RESUMEN
DNA replication, transcription, and translation in eukaryotic cells occur with decreasing but still high fidelity. In contrast, for the estimated 33% of the human proteome that is inserted as transmembrane (TM) proteins, insertion with a non-functional inverted topology is frequent. Correct topology is essential for function and trafficking to appropriate cellular compartments and is controlled principally by responses to charged residues within 15 residues of the inserted TM domain (TMD); the flank with the higher positive charge remains in the cytosol (inside), following the positive inside rule (PIR). Yeast (Saccharomyces cerevisiae) mutants that increase insertion contrary to the PIR were selected. Mutants with strong phenotypes were found only in SPF1 and STE24 (human cell orthologs are ATP13A1 and ZMPSte24) with, at the time, no known relevant functions. Spf1/Atp13A1 is now known to dislocate to the cytosol TM proteins inserted contrary to the PIR, allowing energy-conserving reinsertion. We hypothesize that Spf1 and Ste24 both recognize the short, positively charged ER luminal peptides of TM proteins inserted contrary to the PIR, accepting these peptides into their large membrane-spanning, water-filled cavities through interaction with their many interior surface negative charges. While entry was demonstrated for Spf1, no published evidence directly demonstrates substrate entry to the Ste24 cavity, internal access to its zinc metalloprotease (ZMP) site, or active withdrawal of fragments, which may be essential for function. Spf1 and Ste24 comprise a PIR quality control system that is conserved in all eukaryotes and presumably evolved in prokaryotic progenitors as they gained differentiated membrane functions. About 75% of the PIR is imposed by this quality control system, which joins the UPR, ERAD, and autophagy (ER-phagy) in coordinated, overlapping quality control of ER protein function.
RESUMEN
The human ERG (hERG) K+ channel has a crucial function in cardiac repolarization, and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain variable fragment (scFv) antibodies can modulate hERG function by binding to the PAS domain. Here, we mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface using NMR spectroscopy and mutagenesis and show that scFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. By generating a fluorescently labeled scFv2.12, we demonstrate that association with the full-length hERG channel is state dependent. We detect Förster resonance energy transfer (FRET) with scFv2.12 when the channel gate is open but not when it is closed. In addition, state dependence of scFv2.12 FRET signal disappears when the R56Q mutation, known to destabilize the PAS-CNBh interaction, is introduced in the channel. Altogether, these data are consistent with an extensive structural alteration of the PAS/CNBh assembly when the cytosolic gate opens, likely favoring PAS domain dissociation from the CNBh domain.
Asunto(s)
Canal de Potasio ERG1/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Citosol/metabolismo , Canal de Potasio ERG1/genética , Canal de Potasio ERG1/inmunología , Canales de Potasio Éter-A-Go-Go/inmunología , Canales de Potasio Éter-A-Go-Go/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Activación del Canal Iónico , Síndrome de QT Prolongado/genética , Conformación Molecular , Mutación , Conformación Proteica , Dominios Proteicos/genética , Dominios Proteicos/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Relación Estructura-ActividadRESUMEN
The bacterial K+ homeostasis machinery is widely conserved across bacterial species, and different from that in animals. Dysfunction in components of the machinery has an impact on intracellular turgor, membrane potential, adaptation to changes in both extracellular pH and osmolarity, and in virulence. Using a fluorescence-based liposome flux assay, we have performed a high-throughput screen to identify novel inhibitors of the KtrAB ion channel complex from Bacillus subtilis, a component of the K+ homeostasis machinery that is also present in many bacterial pathogens. The screen identified 41 compounds that inhibited K+ flux and that clustered into eight chemical groups. Many of the identified inhibitors were found to target KtrAB with an in vitro potency in the low µM range. We investigated the mechanisms of inhibition and found that most molecules affected either the membrane component of the channel, KtrB alone or the full KtrAB complex without a preference for the functional conformation of the channel, thus broadening their inhibitory action. A urea derivative molecule that inhibited the membrane component of KtrAB affected cell viability in conditions in which KtrAB activity is essential. With this proof-of-concept study, we demonstrate that targeting components of the K+ homeostasis machinery has the potential as a new antibacterial strategy and that the fluorescence-based flux assay is a robust tool for screening chemical libraries.
RESUMEN
The human human ether-à-go-go-related gene (hERG) potassium channel plays a critical role in the repolarization of the cardiac action potential. Changes in hERG channel function underlie long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. A striking feature of this channel and KCNH channels in general is the presence of an N-terminal Per-Arnt-Sim (PAS) domain. In other proteins, PAS domains bind ligands and modulate effector domains. However, the PAS domains of KCNH channels are orphan receptors. We have uncovered a family of positive modulators of hERG that specifically bind to the PAS domain. We generated two single-chain variable fragments (scFvs) that recognize different epitopes on the PAS domain. Both antibodies increase the rate of deactivation but have different effects on channel activation and inactivation. Importantly, we show that both antibodies, on binding to the PAS domain, increase the total amount of current that permeates the channel during a ventricular action potential and significantly reduce the action potential duration recorded in human cardiomyocytes. Overall, these molecules constitute a previously unidentified class of positive modulators and establish that allosteric modulation of hERG channel function through ligand binding to the PAS domain can be attained.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Canales de Potasio Éter-A-Go-Go/fisiología , Activación del Canal Iónico/efectos de los fármacos , Anticuerpos de Cadena Única/farmacología , Animales , Sitios de Unión/genética , Sitios de Unión/inmunología , Células Cultivadas , Pollos , Estimulación Eléctrica/métodos , Epítopos/genética , Epítopos/inmunología , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/inmunología , Células HEK293 , Humanos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Técnicas de Placa-Clamp , Anticuerpos de Cadena Única/inmunologíaRESUMEN
Members of the ether-à-go-go (EAG) family of voltage-gated K(+) channels are involved in several pathophysiological diseases, and there has been a great interest in screening for drugs that modulate the activity of these channels. Many drugs have been shown to bind in the pore of these channels, blocking ion flux and causing disease pathology. In this report, we present two independent screening campaigns in which we wanted to identify small molecules that bind to either the intracellular cytoplasmic amino terminal Per-Arnt-Sim (PAS) domain from the human EAG-related gene (ERG) channel or the amino or carboxy terminal globular domains from the mouse EAG1 channel, affecting their interaction. We report that in both cases, compounds were identified that showed weak, nonspecific binding. We suggest alternative routes should be pursued in future efforts to identify specific, high-affinity binders to these cytoplasmic domains.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Animales , Sitios de Unión , Canales de Potasio Éter-A-Go-Go/química , Humanos , Ratones , Dominios Proteicos/efectos de los fármacos , Estructura Terciaria de ProteínaRESUMEN
KCNH channels form an important family of voltage gated potassium channels. These channels include a N-terminal Per-Arnt-Sim (PAS) domain with unknown function. In other proteins PAS domains are implicated in cellular responses to environmental queues through small molecule binding or involvement in signaling cascades. To better understand their role we characterized the structural properties of several channel PAS domains. We determined high resolution structures of PAS domains from the mouse EAG (mEAG), drosophila ELK (dELK) and human ERG (hERG) channels and also of the hERG domain without the first nine amino acids. We analyzed these structures for features connected to ligand binding and signaling in other PAS domains. In particular, we have found cavities in the hERG and mEAG structures that share similarities with the ligand binding sites from other PAS domains. These cavities are lined by polar and apolar chemical groups and display potential flexibility in their volume. We have also found that the hydrophobic patch on the domain ß-sheet is a conserved feature and appears to drive the formation of protein-protein contacts. In addition, the structures of the dELK domain and of the truncated hERG domain revealed the presence of N-terminal helices. These helices are equivalent to the helix described in the hERG NMR structures and are known to be important for channel function. Overall, these channel domains retain many of the PAS domain characteristics known to be important for cell signaling.
Asunto(s)
Proteínas de Drosophila/química , Canales de Potasio Éter-A-Go-Go/química , Modelos Moleculares , Canales de Potasio/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/metabolismo , Canales de Potasio Éter-A-Go-Go/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Mutación , Canales de Potasio/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Inherited human long-QT2 syndrome (LQTS) results from mutations in the gene encoding the HERG channel. Several LQT2-associated mutations have been mapped to the amino terminal cytoplasmic Per-Arnt-Sim (PAS) domain of the HERG1a channel subunit. Here we have characterized the trafficking properties of some LQT2-associated PAS domain mutants and analyzed rescue of the trafficking mutants by low temperature (27°C) or by the pore blocker drug E4031. We show that the LQT2-associated mutations in the PAS domain of the HERG channel display molecular properties that are distinct from the properties of LQT2-associated mutations in the trans-membrane region. Unlike the latter, many of the tested PAS domain LQT2-associated mutations do not result in trafficking deficiency of the channel. Moreover, the majority of the PAS domain mutations that cause trafficking deficiencies are not rescued by a pore blocking drug. We have also explored the in vitro folding stability properties of isolated mutant PAS domain proteins using a thermal unfolding fluorescence assay and a chemical unfolding assay.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/genética , Síndrome de QT Prolongado/genética , Mutación , Dicroismo Circular , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/química , Glicósido Hidrolasas/metabolismo , Células HEK293 , Humanos , Fenotipo , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Temperatura , Termodinámica , Rayos UltravioletaRESUMEN
Herpes simplex virus (HSV) capsids assemble, mature and package their viral genome in the nucleoplasm. They then exit the nucleus into the cytoplasm, where they acquire their final tegument and envelope. The molecular mechanism of cytoplasmic envelopment is unclear, but evidence suggests that the viral glycoprotein tails play an important role in the recruitment of tegument and capsids at the final envelopment site. However, due to redundancy in protein-protein interactions among the viral glycoproteins, genetic analysis of the role of individual glycoproteins in assembly has been difficult. To overcome this problem, a glutathione S-transferase fusion protein-binding assay was used in this study to test the interaction between the cytoplasmic tail of one specific viral glycoprotein, gD, and tegument proteins. The study demonstrated that the 38 kDa tegument protein VP22 bound specifically to the gD tail. This association was dependent on arginine and lysine residues at positions 5 and 6 in the gD tail. In addition, HSV-1 capsids bound the gD tail and exhibited a similar sequence dependence. It is concluded that VP22 may serve as a linker protein, mediating the interaction of the HSV capsid with gD.
Asunto(s)
Cápside/metabolismo , Simplexvirus/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/genética , Células COS , Cápside/ultraestructura , Chlorocebus aethiops , Citoplasma/virología , Lisina/genética , Datos de Secuencia Molecular , Peso Molecular , Mutación , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas Estructurales Virales/química , Ensamble de VirusRESUMEN
During Herpes simplex virus envelopment, capsids, tegument polypeptides, and membrane proteins assemble at the site of budding and a cellular lipid bilayer becomes refashioned into a spherical envelope. Though the molecular interactions driving these events are poorly understood, several lines of evidence suggest that associations between envelope protein cytoplasmic tails and tegument polypeptides may play important roles. Consistent with this hypothesis, we show here that a fusion of the cytoplasmic tail of gH with Glutathione-S-Transferase binds to VP16 in a temperature-dependent manner. VP16 prepared by in vitro translation behaves in a similar fashion, demonstrating that the interaction is not dependent on other viral polypeptides. Mutational analysis of the gH tail has also enabled us to identify amino acid residues critical for VP16 binding in vitro. A fusion protein in which the gH tail is fused to the carboxy-terminus of GFP coimmunoprecipitates with VP16 in infected cells, indicating that VP16 can interact with the gH tail in vivo.
Asunto(s)
Citoplasma/metabolismo , Etopósido/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas del Envoltorio Viral/genética , Ensamble de VirusRESUMEN
Yeast protein insertion orientation (PIO) mutants were isolated by selecting for growth on sucrose in cells in which the only source of invertase is a C-terminal fusion to a transmembrane protein. Only the fraction with an exocellular C terminus can be processed to secreted invertase and this fraction is constrained to 2-3% by a strong charge difference signal. Identified pio mutants increased this to 9-12%. PIO1 is SPF1, encoding a P-type ATPase located in the endoplasmic reticulum (ER) or Golgi. spf1-null mutants are modestly sensitive to EGTA. Sensitivity is considerably greater in an spf1 pmr1 double mutant, although PIO is not further disturbed. Pmr1p is the Golgi Ca(2+) ATPase and Spf1p may be the equivalent ER pump. PIO2 is STE24, a metalloprotease anchored in the ER membrane. Like Spf1p, Ste24p is expressed in all yeast cell types and belongs to a highly conserved protein family. The effects of ste24- and spf1-null mutations on invertase secretion are additive, cell generation time is increased 60%, and cells become sensitive to cold and to heat shock. Ste24p and Rce1p cleave the C-AAX bond of farnesylated CAAX box proteins. The closest paralog of SPF1 is YOR291w. Neither rce1-null nor yor291w-null mutations affected PIO or the phenotype of spf1- or ste24-null mutants. Mutations in PIO3 (unidentified) cause a weaker Pio phenotype, enhanced by a null mutation in BMH1, one of two yeast 14-3-3 proteins.