RESUMEN
Electrical signaling is essential for all fast processes in biology, but its molecular mechanisms have been uncertain. This review article focuses on studies of bacterial sodium channels in order to home in on the essential molecular and chemical mechanisms underlying transmembrane ion conductance and voltage-dependent gating without the overlay of complex protein interactions and regulatory mechanisms in mammalian sodium channels. This minimalist approach has yielded a nearly complete picture of sodium channel function at the atomic level that are mostly conserved in mammalian sodium channels, including sodium selectivity and conductance, voltage sensing and activation, electromechanical coupling to pore opening and closing, slow inactivation, and pathogenic dysfunction in a debilitating channelopathy. Future studies of nature's simplest sodium channels may continue to yield key insights into the fundamental molecular and chemical principles of their function and further elucidate the chemical basis of electrical signaling.
Asunto(s)
Bacterias , Canales de Sodio , Canales de Sodio/metabolismo , Canales de Sodio/química , Bacterias/metabolismo , Humanos , Animales , Transducción de Señal , Activación del Canal IónicoRESUMEN
The poison dart toxin batrachotoxin is exceptional for its high potency and toxicity, and for its multifaceted modification of the function of voltage-gated sodium channels. By using cryogenic electron microscopy, we identify two homologous, but nonidentical receptor sites that simultaneously bind two molecules of toxin, one at the interface between Domains I and IV, and the other at the interface between Domains III and IV of the cardiac sodium channel. Together, these two bound toxin molecules stabilize α/π helical conformation in the S6 segments that gate the pore, and one of the bound BTX-B molecules interacts with the crucial Lys1421 residue that is essential for sodium conductance and selectivity via an apparent water-bridged hydrogen bond. Overall, our structure provides insight into batrachotoxin's potency, efficacy, and multifaceted functional effects on voltage-gated sodium channels via a dual receptor site mechanism.
Asunto(s)
Venenos , Canales de Sodio Activados por Voltaje , Batracotoxinas/metabolismo , Sitios de Unión , Conformación Molecular , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
Voltage-gated sodium channels in peripheral nerves conduct nociceptive signals from nerve endings to the spinal cord. Mutations in voltage-gated sodium channel NaV1.7 are responsible for a number of severe inherited pain syndromes, including inherited erythromelalgia (IEM). Here, we describe the negative shifts in the voltage dependence of activation in the bacterial sodium channel NaVAb as a result of the incorporation of four different IEM mutations in the voltage sensor, which recapitulate the gain-of-function effects observed with these mutations in human NaV1.7. Crystal structures of NaVAb with these IEM mutations revealed that a mutation in the S1 segment of the voltage sensor facilitated the outward movement of S4 gating charges by widening the pathway for gating charge translocation. In contrast, mutations in the S4 segments modified hydrophobic interactions with surrounding amino acid side chains or membrane phospholipids that would enhance the outward movement of the gating charges. These results provide key structural insights into the mechanisms by which these IEM mutations in the voltage sensors can facilitate outward movements of the gating charges in the S4 segment and cause hyperexcitability and severe pain in IEM. Our work gives new insights into IEM pathogenesis at the near-atomic level and provides a molecular model for mutation-specific therapy of this debilitating disease.
Asunto(s)
Eritromelalgia , Canal de Sodio Activado por Voltaje NAV1.7 , Humanos , Eritromelalgia/genética , Eritromelalgia/metabolismo , Eritromelalgia/patología , Modelos Moleculares , Mutación , Canal de Sodio Activado por Voltaje NAV1.7/genética , Canal de Sodio Activado por Voltaje NAV1.7/química , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Dolor/genética , Dolor/metabolismo , Dolor/patologíaRESUMEN
Gain-of-function mutations in voltage-gated sodium channel NaV1.7 cause severe inherited pain syndromes, including inherited erythromelalgia (IEM). The structural basis of these disease mutations, however, remains elusive. Here, we focused on three mutations that all substitute threonine residues in the alpha-helical S4-S5 intracellular linker that connects the voltage sensor to the pore: NaV1.7/I234T, NaV1.7/I848T, and NaV1.7/S241T in order of their positions in the amino acid sequence within the S4-S5 linkers. Introduction of these IEM mutations into the ancestral bacterial sodium channel NaVAb recapitulated the pathogenic gain-of-function of these mutants by inducing a negative shift in the voltage dependence of activation and slowing the kinetics of inactivation. Remarkably, our structural analysis reveals a common mechanism of action among the three mutations, in which the mutant threonine residues create new hydrogen bonds between the S4-S5 linker and the pore-lining S5 or S6 segment in the pore module. Because the S4-S5 linkers couple voltage sensor movements to pore opening, these newly formed hydrogen bonds would stabilize the activated state substantially and thereby promote the 8 to 18 mV negative shift in the voltage dependence of activation that is characteristic of the NaV1.7 IEM mutants. Our results provide key structural insights into how IEM mutations in the S4-S5 linkers may cause hyperexcitability of NaV1.7 and lead to severe pain in this debilitating disease.
Asunto(s)
Eritromelalgia , Canales de Sodio Activados por Voltaje , Humanos , Canal de Sodio Activado por Voltaje NAV1.7/genética , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Dolor/genética , Dolor/metabolismo , Mutación , Eritromelalgia/genética , Eritromelalgia/metabolismo , Eritromelalgia/patología , Canales de Sodio Activados por Voltaje/genética , Treonina/genéticaRESUMEN
Voltage-gated ion channels are important drug targets because they play crucial physiological roles in both excitable and non-excitable cells. About 15% of clinical drugs used for treating human diseases target ion channels. However, most of these drugs do not provide sufficient specificity to a single subtype of the channels and their off-target side effects can be serious and sometimes fatal. Recent advancements in imaging techniques have enabled us for the first time to visualize unique and hidden parts of voltage-gated sodium channels in different structural conformations, and to develop drugs that further target a selected functional state in each channel subtype with the potential for high precision and low toxicity. In this review we describe the druggability of voltage-gated sodium channels in distinct functional states, which could potentially be used to selectively target the channels. We review classical drug receptors in the channels that have recently been structurally characterized by cryo-electron microscopy with natural neurotoxins and clinical drugs. We further examine recent drug discoveries for voltage-gated sodium channels and discuss opportunities to use distinct, state-dependent receptor sites in the voltage sensors as unique drug targets. Finally, we explore potential new receptor sites that are currently unknown for sodium channels but may be valuable for future drug discovery. The advancement presented here will help pave the way for drug development that selectively targets voltage-gated sodium channels.
RESUMEN
Voltage-gated sodium channels initiate electrical signals and are frequently targeted by deadly gating-modifier neurotoxins, including tarantula toxins, which trap the voltage sensor in its resting state. The structural basis for tarantula-toxin action remains elusive because of the difficulty of capturing the functionally relevant form of the toxin-channel complex. Here, we engineered the model sodium channel NaVAb with voltage-shifting mutations and the toxin-binding site of human NaV1.7, an attractive pain target. This mutant chimera enabled us to determine the cryoelectron microscopy (cryo-EM) structure of the channel functionally arrested by tarantula toxin. Our structure reveals a high-affinity resting-state-specific toxin-channel interaction between a key lysine residue that serves as a "stinger" and penetrates a triad of carboxyl groups in the S3-S4 linker of the voltage sensor. By unveiling this high-affinity binding mode, our studies establish a high-resolution channel-docking and resting-state locking mechanism for huwentoxin-IV and provide guidance for developing future resting-state-targeted analgesic drugs.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.7/química , Venenos de Araña/química , Sustitución de Aminoácidos , Animales , Humanos , Mutación Missense , Canal de Sodio Activado por Voltaje NAV1.7/genética , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Células Sf9 , SpodopteraRESUMEN
Electrical signaling was a dramatic development in evolution, allowing complex single-cell organisms like Paramecium to coordinate movement and early metazoans like worms and jellyfish to send regulatory signals rapidly over increasing distances. But how are electrical signals generated in biology? In fact, voltage-gated sodium channels conduct sodium currents that initiate electrical signals in all kingdoms of life, from bacteria to man. They are responsible for generating the action potential in vertebrate nerve and muscle, neuroendocrine cells, and other cell types1,2. Because of the high level of conservation of their core structure, it is likely that their fundamental mechanisms of action are conserved as well. Here we describe the complete cycle of conformational changes that a bacterial sodium channel undergoes as it transitions from resting to activated/open and inactivated/closed states, based on high-resolution structural studies of a single sodium channel. We further relate this conformational cycle of the ancestral sodium channel to the function of its vertebrate orthologs. The strong conservation of amino acid sequence and three-dimensional structure suggests that this model, at a fundamental level, is relevant for both prokaryotic and eukaryotic sodium channels, as well as voltage-gated calcium and potassium channels.
Asunto(s)
Potenciales de Acción/fisiología , Bacterias/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/química , Células Procariotas/metabolismo , Secuencia de Aminoácidos , Animales , Bacterias/genética , Secuencia Conservada , Evolución Molecular , Expresión Génica , Humanos , Modelos Moleculares , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Paramecium/genética , Paramecium/metabolismo , Células Procariotas/citología , Estructura Secundaria de ProteínaRESUMEN
Voltage-gated sodium (NaV) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state exists only at deeply negative membrane potentials. To stabilize the resting conformation, we inserted voltage-shifting mutations and introduced a disulfide crosslink in the VS of the ancestral bacterial sodium channel NaVAb. Here, we present a cryo-EM structure of the resting state and a complete voltage-dependent gating mechanism. The S4 segment of the VS is drawn intracellularly, with three gating charges passing through the transmembrane electric field. This movement forms an elbow connecting S4 to the S4-S5 linker, tightens the collar around the S6 activation gate, and prevents its opening. Our structure supports the classical "sliding helix" mechanism of voltage sensing and provides a complete gating mechanism for voltage sensor function, pore opening, and activation-gate closure based on high-resolution structures of a single sodium channel protein.
Asunto(s)
Potenciales de Acción/fisiología , Membrana Externa Bacteriana/metabolismo , Escherichia coli/metabolismo , Activación del Canal Iónico/fisiología , Canales de Sodio Activados por Voltaje/metabolismo , Animales , Línea Celular , Microscopía por Crioelectrón , Cristalografía por Rayos X , Mutación , Conformación Proteica en Hélice alfa , Sodio/metabolismo , Spodoptera/citología , Canales de Sodio Activados por Voltaje/químicaRESUMEN
Electrical signals generated by minute currents of ions moving across cell membranes are central to all rapid processes in biology. Initiation and propagation of electrical signals requires voltage-gated sodium (NaV) and calcium (CaV) channels. These channels contain a tetramer of membrane-bound subunits or domains comprising a voltage sensor and a pore module. Voltage-dependent activation occurs as membrane depolarization drives outward movements of positive gating changes in the voltage sensor via a sliding-helix mechanism, which leads to a conformational change in the pore module that opens its intracellular activation gate. A unique negatively charged site in the selectivity filter conducts hydrated Na+ or Ca2+ rapidly and selectively. Ion conductance is terminated by voltage-dependent inactivation, which causes asymmetric pore collapse. This Review focuses on recent advances in structure and function of NaV and CaV channels that expand our current understanding of the chemical basis for electrical signaling mechanisms conserved from bacteria to humans.
Asunto(s)
Conductividad Eléctrica , Activación del Canal Iónico , Canales Iónicos/química , Canales Iónicos/metabolismo , Calcio/metabolismo , Humanos , Modelos Moleculares , Potasio/metabolismo , Sodio/metabolismoRESUMEN
The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Protones , Simportadores/química , Xilosa/química , Arginina/química , Ácido Aspártico/química , Transporte Biológico , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Expresión Génica , Enlace de Hidrógeno , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Fosfatidilcolinas/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Simportadores/genética , TermodinámicaRESUMEN
Mineral nitrogen in nature is often found in the form of nitrate (NO3(-)). Numerous microorganisms evolved to assimilate nitrate and use it as a major source of mineral nitrogen uptake. Nitrate, which is central in nitrogen metabolism, is first reduced to nitrite (NO2(-)) through a two-electron reduction reaction. The accumulation of cellular nitrite can be harmful because nitrite can be reduced to the cytotoxic nitric oxide. Instead, nitrite is rapidly removed from the cell by channels and transporters, or reduced to ammonium or dinitrogen through the action of assimilatory enzymes. Despite decades of effort no structure is currently available for any nitrate transport protein and the mechanism by which nitrate is transported remains largely unknown. Here we report the structure of a bacterial nitrate/nitrite transport protein, NarK, from Escherichia coli, with and without substrate. The structures reveal a positively charged substrate-translocation pathway lacking protonatable residues, suggesting that NarK functions as a nitrate/nitrite exchanger and that protons are unlikely to be co-transported. Conserved arginine residues comprise the substrate-binding pocket, which is formed by association of helices from the two halves of NarK. Key residues that are important for substrate recognition and transport are identified and related to extensive mutagenesis and functional studies. We propose that NarK exchanges nitrate for nitrite by a rocker switch mechanism facilitated by inter-domain hydrogen bond networks.
Asunto(s)
Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/metabolismo , Escherichia coli/química , Nitratos/metabolismo , Nitritos/metabolismo , Proteínas de Transporte de Anión/genética , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Transportadores de Nitrato , Conformación Proteica , ProtonesRESUMEN
Electron crystallography is arguably the only electron cryomicroscopy (cryo EM) technique able to deliver atomic resolution data (better then 3 Å) for membrane proteins embedded in a membrane. The progress in hardware improvements and sample preparation for diffraction analysis resulted in a number of recent examples where increasingly higher resolutions were achieved. Other chapters in this book detail the improvements in hardware and delve into the intricate art of sample preparation for microscopy and electron diffraction data collection and processing. In this chapter, we describe in detail the protocols for molecular replacement for electron diffraction studies. The use of a search model for phasing electron diffraction data essentially eliminates the need of acquiring image data rendering it immune to aberrations from drift and charging effects that effectively lower the attainable resolution.
Asunto(s)
Microscopía por Crioelectrón/métodos , Cristalografía/métodos , Proteínas de la Membrana/química , Modelos Moleculares , Biología Computacional/instrumentación , Biología Computacional/métodos , Bases de Datos de Proteínas , Programas InformáticosRESUMEN
Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography.
Asunto(s)
Membrana Celular/química , Cristalografía/métodos , Proteínas de la Membrana/química , Acuaporina 1/química , Archaea/química , Bacteriorodopsinas/química , Membrana Celular/fisiología , Microscopía por Crioelectrón , Cristalografía/instrumentación , Detergentes/química , Diálisis/instrumentación , Procesamiento Automatizado de Datos , Humanos , Procesamiento de Imagen Asistido por Computador , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana/química , Conformación Proteica , Electricidad EstáticaRESUMEN
In electron crystallography, membrane protein structure is determined from two-dimensional crystals where the protein is embedded in a membrane. Once large and well-ordered 2D crystals are grown, one of the bottlenecks in electron crystallography is the collection of image data to directly provide experimental phases to high resolution. Here, we describe an approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We use the strengths of electron crystallography in rapidly obtaining accurate experimental phase information from low-resolution images and accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α helix fragments and extended to high resolution using phases from the fragments. Phases were further improved by density modifications followed by fragment expansion and structure refinement against the high-resolution diffraction data. Using this approach, structures of three membrane proteins were determined rapidly and accurately to atomic resolution without high-resolution image data.
Asunto(s)
Cristalografía por Rayos X/métodos , Proteínas de la Membrana/química , Imagen Molecular/métodos , Fragmentos de Péptidos/química , Cristalización , Electrones , Ligandos , Modelos Moleculares , Conformación ProteicaRESUMEN
Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. Cellular 2OG concentrations increase during nitrogen starvation. We recently identified a family of 2OG-sensing proteins--the nitrogen regulatory protein NrpR--that bind DNA and repress transcription of nitrogen assimilation genes. We used X-ray crystallography to determine the structure of NrpR regulatory domain. We identified the NrpR 2OG-binding cleft and show that residues predicted to interact directly with 2OG are conserved among diverse classes of 2OG-binding proteins. We show that high levels of 2OG inhibit NrpRs ability to bind DNA. Electron microscopy analyses document that NrpR adopts different quaternary structures in its inhibited 2OG-bound state compared with its active apo state. Our results indicate that upon 2OG release, NrpR repositions its DNA-binding domains correctly for optimal interaction with DNA thereby enabling gene repression.
Asunto(s)
Regulación de la Expresión Génica Arqueal/genética , Ácidos Cetoglutáricos/metabolismo , Methanococcus/química , Modelos Moleculares , Simulación de Dinámica Molecular , Proteínas PII Reguladoras del Nitrógeno/química , Conformación Proteica , Factores de Transcripción/química , Microscopía Electrónica , Nitrógeno/metabolismo , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The response regulator DosR is essential for promoting long-term survival of Mycobacterium tuberculosis under low oxygen conditions in a dormant state and may be responsible for latent tuberculosis in one-third of the world's population. Here, we report crystal structures of full-length unphosphorylated DosR at 2.2 A resolution and its C-terminal DNA-binding domain at 1.7 A resolution. The full-length DosR structure reveals several features never seen before in other response regulators. The N-terminal domain of the full-length DosR structure has an unexpected (beta alpha)(4) topology instead of the canonical (beta alpha)(5) fold observed in other response regulators. The linker region adopts a unique conformation that contains two helices forming a four-helix bundle with two helices from another subunit, resulting in dimer formation. The C-terminal domain in the full-length DosR structure displays a novel location of helix alpha 10, which allows Gln199 to interact with the catalytic Asp54 residue of the N-terminal domain. In contrast, the structure of the DosR C-terminal domain alone displays a remarkable unstructured conformation for helix alpha 10 residues, different from the well-defined helical conformations in all other known structures, indicating considerable flexibility within the C-terminal domain. Our structures suggest a mode of DosR activation by phosphorylation via a helix rearrangement mechanism.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Dimerización , Datos de Secuencia Molecular , Fosforilación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
The iron-dependent regulator IdeR is a key transcriptional regulator of iron uptake in Mycobacterium tuberculosis. In order to increase our insight into the role of the SH3-like third domain of this essential regulator, the metal-binding and DNA-binding properties of two-domain IdeR (2D-IdeR) whose SH3-like domain has been truncated were characterized. The equilibrium dissociation constants for Co2+ and Ni2+ activation of 2D-IdeR for binding to the fxbA operator and the DNA-binding affinities of 2D-IdeR in the presence of excess metal ions were estimated using fluorescence spectroscopy. 2D-IdeR binds to fxbA operator DNA with similar affinity as full-length IdeR in the presence of excess metal ion. However, the Ni2+ concentrations required to activate 2D-IdeR for DNA binding appear to be smaller than that for full-length IdeR while the concentration of Co2+ required for activation remains the same. We have determined the crystal structures of Ni2+-activated 2D-IdeR at 1.96 A resolution and its double dimer complex with the mbtA-mbtB operator DNA in two crystal forms at 2.4 A and 2.6 A, the highest resolutions for DNA complexes for any structures of iron-dependent regulator family members so far. The 2D-IdeR-DNA complex structures confirm the specificity of Ser37 and Pro39 for thymine bases and suggest preferential contacts of Gln43 to cytosine bases of the DNA. In addition, our 2D-IdeR structures reveal a remarkable property of the TEV cleavage sequence remaining after removal of the C-terminal His6. This C-terminal tail promotes crystal contacts by forming a beta-sheet with the corresponding tail of neighboring subunits in two unrelated structures of 2D-IdeR, one with and one without DNA. The contact-promoting properties of this C-terminal TEV cleavage sequence may be beneficial for crystallizing other proteins.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Cristalografía por Rayos X , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Dimerización , Cinética , Sustancias Macromoleculares , Metales/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/genética , Conformación de Ácido Nucleico , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genéticaRESUMEN
On encountering low oxygen conditions, DosR activates the transcription of 47 genes, promoting long-term survival of Mycobacterium tuberculosis in a non-replicating state. Here, we report the crystal structures of the DosR C-terminal domain and its complex with a consensus DNA sequence of the hypoxia-induced gene promoter. The DosR C-terminal domain contains four alpha-helices and forms tetramers consisting of two dimers with non-intersecting dyads. In the DNA-bound structure, each DosR C-terminal domain in a dimer places its DNA-binding helix deep into the major groove, causing two bends in the DNA. DosR makes numerous protein-DNA base contacts using only three amino acid residues per subunit: Lys179, Lys182, and Asn183. The DosR tetramer is unique among response regulators with known structures.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/fisiología , Oxígeno/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Cristalografía por Rayos X , ADN Bacteriano/química , ADN Bacteriano/genética , Dimerización , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Hipoxia/metabolismo , Hipoxia/microbiología , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Conformación de Ácido Nucleico , Oxígeno/farmacología , Unión Proteica , Estructura Cuaternaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína , Activación TranscripcionalRESUMEN
The iron-dependent regulator (IdeR) protein in Mycobacterium tuberculosis, and its better characterized homologue, the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae, are iron-dependent regulatory proteins that control gene expression in response to iron availability in bacteria. IdeR regulates several genes required for iron uptake and storage including those involved in the synthesis of transition metal chelators called siderophores that are linked to the M. tuberculosis virulence. In this study, the metal ion and binding affinities for IdeR binding to an fxbA operator duplex DNA were estimated using fluorescence assays. The Fe(2+), Co(2+), and Ni(2+) affinities of the two metal ion binding sites in IdeR that are involved in the activation of the regulator DNA binding process in vitro were independently estimated. Binding to the two metal ion binding sites is apparently cooperative and the two affinities differ significantly. Occupation of the first metal ion binding site causes dimerization of IdeR, and the metal ion affinity is about 4 microM for Ni(2+) and much less for Fe(2+) and Co(2+). Binding of the second metal ion fully activates IdeR for binding to the fxbA operator. The equilibrium metal ion dissociation constants for IdeR-fxbA operator binding are approximately 9 microM for Fe(2+), 13 microM for Ni(2+), and 23 microM for Co(2+). Interestingly, the natural IdeR cofactor, Fe(2+), shows high affinities toward both binding sites. These results provide insight into the possible roles for each metal binding site in IdeR activation.
Asunto(s)
Proteínas Bacterianas/fisiología , Mycobacterium tuberculosis/metabolismo , Proteínas Represoras/fisiología , Anisotropía , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , ADN/química , Proteínas de Unión al ADN/metabolismo , Dimerización , Relación Dosis-Respuesta a Droga , Iones/química , Hierro/química , Hierro/metabolismo , Cinética , Metales/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Níquel/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Represoras/química , Espectrometría de Fluorescencia , Triptófano/química , Dominios Homologos srcRESUMEN
The iron-dependent regulator (IdeR) is an essential protein in Mycobacterium tuberculosis that regulates iron uptake in this major pathogen. Under high iron concentrations, IdeR binds to several operator regions and represses transcription of target genes. Here, we report the first crystal structure of cobalt-activated IdeR bound to the mbtA-mbtB operator at 2.75 A resolution. IdeR binds to the DNA as a "double-dimer" complex with two dimers on opposite sides of the DNA duplex with the dimer axes deviating approximately 157 degrees. The asymmetric unit contains two such double-dimer complexes with a total molecular mass of 240 kDa. Two metal-binding sites are fully occupied with the SH3-like third domain adopting a "wedge" position to interact with the two other domains, and providing two ligands for the metal site 1 in all eight subunits per asymmetric unit. A putative sodium ion is observed to mediate interactions between Asp35 and DNA. There is a conformational change in the DNA-binding domain caused by a 6-9 degrees rotation of the helix-turn-helix motif with respect to the rest of the molecule upon binding to the DNA. Ser37 and Pro39 make specific interactions with conserved thymine bases while Gln43 makes non-specific contacts with different types of bases in different subunits. A "p1s2C3T4a5" base recognition pattern is proposed to be the basis for key interactions for each IdeR subunit with the DNA in the IdeR-DNA complex structure.