RESUMEN
The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near-atomic resolution. Here, we report the development of methods to account for local variations in defocus and beam-induced drift, and the implementation of a data-driven dose compensation scheme that significantly improves the extraction of high-resolution information recorded during exposure of the specimen to the electron beam. These advances enable determination of a cryo-EM density map for ß-galactosidase bound to the inhibitor phenylethyl ß-D-thiogalactopyranoside where the ordered regions are resolved at a level of detail seen in X-ray maps at â¼ 1.5 Å resolution. Using this density map in conjunction with constrained molecular dynamics simulations provides a measure of the local flexibility of the non-covalently bound inhibitor and offers further opportunities for structure-guided inhibitor design.
Asunto(s)
Tiogalactósidos/farmacología , beta-Galactosidasa/química , beta-Galactosidasa/metabolismo , Sitios de Unión , Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X , Diseño de Fármacos , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ â¼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.
Asunto(s)
Descubrimiento de Drogas , Glutamato Deshidrogenasa/ultraestructura , Isocitrato Deshidrogenasa/ultraestructura , L-Lactato Deshidrogenasa/ultraestructura , Aminoquinolinas/química , Aminoquinolinas/farmacología , Animales , Bovinos , Pollos , Microscopía por Crioelectrón , Cristalografía por Rayos X , Glutamato Deshidrogenasa/antagonistas & inhibidores , Glutamato Deshidrogenasa/química , Humanos , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Isocitrato Deshidrogenasa/química , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/química , Modelos Moleculares , Conformación Proteica , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.
Asunto(s)
Microscopía por Crioelectrón/métodos , Glutamato Deshidrogenasa/ultraestructura , Animales , Bovinos , Cristalografía por Rayos X , Glutamato Deshidrogenasa/química , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Ligandos , Mamíferos , Modelos Moleculares , NAD/química , NAD/metabolismo , Estructura Cuaternaria de ProteínaRESUMEN
p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo-electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)-bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5'-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/química , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Adenosina Difosfato/química , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Regulación Alostérica , Sitios de Unión , Microscopía por Crioelectrón , Inhibidores Enzimáticos , Humanos , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
Cryo-electron microscopy (cryo-EM) is rapidly emerging as a powerful tool for protein structure determination at high resolution. Here we report the structure of a complex between Escherichia coli ß-galactosidase and the cell-permeant inhibitor phenylethyl ß-D-thiogalactopyranoside (PETG), determined by cryo-EM at an average resolution of ~2.2 angstroms (Å). Besides the PETG ligand, we identified densities in the map for ~800 water molecules and for magnesium and sodium ions. Although it is likely that continued advances in detector technology may further enhance resolution, our findings demonstrate that preparation of specimens of adequate quality and intrinsic protein flexibility, rather than imaging or image-processing technologies, now represent the major bottlenecks to routinely achieving resolutions close to 2 Å using single-particle cryo-EM.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Tiogalactósidos/química , beta-Galactosidasa/química , Dominio Catalítico , Microscopía por Crioelectrón , Cristalografía por Rayos X , Agua/químicaRESUMEN
Poor partitioning of macromolecules into the holes of holey carbon support grids frequently limits structural determination by single particle cryo-electron microscopy (cryo-EM). Here, we present a method to deposit, on gold-coated carbon grids, a self-assembled monolayer whose surface properties can be controlled by chemical modification. We demonstrate the utility of this approach to drive partitioning of ionotropic glutamate receptors into the holes, thereby enabling 3D structural analysis using cryo-EM methods.
Asunto(s)
Proteínas Bacterianas/química , Chaperonina 60/química , Microscopía por Crioelectrón/instrumentación , Receptores AMPA/química , Receptores de Ácido Kaínico/química , Animales , Carbono/química , Oro/química , Porosidad , Ratas , Humectabilidad , Receptor de Ácido Kaínico GluK2RESUMEN
We report the solution structure of Escherichia coli ß-galactosidase (â¼465 kDa), solved at â¼3.2-Å resolution by using single-particle cryo-electron microscopy (cryo-EM). Densities for most side chains, including those of residues in the active site, and a catalytic Mg(2+) ion can be discerned in the map obtained by cryo-EM. The atomic model derived from our cryo-EM analysis closely matches the 1.7-Å crystal structure with a global rmsd of â¼0.66 Å. There are significant local differences throughout the protein, with clear evidence for conformational changes resulting from contact zones in the crystal lattice. Inspection of the map reveals that although densities for residues with positively charged and neutral side chains are well resolved, systematically weaker densities are observed for residues with negatively charged side chains. We show that the weaker densities for negatively charged residues arise from their greater sensitivity to radiation damage from electron irradiation as determined by comparison of density maps obtained by using electron doses ranging from 10 to 30 e(-)/Å(2). In summary, we establish that it is feasible to use cryo-EM to determine near-atomic resolution structures of protein complexes (<500 kDa) with low symmetry, and that the residue-specific radiation damage that occurs with increasing electron dose can be monitored by using dose fractionation tools available with direct electron detector technology.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestructura , beta-Galactosidasa/química , beta-Galactosidasa/ultraestructura , Fenómenos Biofísicos , Dominio Catalítico , Microscopía por Crioelectrón , Cristalografía por Rayos X , Escherichia coli/enzimología , Modelos Moleculares , Conformación Proteica , Estructura Cuaternaria de Proteína , Electricidad EstáticaRESUMEN
PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin's ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.
Asunto(s)
Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Sustitución de Aminoácidos , Animales , Línea Celular , Drosophila melanogaster , Activación Enzimática/fisiología , Humanos , Mutación Missense , Fosforilación/fisiología , Proteínas Quinasas/genética , Estructura Terciaria de Proteína , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8-11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas con Dominio LIM/metabolismo , Mutación Missense , Miofibrillas/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/patología , Actinina/genética , Actinina/metabolismo , Actinas/genética , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos , Animales , Línea Celular , Conectina/genética , Conectina/metabolismo , Humanos , Proteínas con Dominio LIM/genética , Ratones , Proteínas de Microfilamentos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miofibrillas/genética , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/metabolismo , Miopatías Estructurales Congénitas/patología , Estructura Terciaria de ProteínaRESUMEN
Genetic studies indicate that the mitochondrial kinase PINK1 and the RING-between-RING E3 ubiquitin ligase Parkin function in the same pathway. In concurrence, mechanistic studies show that PINK1 can recruit Parkin from the cytosol to the mitochondria, increase the ubiquitination activity of Parkin, and induce Parkin-mediated mitophagy. Here, we used a cell-free assay to recapitulate PINK1-dependent activation of Parkin ubiquitination of a validated mitochondrial substrate, mitofusin 1. We show that PINK1 activated the formation of a Parkin-ubiquitin thioester intermediate, a hallmark of HECT E3 ligases, both in vitro and in vivo. Parkin HECT-like ubiquitin ligase activity was essential for PINK1-mediated Parkin translocation to mitochondria and mitophagy. Using an inactive Parkin mutant, we found that PINK1 stimulated Parkin self-association and complex formation upstream of mitochondrial translocation. Self-association occurred independent of ubiquitination activity through the RING-between-RING domain, providing mechanistic insight into how PINK1 activates Parkin.
Asunto(s)
Mitocondrias/enzimología , Proteínas Quinasas/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Sistema Libre de Células , Citosol/enzimología , Células HeLa , Humanos , Modelos Biológicos , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The Bcl-2 family member Bax translocates from the cytosol to mitochondria, where it oligomerizes and permeabilizes the mitochondrial outer membrane to promote apoptosis. Bax activity is counteracted by prosurvival Bcl-2 proteins, but how they inhibit Bax remains controversial because they neither colocalize nor form stable complexes with Bax. We constrained Bax in its native cytosolic conformation within cells using intramolecular disulfide tethers. Bax tethers disrupt interaction with Bcl-x(L) in detergents and cell-free MOMP activity but unexpectedly induce Bax accumulation on mitochondria. Fluorescence loss in photobleaching (FLIP) reveals constant retrotranslocation of WT Bax, but not tethered Bax, from the mitochondria into the cytoplasm of healthy cells. Bax retrotranslocation depends on prosurvival Bcl-2 family proteins, and inhibition of retrotranslocation correlates with Bax accumulation on the mitochondria. We propose that Bcl-x(L) inhibits and maintains Bax in the cytosol by constant retrotranslocation of mitochondrial Bax.
Asunto(s)
Citosol/metabolismo , Mitocondrias/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Apoptosis , Línea Celular Tumoral , Humanos , Conformación Proteica , Pliegue de Proteína , Transporte de Proteínas , Proteína X Asociada a bcl-2/químicaRESUMEN
In mammals, fusion of the mitochondrial outer membrane is controlled by two DRPs, MFN1 and MFN2, that function in place of a single outer membrane DRP, Fzo1 in yeast. We addressed the significance of two mammalian outer membrane fusion DRPs using an in vitro mammalian mitochondrial fusion assay. We demonstrate that heterotypic MFN1-MFN2 trans complexes possess greater efficacy in fusion as compared to homotypic MFN1 or MFN2 complexes. In addition, we show that the soluble form of the proapoptotic Bcl2 protein, Bax, positively regulates mitochondrial fusion exclusively through homotypic MFN2 trans complexes. Together, these data demonstrate functional and regulatory distinctions between MFN1 and MFN2 and provide insight into their unique physiological roles.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/fisiología , Proteína X Asociada a bcl-2/fisiología , Animales , Células Cultivadas , Ratones , Mitocondrias/ultraestructura , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Solubilidad , Proteína X Asociada a bcl-2/químicaRESUMEN
Endophilins participate in membrane scission events that occur during endocytosis and intracellular organelle biogenesis through the combined activity of an N-terminal BAR domain that interacts with membranes and a C-terminal SH3 domain that mediates protein binding. Endophilin B1 (Endo B1) was identified to bind Bax, a Bcl-2 family member that promotes apoptosis, through yeast two-hybrid protein screens. Although Endo B1 does not bind Bax in healthy cells, during apoptosis, Endo B1 interacts transiently with Bax and promotes cytochrome c release from mitochondria. To explore the molecular mechanism of action of Endo B1, we have analyzed its interaction with Bax in cell-free systems. Purified recombinant Endo B1 in solution displays a Stokes radius indicating a tetrameric quarternary structure. However, when incubated with purified Bax, it assembles into oligomers more than 4-fold greater in molecular weight. Although Endo B1 oligomerization is induced by Bax, Bax does not stably associate with the high molecular weight Endo B1 complex. Endo B1 oligomerization requires its C-terminal Src homology 3 domain and is not induced by Bcl-xL. Endo B1 combined with Bax reduces the size and changes the morphology of giant unilamellar vesicles by inducing massive vesiculation of liposomes. This activity of purified Bax protein to induce cell-free assembly of Endo B1 may reflect its activity in cells that regulates apoptosis and/or mitochondrial fusion.
Asunto(s)
Aciltransferasas/química , Lípidos de la Membrana/química , Proteína X Asociada a bcl-2/química , Apoptosis , Catálisis , Sistema Libre de Células , ADN Complementario/metabolismo , Endocitosis , Humanos , Liposomas/química , Microscopía Fluorescente/métodos , Modelos Estadísticos , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteína bcl-X/químicaRESUMEN
A number of bacterial metal transporters belong to the cluster 9 family of ABC transporters. The residues in the periplasmic domain thought to be involved in metal binding seem highly conserved and yet the transporters have varying metal specificity. To solve this seeming paradox and ascertain how metal specificity is exacted, the structure of ZnuA, the periplasmic domain of a zinc transporter from Synechocystis 6803, has been determined to a resolution of 1.9A. In previously determined structures of homologous proteins, four residues chelate the bound metal. From sequence alignments of the cluster 9 metal transporters, the fourth residue in this metal-binding site, an aspartate, is also present in the appropriate position in the ZnuA sequence. However, this result is misleading, since our structural data indicate that zinc binds via only three histidine residues and the aspartate is replaced by a large hydrophobic cavity. We propose that ZnuA binds zinc over manganese by providing only three ligating residues. ZnuA has a highly charged and mobile loop that protrudes from the protein in the vicinity of the metal-binding site. Similar loops are found in other types of zinc transporters but not manganese transporters. Therefore, we propose that the function of this domain is to act as a zinc chaperone to facilitate acquisition. Therefore, while Mn2+ transporters can bind Zn2+ in vitro they may not be able to acquire it in vivo without this structure because of the low concentration of free Zn2+.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Cianobacterias/metabolismo , Zinc/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Portadoras/química , Cianobacterias/química , Datos de Secuencia Molecular , Estructura Terciaria de ProteínaRESUMEN
Glutamate dehydrogenase (GDH) is found in all organisms and catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate. Unlike GDH from bacteria, mammalian GDH exhibits negative cooperativity with respect to coenzyme, activation by ADP, and inhibition by GTP. Presented here are the structures of apo bovine GDH, bovine GDH complexed with ADP, and the R463A mutant form of human GDH (huGDH) that is insensitive to ADP activation. In the absence of active site ligands, the catalytic cleft is in the open conformation, and the hexamers form long polymers in the crystal cell with more interactions than found in the abortive complex crystals. This is consistent with the fact that ADP promotes aggregation in solution. ADP is shown to bind to the second, inhibitory, NADH site yet causes activation. The beta-phosphates of the bound ADP interact with R459 (R463 in huGDH) on the pivot helix. The structure of the ADP-resistant, R463A mutant of human GDH is identical to native GDH with the exception of the truncated side chain on the pivot helix. Together, these results strongly suggest that ADP activates by facilitating the opening of the catalytic cleft. From alignment of GDH from various sources, it is likely that the antenna evolved in the protista prior to the formation of purine regulatory sites. This suggests that there was some selective advantage of the antenna itself and that animals evolved new functions for GDH through the addition of allosteric regulation.