RESUMO
ATP-sensitive potassium (KATP) channels function as metabolic sensors that link cell membrane excitability to the cellular energy status by controlling potassium ion (K+) flow across the cell membrane according to intracellular ATP and ADP concentrations. As such, KATP channels influence a broad spectrum of physiological processes, including insulin secretion and cardiovascular functions. KATP channels are hetero-octamers, consisting of four inward rectifier potassium channel subunits, Kir6.1 or Kir6.2, and four sulfonylurea receptors (SURs), SUR1, SUR2A, or SUR2B. Different Kir6 and SUR isoforms assemble into KATP channel subtypes with distinct tissue distributions and physiological functions. Mutations in the genes encoding KATP channel subunits underlie various human diseases. Targeted treatment for these diseases requires subtype-specific KATP channel modulators. Rubidium ions (Rb+) also pass through KATP channels, and Rb+ efflux assays can be used to assess KATP channel function and activity. Flame atomic absorption spectroscopy (Flame-AAS) combined with microsampling can measure Rb+ in small volume, which provides an efficient tool to screen for compounds that alter KATP channel activity in Rb+ efflux assays. In this chapter, we describe a detailed protocol for Rb+ efflux assays designed to identify new KATP channel modulators with potential therapeutic utilities.
Assuntos
Canais KATP , Rubídio , Canais KATP/metabolismo , Canais KATP/genética , Humanos , Rubídio/metabolismo , Receptores de Sulfonilureias/metabolismo , Receptores de Sulfonilureias/genética , Animais , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genéticaRESUMO
ATP-sensitive potassium (KATP) channels, composed of four pore-lining Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1) subunits, control insulin secretion in pancreatic ß-cells. KATP channel opening is stimulated by PIP2 and inhibited by ATP. Mutations that increase channel opening by PIP2 reduce ATP inhibition and cause neonatal diabetes. Although considerable evidence has implicated a role for PIP2 in KATP channel function, previously solved open-channel structures have lacked bound PIP2, and mechanisms by which PIP2 regulates KATP channels remain unresolved. Here, we report the cryoEM structure of a KATP channel harboring the neonatal diabetes mutation Kir6.2-Q52R, in the open conformation, bound to amphipathic molecules consistent with natural C18:0/C20:4 long-chain PI(4,5)P2 at two adjacent binding sites between SUR1 and Kir6.2. The canonical PIP2 binding site is conserved among PIP2-gated Kir channels. The non-canonical PIP2 binding site forms at the interface of Kir6.2 and SUR1. Functional studies demonstrate both binding sites determine channel activity. Kir6.2 pore opening is associated with a twist of the Kir6.2 cytoplasmic domain and a rotation of the N-terminal transmembrane domain of SUR1, which widens the inhibitory ATP binding pocket to disfavor ATP binding. The open conformation is particularly stabilized by the Kir6.2-Q52R residue through cation-π bonding with SUR1-W51. Together, these results uncover the cooperation between SUR1 and Kir6.2 in PIP2 binding and gating, explain the antagonistic regulation of KATP channels by PIP2 and ATP, and provide a putative mechanism by which Kir6.2-Q52R stabilizes an open channel to cause neonatal diabetes.
Assuntos
Diabetes Mellitus , Canais de Potássio Corretores do Fluxo de Internalização , Recém-Nascido , Humanos , Receptores de Sulfonilureias/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Sítios de Ligação , Trifosfato de Adenosina/metabolismo , Canais KATP/genética , Canais KATP/metabolismoRESUMO
KATP channels are ligand-gated potassium channels that couple cellular energetics with membrane potential to regulate cell activity. Each channel is an eight subunit complex comprising four central pore-forming Kir6 inward rectifier potassium channel subunits surrounded by four regulatory subunits known as the sulfonylurea receptor, SUR, which confer homeostatic metabolic control of KATP gating. SUR is an ATP binding cassette (ABC) protein family homolog that lacks membrane transport activity but is essential for KATP expression and function. For more than four decades, understanding the structure-function relationship of Kir6 and SUR has remained a central objective of clinical significance. Here, we review progress in correlating the wealth of functional data in the literature with recent KATP cryoEM structures.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Receptores de Sulfonilureias/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Potenciais da Membrana , Trifosfato de Adenosina/metabolismo , Canais KATP/genéticaRESUMO
ATP-sensitive potassium (K ATP ) channels, composed of four pore-lining Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1) subunits, control insulin secretion in pancreatic ß-cells. K ATP channel opening is stimulated by PIP 2 and inhibited by ATP. Mutations that increase channel opening by PIP 2 reduce ATP inhibition and cause neonatal diabetes. Although considerable evidence has indicated PIP 2 in K ATP channel function, previously solved open-channel structures have lacked bound PIP 2 , and mechanisms by which PIP 2 regulates K ATP channels remain unresolved. Here, we report the cryoEM structure of a K ATP channel harboring the neonatal diabetes mutation Kir6.2-Q52R, bound to natural C18:0/C20:4 long-chain PIP 2 in an open conformation. The structure reveals two adjacent PIP 2 molecules between SUR1 and Kir6.2. The first PIP 2 binding site is conserved among PIP 2 -gated Kir channels. The second site forms uniquely in K ATP at the interface of Kir6.2 and SUR1. Functional studies demonstrate both binding sites determine channel activity. Kir6.2 pore opening is associated with a twist of the Kir6.2 cytoplasmic domain and a rotation of the N-terminal transmembrane domain of SUR1, which widens the inhibitory ATP binding pocket to disfavor ATP binding. The open conformation is particularly stabilized by the Kir6.2-Q52R residue through cation-π bonding with SUR1-W51. Together, these results uncover the cooperation between SUR1 and Kir6.2 in PIP 2 binding and gating, explain the antagonistic regulation of K ATP channels by PIP 2 and ATP, and provide the mechanism by which Kir6.2-Q52R stabilizes an open channel to cause neonatal diabetes.
RESUMO
Gated by intracellular ATP and ADP, ATP-sensitive potassium (KATP) channels couple cell energetics with membrane excitability in many cell types, enabling them to control a wide range of physiological processes based on metabolic demands. The KATP channel is a complex of four potassium channel subunits from the Kir channel family, Kir6.1 or Kir6.2, and four sulfonylurea receptor subunits, SUR1, SUR2A, or SUR2B, from the ATP-binding cassette (ABC) transporter family. Dysfunction of KATP channels underlies several human diseases. The importance of these channels in human health and disease has made them attractive drug targets. How the channel subunits interact with one another and how the ligands interact with the channel to regulate channel activity have been long-standing questions in the field. In the past 5 yr, a steady stream of high-resolution KATP channel structures has been published using single-particle cryo-electron microscopy (cryo-EM). Here, we review the advances these structures bring to our understanding of channel regulation by physiological and pharmacological ligands.
Assuntos
Trifosfato de Adenosina , Canais KATP , Humanos , Microscopia Crioeletrônica , Ligantes , Receptores de SulfonilureiasRESUMO
Regulation of pancreatic KATP channels involves orchestrated interactions of their subunits, Kir6.2 and SUR1, and ligands. Previously we reported KATP channel cryo-EM structures in the presence and absence of pharmacological inhibitors and ATP, focusing on the mechanisms by which inhibitors act as pharmacological chaperones of KATP channels (Martin et al., 2019). Here we analyzed the same cryo-EM datasets with a focus on channel conformational dynamics to elucidate structural correlates pertinent to ligand interactions and channel gating. We found pharmacological inhibitors and ATP enrich a channel conformation in which the Kir6.2 cytoplasmic domain is closely associated with the transmembrane domain, while depleting one where the Kir6.2 cytoplasmic domain is extended away into the cytoplasm. This conformational change remodels a network of intra- and inter-subunit interactions as well as the ATP and PIP2 binding pockets. The structures resolved key contacts between the distal N-terminus of Kir6.2 and SUR1's ABC module involving residues implicated in channel function and showed a SUR1 residue, K134, participates in PIP2 binding. Molecular dynamics simulations revealed two Kir6.2 residues, K39 and R54, that mediate both ATP and PIP2 binding, suggesting a mechanism for competitive gating by ATP and PIP2.
Assuntos
Canais KATP , Trifosfato de Adenosina/metabolismo , Humanos , Canais KATP/química , Ligantes , Pâncreas , Conformação ProteicaRESUMO
Vascular tone is dependent on smooth muscle KATP channels comprising pore-forming Kir6.1 and regulatory SUR2B subunits, in which mutations cause Cantú syndrome. Unique among KATP isoforms, they lack spontaneous activity and require Mg-nucleotides for activation. Structural mechanisms underlying these properties are unknown. Here, we determined cryogenic electron microscopy structures of vascular KATP channels bound to inhibitory ATP and glibenclamide, which differ informatively from similarly determined pancreatic KATP channel isoform (Kir6.2/SUR1). Unlike SUR1, SUR2B subunits adopt distinct rotational "propeller" and "quatrefoil" geometries surrounding their Kir6.1 core. The glutamate/aspartate-rich linker connecting the two halves of the SUR-ABC core is observed in a quatrefoil-like conformation. Molecular dynamics simulations reveal MgADP-dependent dynamic tripartite interactions between this linker, SUR2B, and Kir6.1. The structures captured implicate a progression of intermediate states between MgADP-free inactivated, and MgADP-bound activated conformations wherein the glutamate/aspartate-rich linker participates as mobile autoinhibitory domain, suggesting a conformational pathway toward KATP channel activation.
Assuntos
Difosfato de Adenosina/metabolismo , Canais KATP/ultraestrutura , Receptores de Sulfonilureias/ultraestrutura , Trifosfato de Adenosina/metabolismo , Cardiomegalia/metabolismo , Humanos , Hipertricose/metabolismo , Canais KATP/genética , Canais KATP/metabolismo , Músculo Liso/metabolismo , Osteocondrodisplasias/metabolismo , Pâncreas/metabolismo , Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Relação Estrutura-Atividade , Receptores de Sulfonilureias/genética , Receptores de Sulfonilureias/metabolismoRESUMO
ATP-sensitive potassium (KATP) channels are multimeric protein complexes made of four inward rectifying potassium channel (Kir6.x) subunits and four ABC protein sulfonylurea receptor (SURx) subunits. Kir6.x subunits form the potassium ion conducting pore of the channel, and SURx functions to regulate Kir6.x. Kir6.x and SURx are uniquely dependent on each other for expression and function. In pancreatic ß-cells, channels comprising SUR1 and Kir6.2 mediate glucose-stimulated insulin secretion and are the targets of antidiabetic sulfonylureas. Mutations in genes encoding SUR1 or Kir6.2 are linked to insulin secretion disorders, with loss- or gain-of-function mutations causing congenital hyperinsulinism or neonatal diabetes mellitus, respectively. Defects in the KATP channel in other tissues underlie human diseases of the cardiovascular and nervous systems. Key to understanding how channels are regulated by physiological and pharmacological ligands and how mutations disrupt channel assembly or gating to cause disease is the ability to observe structural changes associated with subunit interactions and ligand binding. While recent advances in the structural method of single-particle cryo-electron microscopy (cryoEM) offers direct visualization of channel structures, success of obtaining high-resolution structures is dependent on highly concentrated, homogeneous KATP channel particles. In this chapter, we describe a method for expressing KATP channels in mammalian cell culture, solubilizing the channel in detergent micelles and purifying KATP channels using an affinity tag to the SURx subunit for cryoEM structural studies.
Assuntos
Canais KATP , Canais de Potássio Corretores do Fluxo de Internalização , Trifosfato de Adenosina , Animais , Microscopia Crioeletrônica , Humanos , Recém-Nascido , Canais KATP/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Receptores de Sulfonilureias/genéticaRESUMO
The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein dynamics, either alone or as part of a Förster resonance energy transfer (FRET) or photo-induced electron transfer (eT) probe pair. We have previously reported the genetic incorporation of Acd by an aminoacyl tRNA synthetase (RS). However, this RS, developed from a library of permissive RSs, also incorporates N-phenyl-aminophenylalanine (Npf), a trace byproduct of one Acd synthetic route. We have performed negative selections in the presence of Npf and analyzed the selectivity of the resulting AcdRSs by in vivo protein expression and detailed kinetic analyses of the purified RSs. We find that selection conferred a â¼50-fold increase in selectivity for Acd over Npf, eliminating incorporation of Npf contaminants, and allowing one to use a high yielding Acd synthetic route for improved overall expression of Acd-containing proteins. More generally, our report also provides a cautionary tale on the use of permissive RSs, as well as a strategy for improving selectivity for the target amino acid.
Assuntos
Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Aminoacil-tRNA Sintetases/química , Domínio Catalítico , Transferência Ressonante de Energia de Fluorescência , Cinética , Modelos Moleculares , Ligação ProteicaRESUMO
In mammals, the non-heme iron enzyme cysteine dioxygenase (CDO) helps regulate Cys levels through converting Cys to Cys sulfinic acid. Its activity is in part modulated by the formation of a Cys93-Tyr157 crosslink that increases its catalytic efficiency over 10-fold. Here, 21 high-resolution mammalian CDO structures are used to gain insight into how the Cys-Tyr crosslink promotes activity and how select competitive inhibitors bind. Crystal structures of crosslink-deficient C93A and Y157F variants reveal similar ~1.0-Å shifts in the side chain of residue 157, and both variant structures have a new chloride ion coordinating the active site iron. Cys binding is also different from wild-type CDO, and no Cys-persulfenate forms in the C93A or Y157F active sites at pH6.2 or 8.0. We conclude that the crosslink enhances activity by positioning the Tyr157 hydroxyl to enable proper Cys binding, proper oxygen binding, and optimal chemistry. In addition, structures are presented for homocysteine (Hcy), D-Cys, thiosulfate, and azide bound as competitive inhibitors. The observed binding modes of Hcy and D-Cys clarify why they are not substrates, and the binding of azide shows that in contrast to what has been proposed, it does not bind in these crystals as a superoxide mimic.
Assuntos
Cisteína Dioxigenase/química , Cisteína Dioxigenase/metabolismo , Inibidores Enzimáticos/metabolismo , Animais , Cristalografia por Raios X , Cisteína/genética , Cisteína/metabolismo , Cisteína Dioxigenase/genética , Mamíferos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica , Tirosina/genética , Tirosina/metabolismoRESUMO
In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only â¼15-30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue ("Arg-type" enzymes) and some having a Gln substituted for this Arg ("Gln-type" enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis "Arg-type" enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha "Gln-type" CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among "Gln-type" CDO enzymes, we conclude that the "Gln-type" CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.
Assuntos
Bacillus subtilis/enzimologia , Cisteína Dioxigenase/química , Ralstonia/enzimologia , Ácido 3-Mercaptopropiônico/metabolismo , Sequência de Aminoácidos , Animais , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Cisteína Dioxigenase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Ralstonia/química , Ralstonia/metabolismo , Ratos , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
The Escherichia coli sulfur starvation utilization (ssu) operon includes a two-component monooxygenase system consisting of a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent flavin mononucleotide (FMN) reductase, SsuE, and a monooxygenase, SsuD. SsuE is part of the flavodoxin-like superfamily, and we report here the crystal structures of its apo, FMN-bound, and FMNH2-bound forms at â¼2 Å resolution. In the crystals, SsuE forms a tetramer that is a dimer of dimers similar to those seen for homologous FMN reductases, quinone reductases, and the WrbA family of enzymes. A π-helix present at the tetramer building interface is unique to the reductases from two-component monooxygenase systems. Analytical ultracentrifugation studies of SsuE confirm a dimer-tetramer equilibrium exists in solution, with FMN binding favoring the dimer. As the active site includes residues from both subunits, at least a dimeric association is required for the function of SsuE. The structures show that one FMN binds tightly in a deeply held site, which makes available a second binding site, in which either a second FMN or the nicotinamide of NADPH can bind. The FMNH2-bound structure shows subtle changes consistent with its binding being weaker than that of FMN. Combining this information with published kinetic studies, we propose a general catalytic cycle for two-component reductases of the flavodoxin-like superfamily, by which the enzyme can potentially provide FMNH2 to its partner monooxygenase by different routes depending on the FMN concentration and the presence of a partner monooxygenase.
Assuntos
Proteínas de Escherichia coli/química , FMN Redutase/química , Flavodoxina/química , Sequência de Aminoácidos , Apoproteínas/química , Biocatálise , Domínio Catalítico , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Proteínas Recombinantes/químicaRESUMO
Genetic code expansion has provided the ability to site-specifically incorporate a multitude of noncanonical amino acids (ncAAs) into proteins for a wide variety of applications, but low ncAA incorporation efficiency can hamper the utility of this powerful technology. When investigating proteins containing the post-translational modification 3-nitro-tyrosine (nitroTyr), we developed second-generation amino-acyl tRNA synthetases (RS) that incorporate nitroTyr at efficiencies roughly an order of magnitude greater than those previously reported and that advanced our ability to elucidate the role of elevated cellular nitroTyr levels in human disease (e.g., Franco, M. et al. Proc. Natl. Acad. Sci. U.S.A 2013 , 110 , E1102 ). Here, we explore the origins of the improvement achieved in these second-generation RSs. Crystal structures of the most efficient of these synthetases reveal the molecular basis for the enhanced efficiencies observed in the second-generation nitroTyr-RSs. Although Tyr is not detectably incorporated into proteins when expression media is supplemented with 1 mM nitroTyr, a major difference between the first- and second-generation RSs is that the second-generation RSs have an active site more compatible with Tyr binding. This feature of the second-generation nitroTyr-RSs appears to be the result of using less stringent criteria when selecting from a library of mutants. The observation that a different selection strategy performed on the same library of mutants produced nitroTyr-RSs with dramatically improved efficiencies suggests the optimization of established selection protocols could lead to notable improvements in ncAA-RS efficiencies and thus the overall utility of this technology.
Assuntos
Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/metabolismo , Tirosina/química , Domínio Catalítico/genética , Linhagem Celular , Cristalografia por Raios X , Código Genético , Humanos , Mutação , Estrutura Secundária de Proteína , Tirosina/genética , Tirosina/metabolismo , Tirosina-tRNA Ligase/genéticaRESUMO
Mammalian cysteine dioxygenase (CDO) is a mononuclear non-heme iron protein that catalyzes the conversion of cysteine (Cys) to cysteine sulfinic acid by an unclarified mechanism. One structural study revealed that a Cys-persulfenate (or Cys-persulfenic acid) formed in the active site, but quantum mechanical calculations have been used to support arguments that it is not an energetically feasible reaction intermediate. Here, we report a series of high-resolution structures of CDO soaked with Cys at pH values from 4 to 9. Cys binding is minimal at pH≤5 and persulfenate formation is consistently seen at pH values between 5.5 and 7. Also, a structure determined using laboratory-based X-ray diffraction shows that the persulfenate, with an apparent average O-O separation distance of ~1.8Å, is not an artifact of synchrotron radiation. At pH≥8, the active-site iron shifts from 4- to 5-coordinate, and Cys soaks reveal a complex with Cys, but no dioxygen, bound. This 'Cys-only' complex differs in detail from a previously published 'Cys-only' complex, which we reevaluate and conclude is not reliable. The high-resolution structures presented here do not resolve the CDO mechanism but do imply that an iron-bound persulfenate (or persulfenic acid) is energetically accessible in the CDO active site, and that CDO active-site chemistry in the crystals is influenced by protonation/deprotonation events with effective pKa values near ~5.5 and ~7.5 that influence Cys binding and oxygen binding/reactivity, respectively. Furthermore, this work provides reliable ligand-bound models for guiding future mechanistic considerations.
Assuntos
Cisteína Dioxigenase/química , Cisteína Dioxigenase/metabolismo , Cisteína/análogos & derivados , Cisteína/química , Cisteína/metabolismo , Animais , Domínio Catalítico , Cristalografia por Raios X/métodos , Concentração de Íons de Hidrogênio , Ferro/metabolismo , Ligação Proteica , Conformação Proteica , Ratos , Difração de Raios X/métodosRESUMO
The planarity of peptide bonds is an assumption that underlies decades of theoretical modeling of proteins. Peptide bonds strongly deviating from planarity are considered very rare features of protein structure that occur for functional reasons. Here, empirical analyses of atomic-resolution protein structures reveal that trans peptide groups can vary by more than 25° from planarity and that the true extent of nonplanarity is underestimated even in 1.2 Å resolution structures. Analyses as a function of the Ï,ψ-backbone dihedral angles show that the expected value deviates by ± 8° from planar as a systematic function of conformation, but that the large majority of variation in planarity depends on tertiary effects. Furthermore, we show that those peptide bonds in proteins that are most nonplanar, deviating by over 20° from planarity, are not strongly associated with active sites. Instead, highly nonplanar peptides are simply integral components of protein structure related to local and tertiary structural features that tend to be conserved among homologs. To account for the systematic Ï,ψ-dependent component of nonplanarity, we present a conformation-dependent library that can be used in crystallographic refinement and predictive protein modeling.