RESUMEN
ATP-binding cassette subfamily B member 7 (ABCB7) is localized in the inner membrane of mitochondria, playing a critical role in iron metabolism. Here, we determined the structure of the nonhydrolyzable ATP analog adenosine-5'-(ß-γ-imido) triphosphate (AMP-PNP) bound human ABCB7 at 3.3 Å by single-particle electron cryo-microscopy (cryo-EM). The AMP-PNP-bound human ABCB7 shows an inverted V-shaped homodimeric architecture with an inward-facing open conformation. One AMP-PNP molecule and Mg2+ were identified in each nucleotide-binding domain (NBD) of the hABCB7 monomer. Moreover, four disease-causing missense mutations of human ABCB7 have been mapped to the structure, creating a hotspot map for X-linked sideroblastic anemia and ataxia disease. Our results provide a structural basis for further understanding the transport mechanism of the mitochondrial ABC transporter.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Anemia Sideroblástica , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Adenilil Imidodifosfato/metabolismo , Anemia Sideroblástica/genética , Anemia Sideroblástica/metabolismo , Microscopía por Crioelectrón , Humanos , Mitocondrias/metabolismoRESUMEN
Cellular RNAs depend on proteins for efficient folding to specific functional structures and for transitions between functional structures. This dependence arises from intrinsic properties of RNA structure. Specifically, RNAs possess stable local structure, largely in the form of helices, and there are abundant opportunities for RNAs to form alternative helices and tertiary contacts and therefore to populate alternative structures. Proteins with RNA chaperone activity, either ATP-dependent or ATP-independent, can promote structural transitions by interacting with single-stranded RNA (ssRNA) to compete away partner interactions and then release ssRNA so that it can form new interactions. In this chapter we review the basic properties of RNA and the proteins that function as chaperones and remodelers. We then use these properties as a foundation to explore key points for the design and interpretation of experiments that probe RNA rearrangements and their acceleration by proteins.
Asunto(s)
Proteínas , ARN , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Conformación de Ácido Nucleico , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas/química , Proteínas/metabolismo , ARN/química , ARN/metabolismoRESUMEN
The P-glycoprotein (Pgp) transporter plays a central role in drug disposition by effluxing a chemically diverse range of drugs from cells through conformational changes and ATP hydrolysis. A number of drugs are known to activate ATP hydrolysis of Pgp, but coupling between ATP and drug binding is not well understood. The cardiovascular drug verapamil is one of the most widely studied Pgp substrates and therefore, represents an ideal drug to investigate the drug-induced ATPase activation of Pgp. As previously noted, verapamil-induced Pgp-mediated ATP hydrolysis kinetics was biphasic at saturating ATP concentrations. However, at subsaturating ATP concentrations, verapamil-induced ATPase activation kinetics became monophasic. To further understand this switch in kinetic behavior, the Pgp-coupled ATPase activity kinetics was checked with a panel of verapamil and ATP concentrations and fit with the substrate inhibition equation and the kinetic fitting software COPASI. The fits suggested that cooperativity between ATP and verapamil switched between low and high verapamil concentration. Fluorescence spectroscopy of Pgp revealed that cooperativity between verapamil and a non-hydrolyzable ATP analog leads to distinct global conformational changes of Pgp. NMR of Pgp reconstituted in liposomes showed that cooperativity between verapamil and the non-hydrolyzable ATP analog modulate each other's interactions. This information was used to produce a conformationally-gated model of drug-induced activation of Pgp-mediated ATP hydrolysis.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/agonistas , Adenosina Trifosfato/metabolismo , Antiarrítmicos/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Modelos Moleculares , Verapamilo/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/química , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Algoritmos , Animales , Antiarrítmicos/química , Antiarrítmicos/farmacología , Sitios de Unión , Biocatálisis/efectos de los fármacos , Bloqueadores de los Canales de Calcio/química , Bloqueadores de los Canales de Calcio/farmacología , Simulación por Computador , Hidrólisis/efectos de los fármacos , Ligandos , Liposomas , Ratones , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Verapamilo/química , Verapamilo/farmacologíaRESUMEN
In kinesin X-ray crystal structures, the N-terminal region of the α-1 helix is adjacent to the adenine ring of the bound nucleotide, while the C-terminal region of the helix is near the neck-linker (NL). Here, we monitor the displacement of the α-1 helix within a kinesin monomer bound to microtubules (MTs) in the presence or absence of nucleotides using site-directed spin labeling EPR. Kinesin was doubly spin-labeled at the α-1 and α-2 helices, and the resulting EPR spectrum showed dipolar broadening. The inter-helix distance distribution showed that 20% of the spins have a peak characteristic of 1.4-1.7 nm separation, which is similar to what is predicted from the X-ray crystal structure, albeit 80% were beyond the sensitivity limit (>2.5 nm) of the method. Upon MT binding, the fraction of kinesin exhibiting an inter-helix distance of 1.4-1.7 nm in the presence of AMPPNP (a non-hydrolysable ATP analog) and ADP was 20% and 25%, respectively. In the absence of nucleotide, this fraction increased to 40-50%. These nucleotide-induced changes in the fraction of kinesin undergoing displacement of the α-1 helix were found to be related to the fraction in which the NL undocked from the motor core. It is therefore suggested that a shift in the α-1 helix conformational equilibrium occurs upon nucleotide binding and release, and this shift controls NL docking onto the motor core.
Asunto(s)
Cinesinas/química , Cinesinas/metabolismo , Nucleótidos/metabolismo , Marcadores de Spin , Adenosina Trifosfatasas/metabolismo , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Microtúbulos/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Estructura Secundaria de Proteína , RotaciónRESUMEN
Surface plasmon resonance (SPR) spectroscopy is a rapidly developing technique for the study of ligand binding interactions with membrane proteins, which are the major molecular targets for validated drugs and for current and foreseeable drug discovery. SPR is label-free and capable of measuring real-time quantitative binding affinities and kinetics for membrane proteins interacting with ligand molecules using relatively small quantities of materials and has potential to be medium-throughput. The conventional SPR technique requires one binding component to be immobilised on a sensor chip whilst the other binding component in solution is flowed over the sensor surface; a binding interaction is detected using an optical method that measures small changes in refractive index at the sensor surface. This review first describes the basic SPR experiment and the challenges that have to be considered for performing SPR experiments that measure membrane protein-ligand binding interactions, most importantly having the membrane protein in a lipid or detergent environment that retains its native structure and activity. It then describes a wide-range of membrane protein systems for which ligand binding interactions have been characterised using SPR, including the major drug targets G protein-coupled receptors, and how challenges have been overcome for achieving this. Finally it describes some recent advances in SPR-based technology and future potential of the technique to screen ligand binding in the discovery of drugs. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.
Asunto(s)
Descubrimiento de Drogas , Proteínas de la Membrana/metabolismo , Resonancia por Plasmón de Superficie/métodos , Ligandos , Unión Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
The function of F1-ATPase relies critically on the intrinsic ability of its catalytic and noncatalytic subunits to interact with nucleotides. Therefore, the study of isolated subunits represents an opportunity to dissect elementary energetic contributions that drive the enzyme's rotary mechanism. In this study we have calorimetrically characterized the association of adenosine nucleotides to the isolated noncatalytic α-subunit. The resulting recognition behavior was compared with that previously reported for the isolated catalytic ß-subunit (N.O. Pulido, G. Salcedo, G. Pérez-Hernández, C. José-Núñez, A. Velázquez-Campoy, E. García-Hernández, Energetic effects of magnesium in the recognition of adenosine nucleotides by the F1-ATPase ß subunit, Biochemistry 49 (2010) 5258-5268). The two subunits exhibit nucleotide-binding thermodynamic signatures similar to each other, characterized by enthalpically-driven affinities in the µM range. Nevertheless, contrary to the catalytic subunit that recognizes MgATP and MgADP with comparable strength, the noncatalytic subunit much prefers the triphosphate nucleotide. Besides, the α-subunit depends more on Mg(II) for stabilizing the interaction with ATP, while both subunits are rather metal-independent for ADP recognition. These binding behaviors are discussed in terms of the properties that the two subunits exhibit in the whole enzyme.
Asunto(s)
Adenosina/química , Dominio Catalítico , Metabolismo Energético , ATPasas de Translocación de Protón/química , Adenosina/metabolismo , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Calorimetría , Proteínas de Unión al ADN/química , Escherichia coli/enzimología , Cinética , Magnesio/química , Magnesio/metabolismo , Nucleótidos/metabolismo , ATPasas de Translocación de Protón/aislamiento & purificación , ATPasas de Translocación de Protón/metabolismo , TermodinámicaRESUMEN
We investigated the interacting amino acids critical for the stability and ATP binding of Mycobacterium tuberculosis PII protein through a series of site specific mutagenesis experiments. We assessed the effect of mutants using glutaraldehyde crosslinking and size exclusion chromatography and isothermal titration calorimetry. Mutations in the amino acid pair R60-E62 affecting central electrostatic interaction resulted in insoluble proteins. Multiple sequence alignment of PII orthologs displayed a conserved pattern of charged residues at these positions. Mutation of amino acid D97 to a neutral residue was tolerated whereas positive charge was not acceptable. Mutation of R107 alone had no effect on trimer formation. However, the combination of neutral residues both at positions 97 and 107 was not acceptable even with the pair at 60-62 intact. Reversal of charge polarity could partially restore the interaction. The residues including K90, R101 and R103 with potential to form H-bonds to ATP are conserved throughout across numerous orthologs of PII but when mutated to Alanine, they did not show significant differences in the total free energy change of the interaction as examined through isothermal titration calorimetry. The ATP binding pattern showed anti-cooperativity using three-site binding model. We observed compensatory effect in enthalpy and entropy changes and these may represent structural adjustments to accommodate ATP in the cavity even in absence of some interactions to perform the requisite function. In this respect these small differences between the PII orthologs may have evolved to suite species specific physiological niches.
Asunto(s)
Adenosina Trifosfato/química , Proteínas Bacterianas/química , Modelos Moleculares , Mycobacterium tuberculosis/química , Proteínas PII Reguladoras del Nitrógeno/química , Multimerización de Proteína/fisiología , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Sustitución de Aminoácidos , Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Evolución Molecular , Mutación Missense , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas PII Reguladoras del Nitrógeno/genética , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Kinesin motor proteins comprise an ATPase superfamily that works hand in hand with microtubules in every eukaryote. The mitotic kinesins, by virtue of their potential therapeutic role in cancerous cells, have been a major focus of research for the past 28 years since the discovery of the canonical Kinesin-1 heavy chain. Perhaps the simplest player in mitotic spindle assembly, Kinesin-5 (also known as Kif11, Eg5, or kinesin spindle protein, KSP) is a plus-end-directed motor localized to interpolar spindle microtubules and to the spindle poles. Comprised of a homotetramer complex, its function primarily is to slide anti-parallel microtubules apart from one another. Based on multi-faceted analyses of this motor from numerous laboratories over the years, we have learned a great deal about the function of this motor at the atomic level for catalysis and as an integrated element of the cytoskeleton. These data have, in turn, informed the function of motile kinesins on the whole, as well as spearheaded integrative models of the mitotic apparatus in particular and regulation of the microtubule cytoskeleton in general. We review what is known about how this nanomotor works, its place inside the cytoskeleton of cells, and its small-molecule inhibitors that provide a toolbox for understanding motor function and for anticancer treatment in the clinic.