense , Mutación Puntual , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio/genética , Receptores de Droga/genética , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Difosfato/farmacología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Sustitución de Aminoácidos , Arginina/química , Berilio/farmacología , Sitios de Unión , Diabetes Mellitus Tipo 1/congénito , Fluoruros/farmacología , Humanos , Hidrólisis , Recién Nacido , Insulina/metabolismo , Secreción de Insulina , Activación del Canal Iónico/efectos de los fármacos , Cinética , Modelos Moleculares , Potasio/metabolismo , Canales de Potasio/química , Canales de Potasio/metabolismo , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/metabolismo , Estructura Terciaria de Proteína/genética , Receptores de Droga/antagonistas & inhibidores , Receptores de Droga/química , Receptores de Droga/metabolismo , Relación Estructura-Actividad , Receptores de SulfonilureasRESUMEN
Using extensive atomistic simulations, we show that there is a single experimentally accessible parameter--the sterol tilt--that can be used to determine a sterol's capability to induce order, and thus to promote, e.g., formation of lipid rafts. The observations also facilitate designing new effective sterols.
Asunto(s)
Membranas Artificiales , Esteroles/químicaRESUMEN
The ATP-sensitive potassium (K(ATP)) channel is a hetero-octameric complex that links cell metabolism to membrane electrical activity in many cells, thereby controlling physiological functions such as insulin release, muscle contraction and neuronal activity. It consists of four pore-forming Kir6.2 and four regulatory sulfonylurea receptor (SUR) subunits. SUR2B serves as the regulatory subunit in smooth muscle and some neurones. An integrative approach, combining electron microscopy and homology modelling, has been used to obtain information on the structure of this large (megadalton) membrane protein complex. Single-particle electron microscopy of purified SUR2B tethered to a lipid monolayer revealed that it assembles as a tetramer of four SUR2B subunits surrounding a central hole. In the absence of an X-ray structure, a homology model for SUR2B based on the X-ray structure of the related ABC transporter Sav1866 was used to fit the experimental images. The model indicates that the central hole can readily accommodate the transmembrane domains of the Kir tetramer, suggests a location for the first transmembrane domains of SUR2B (which are absent in Sav1866) and suggests the relative orientation of the SUR and Kir6.2 subunits.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/ultraestructura , Canales de Potasio de Rectificación Interna/ultraestructura , Receptores de Droga/ultraestructura , Transportadoras de Casetes de Unión a ATP/química , Animales , Modelos Moleculares , Canales de Potasio de Rectificación Interna/química , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Ratas , Receptores de Droga/química , Células Sf9 , Homología Estructural de Proteína , Receptores de SulfonilureasRESUMEN
SUR1 is an ATP-binding cassette (ABC) transporter with a novel function. In contrast to other ABC proteins, it serves as the regulatory subunit of an ion channel. The ATP-sensitive (KATP) channel is an octameric complex of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits, and it links cell metabolism to electrical activity in many cell types. ATPase activity at the nucleotide-binding domains of SUR results in an increase in KATP channel open probability. Conversely, ATP binding to Kir6.2 closes the channel. Metabolic regulation is achieved by the balance between these two opposing effects. Precisely how SUR1 talks to Kir6.2 remains unclear, but recent studies have identified some residues and domains that are involved in both physical and functional interactions between the two proteins. The importance of these interactions is exemplified by the fact that impaired regulation of Kir6.2 by SUR1 results in human disease, with loss-of-function SUR1 mutations causing congenital hyperinsulinism and gain-of-function SUR1 mutations leading to neonatal diabetes. This paper reviews recent data on the regulation of Kir6.2 by SUR1 and considers the molecular mechanisms by which SUR1 mutations produce disease.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Receptores de Droga/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Diabetes Mellitus/genética , Humanos , Modelos Moleculares , Mutación , Canales de Potasio de Rectificación Interna/genética , Conformación Proteica , Receptores de Droga/genética , Receptores de SulfonilureasRESUMEN
The effects of cholesterol (Chol) on phospholipid bilayers include ordering of the fatty acyl chains, condensing of the lipids in the bilayer plane, and promotion of the liquid-ordered phase. These effects depend on the type of phospholipids in the bilayer and are determined by the nature of the underlying molecular interactions. As for Chol, it has been shown to interact more favorably with sphingomyelin than with most phosphatidylcholines, which in given circumstances leads to formation of lateral domains. However, the exact origin and nature of Chol-phospholipid interactions have recently been subjects of speculation. We examine interactions between Chol, palmitoylsphingomyelin (PSM) and palmitoyl-oleoyl-phosphatidylcholine (POPC) in hydrated lipid bilayers by extensive atom-scale molecular dynamics simulations. We employ a tailored lipid configuration: Individual PSM and Chol monomers, as well as PSM-Chol dimers, are embedded in a POPC lipid bilayer in the liquid crystalline phase. Such a setup allows direct comparison of dimeric and monomeric PSMs and Chol, which ultimately shows how the small differences in PSM and POPC structure can lead to profoundly different interactions with Chol. Our analysis shows that direct hydrogen bonding between PSM and Chol does not provide an adequate explanation for their putative specific interaction. Rather, a combination of charge-pairing, hydrophobic, and van der Waals interactions leads to a lower tilt in PSM neighboring Chol than in Chol with only POPC neighbors. This implies improved Chol-induced ordering of PSM's chains over POPC's chains. These findings are discussed in the context of the hydrophobic mismatch concept suggested recently.