RESUMEN
Neurotransmitter:sodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of small-molecule neurotransmitters from the synaptic cleft. The symporters for the biogenic amines serotonin (SERT), dopamine (DAT), and norepinephrine (NET) are targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric ailments. LeuT, a thermostable eubacterial NSS homolog, has been exploited as a model protein for NSS members to canvass the conformational mechanism of transport with a combination of X-ray crystallography, cysteine accessibility, and solution spectroscopy. Despite yielding remarkable insights, these studies have primarily been conducted with protein in the detergent-solubilized state rather than embedded in a membrane mimic. In addition, solution spectroscopy has required site-specific labeling of nonnative cysteines, a labor-intensive process occasionally resulting in diminished transport and/or binding activity. Here, we overcome these limitations by reconstituting unlabeled LeuT in phospholipid bilayer nanodiscs, subjecting them to hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), and facilitating interpretation of the data with molecular dynamics simulations. The data point to changes of accessibility and dynamics of structural elements previously implicated in the transport mechanism, in particular transmembrane helices (TMs) 1a and 7 as well as extracellular loops (ELs) 2 and 4. The results therefore illuminate the value of this strategy for interrogating the conformational mechanism of the more clinically significant mammalian membrane proteins including SERT and DAT, neither of which tolerates complete removal of endogenous cysteines, and whose activity is heavily influenced by neighboring lipids.
Asunto(s)
Dopamina/química , Neurotransmisores/química , Serotonina/química , Proteínas Cotransportadoras de Sodio-Fosfato/química , Aminas Biogénicas/química , Aminas Biogénicas/metabolismo , Cristalografía por Rayos X , Cisteína/química , Dopamina/metabolismo , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Simulación de Dinámica Molecular , Neurotransmisores/metabolismo , Norepinefrina/química , Norepinefrina/metabolismo , Serotonina/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato/metabolismoRESUMEN
The GABA transporter GAT-1 mediates electrogenic transport of its substrate together with sodium and chloride. It is a member of the neurotransmitter:sodium:symporters, which are crucial for synaptic transmission. Compared with all other neurotransmitter:sodium:symporters, GAT-1 and other members of the GABA transporter subfamily all contain an extra amino acid residue at or near a conserved glycine in transmembrane segment 10. Therefore, we studied the functional impact of deletion and replacement mutants of Gly-457 and its two adjacent residues in GAT-1. The glycine replacement mutants were devoid of transport activity, but remarkably the deletion mutant was active, as were mutants obtained by deleting positions on either side of Gly-457. However, the inward rectification of GABA-induced transport currents by all three deletion mutants was diminished, and the charge-to-flux ratio was increased by more than 2.5-fold, both of which indicate substantial uncoupled transport. These observations suggest that the deletions render the transporters less tightly packed. Consistent with this interpretation, the inactive G457A mutant was partially rescued by removing the adjacent serine residue. Moreover, the activity of several gating mutants was also partially rescued upon deletion of Gly-457. Structural modeling showed that the stretch surrounding Gly-457 is likely to form a π-helix. Our data indicate that the "extra" residue in transmembrane domain 10 of the GABA transporter GAT-1 provides extra bulk, probably in the form of a π-helix, which is required for stringent gating and tight coupling of ion and substrate fluxes in the GABA transporter family.
Asunto(s)
Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Glicina/genética , Transporte Iónico , Mutagénesis Sitio-Dirigida , Aminoácidos , Secuencia Conservada/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Células HeLa , Humanos , Conformación Proteica , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
The serotonin transporter (SERT) is an integral membrane protein that exploits preexisting sodium-, chloride-, and potassium ion gradients to catalyze the thermodynamically unfavorable movement of synaptic serotonin into the presynaptic neuron. SERT has garnered significant clinical attention partly because it is the target of multiple psychoactive agents, including the antidepressant paroxetine (Paxil), the most potent selective serotonin reuptake inhibitor known. However, the binding site and orientation of paroxetine in SERT remain controversial. To provide molecular insight, we constructed SERT homology models based on the Drosophila melanogaster dopamine transporter and docked paroxetine to these models. We tested the predicted binding configurations with a combination of radioligand binding and flux assays on wild-type and mutant SERTs. Our data suggest that the orientation of paroxetine, specifically its fluorophenyl ring, in SERT's substrate binding site directly depends on this pocket's charge distribution, and thereby provide an avenue toward understanding and enhancing high-affinity antidepressant activity.
Asunto(s)
Paroxetina/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células Cultivadas , Pollos , Cocaína/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Modelos Moleculares , Conformación Molecular , Simulación del Acoplamiento Molecular , Paroxetina/química , Conformación Proteica , Ensayo de Unión Radioligante , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serotonina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Inhibidores Selectivos de la Recaptación de Serotonina/químicaRESUMEN
In LeuT, a prokaryotic homolog of neurotransmitter transporters, Na(+) stabilizes outward-open conformational states. We examined how each of the two LeuT Na(+) binding sites contributes to Na(+)-dependent closure of the cytoplasmic pathway using biochemical and biophysical assays of conformation. Mutating either of two residues that contribute to the Na2 site completely prevented cytoplasmic closure in response to Na(+), suggesting that Na2 is essential for this conformational change, whereas Na1 mutants retained Na(+) responsiveness. However, mutation of Na1 residues also influenced the Na(+)-dependent conformational change in ways that varied depending on the position mutated. Computational analyses suggest those mutants influence the ability of Na1 binding to hydrate the substrate pathway and perturb an interaction network leading to the extracellular gate. Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na(+) and transmembrane helices 1 and 8, whereas Na(+) binding at Na1 influences conformational change through a network of intermediary interactions. The results also provide evidence that N-terminal release and helix motions represent distinct steps in cytoplasmic pathway opening.
Asunto(s)
Sistemas de Transporte de Aminoácidos/química , Organismos Acuáticos/metabolismo , Proteínas Bacterianas/química , Bacterias Gramnegativas/metabolismo , Modelos Moleculares , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/química , Sodio/metabolismo , Sustitución de Aminoácidos , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cisteína/química , Ligandos , Liposomas , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mutación , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/genética , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/metabolismo , Conformación Proteica , Pliegue de Proteína , Estabilidad Proteica , Proteolípidos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Huntington's disease (HD) is caused by the presence of an extended polyglutamine (polyQ) region at the N-terminus of the huntingtin (htt) protein. The presence of flanking sequences adjacent to the polyQ region has been reported to modulate the effects of potentially toxic protein-membrane interactions. In this study, we consider four peptide systems with various combinations of flanking sequences (KKQ35KK, KKQ35P11KK, N17Q35KK, N17Q35P11KK) and use atomistic molecular dynamics simulations to study the interactions with a DOPC lipid bilayer. We observe significant membrane thinning, disorderliness of lipid molecules, and compensation effects between the top and the bottom leaflets of the bilayer depending on the presence of particular flanking sequences. Overall, we find that the presence of the N-17 flanking sequence is crucial for membrane interactions. Polyproline decreases the interaction with the membrane in the absence of N-17, but enhances it when present along N-17.
Asunto(s)
Membrana Dobles de Lípidos/química , Péptidos/química , Secuencia de Aminoácidos , Enlace de Hidrógeno , Membrana Dobles de Lípidos/metabolismo , Simulación de Dinámica Molecular , Péptidos/metabolismo , Fosfatidilcolinas/química , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
We present a multiresolution simulation scheme for the solvent environment where four atomistic water molecules are mapped onto one coarse-grained bead. Soft restraining potentials are used to allow a resolution exchange of four water molecules into a single coarse-grained site. We first study the effect of adding restraining potentials in liquid water using full all-atom simulations. The usage of very soft restraining potentials to bundle four nearest neighbor water molecules does not disrupt the hydrogen bonding patterns in the liquid water. The structural properties of the first solvation shell around hydrophobic, hydrophilic, and ionic solutes are well preserved when soft restraining potentials are added. By modeling a bundle of four water molecules as a single molecule, a smooth transition and free exchange between coarse-grained and all-atom resolution is possible by using the adaptive resolution scheme (AdResS).
RESUMEN
SERCA is a membrane transport protein that has been extensively studied. There are a large number of highly resolved X-ray structures and several hundred mutations that have been characterized functionally. Despite this, the molecular details of the catalytic cycle, a cycle that includes large conformational changes, is not fully understood. In this computational study, we provide molecular dynamics descriptions of conformational changes during the E2âE1 transitions. The motivating point for these calculations was a series of insertion mutants in the A-M3 linker region that led to significant shifts in measured rates between the E2 and E1 states, as shown by experimental characterization. Using coarse-grained dynamic importance sampling within the context of a population shift framework, we sample on the intermediates along the transition pathway to address the mechanism for the conformational changes and the effects of the insertion mutations on the kinetics of the transition. The calculations define an approximation for the relative changes in entropy and enthalpy along the transition. These are found to be important for understanding the experimentally observed differences in rates. In particular, the interactions between cytoplasmic domains, water interactions, and the shifts in protein degrees of freedom with the insertion mutations show mutual compensation for the E2âE1 transitions in wild-type and mutant systems.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Animales , Calcio/metabolismo , Entropía , Cinética , Proteínas de la Membrana/química , Proteínas de Transporte de Membrana/química , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Conejos , TermodinámicaRESUMEN
SERCA is an important model system for understanding the molecular details of conformational change in membrane transport systems. This reflects the large number of solved X-ray structures and the equally large database of mutations that have been assayed. In this computational study, we provide a molecular dynamics description of the conformational changes during the E1P â E2P transitions. This set of states further changes with insertion mutants in the A-M3 linker region. These mutants were experimentally shown to lead to significant shifts in rates between the E1P â E2P states. Using the population shift framework and dynamic importance sampling method along with coarse-grained representations of the protein, lipid, and water, we suggest why these changes are found. The calculations sample on intermediates and suggest that changes in interactions, individual helix interactions, and water behavior are key elements in the molecular compositions that underlie shifts in kinetics. In particular, as the insertion length grows, it attracts more water and disrupts domain interactions, creating changes as well at the sites of key helix interactions between the A-Domain and the P-Domain. This provides a conceptual picture that aids understanding of the experimental results.