RESUMEN
Inositol diphosphates (PP-IPs), also known as inositol pyrophosphates, are high-energy cellular signaling codes involved in nutrient and regulatory responses. We report that the evolutionarily conserved gene product, Vip1, possesses autonomous kinase and pyrophosphatase domains capable of synthesis and destruction of D-1 PP-IPs. Our studies provide atomic-resolution structures of the PP-IP products and unequivocally define that the Vip1 gene product is a highly selective 1-kinase and 1-pyrophosphatase enzyme whose activities arise through distinct active sites. Kinetic analyses of kinase and pyrophosphatase parameters are consistent with Vip1 evolving to modulate levels of 1-IP7 and 1,5-IP8 Individual perturbations in kinase and pyrophosphatase activities in cells result in differential effects on vacuolar morphology and osmotic responses. Analogous to the dual-functional key energy metabolism regulator, phosphofructokinase 2, Vip1 is a kinase and pyrophosphatase switch whose 1-PP-IP products play an important role in a cellular adaptation.
Asunto(s)
Fosfatos de Inositol/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Difosfatos/metabolismo , Fosfatos de Inositol/fisiología , Cinética , Fosforilación , Fosfotransferasas (Aceptor del Grupo Fosfato)/fisiología , Pirofosfatasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
Inositol polyphosphate 1-phosphatase (INPP1) is a prototype member of metal-dependent/lithium-inhibited phosphomonoesterase protein family defined by a conserved three-dimensional core structure. Enzymes within this family function in distinct pathways including inositide signaling, gluconeogenesis, and sulfur assimilation. Using structural and biochemical studies, we report the effect of substrate and lithium on a network of metal binding sites within the catalytic center of INPP1. We find that lithium preferentially occupies a key site involved in metal-activation only when substrate or product is added. Mutation of a conserved residue that selectively coordinates the putative lithium-binding site results in a dramatic 100-fold reduction in the inhibitory constant as compared with wild-type. Furthermore, we report the INPP1/inositol 1,4-bisphosphate complex which illuminates key features of the enzyme active site. Our results provide insights into a structural basis for uncompetitive lithium inhibition and substrate recognition and define a sequence motif for metal binding within this family of regulatory phosphatases.
Asunto(s)
Litio/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Dominio Catalítico , Bovinos , Cristalografía por Rayos X , Gadolinio/metabolismo , Mutación , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/metabolismo , Células Sf9 , Especificidad por SustratoRESUMEN
Hsp90 chaperones contain an N-terminal ATP binding site that has been effectively targeted by competitive inhibitors. Despite the myriad of inhibitors, none to date have been designed to bind specifically to just one of the four mammalian Hsp90 paralogs, which are cytoplasmic Hsp90alpha and beta, endoplasmic reticulum GRP94, and mitochondrial Trap-1. Given that each of the Hsp90 paralogs is responsible for chaperoning a distinct set of client proteins, specific targeting of one Hsp90 paralog may result in higher efficacy and therapeutic control. Specific inhibitors may also help elucidate the biochemical roles of each Hsp90 paralog. Here, we present side-by-side comparisons of the structures of yeast Hsp90 and mammalian GRP94, bound to the pan-Hsp90 inhibitors geldanamycin (Gdm) and radamide. These structures reveal paralog-specific differences in the Hsp90 and GRP94 conformations in response to Gdm binding. We also report significant variation in the pose and disparate binding affinities for the Gdm-radicicol chimera radamide when bound to the two paralogs, which may be exploited in the design of paralog-specific inhibitors.
Asunto(s)
Diseño de Fármacos , Proteínas HSP70 de Choque Térmico , Proteínas HSP90 de Choque Térmico , Proteínas de la Membrana , Acetanilidas/química , Acetanilidas/metabolismo , Animales , Benzoatos/química , Benzoatos/metabolismo , Benzoquinonas/química , Benzoquinonas/metabolismo , Sitios de Unión , Cristalización , Perros , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/metabolismo , Ligandos , Macrólidos/química , Macrólidos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Estructura Terciaria de Proteína , Electricidad Estática , Difracción de Rayos XRESUMEN
Inositol hexakisphosphate kinases (IP6Ks) phosphorylate inositol hexakisphosphate (InsP(6)) to yield 5-diphosphoinositol pentakisphosphate (5-[PP]-InsP(5) or InsP(7)). In this study, we report the characterization of a selective inhibitor, N(2)-(m-(trifluoromethy)lbenzyl) N(6)-(p-nitrobenzyl)purine (TNP), for these enzymes. TNP dose-dependently and selectively inhibited the activity of IP6K in vitro and inhibited InsP(7) and InsP(8) synthesis in vivo without affecting levels of other inositol phosphates. TNP did not inhibit either human or yeast Vip/PPIP5K, a newly described InsP(6)/InsP(7) 1/3-kinase. Overexpression of IP6K1, -2, or -3 in cells rescued TNP inhibition of InsP(7) synthesis. TNP had no effect on the activity of a large number of protein kinases, suggesting that it is selective for IP6Ks. TNP reversibly reduced InsP(7)/InsP(8) levels. TNP in combination with genetic studies was used to implicate the involvement of two pathways for synthesis of InsP(8) in yeast. TNP induced a fragmented vacuole phenotype in yeast, consistent with inhibition of Kcs1, a Saccharomyces cerevisiae IP6K. In addition, it also inhibited insulin release from Min6 cells in a dose-dependent manner further implicating InsP(7) in this process. TNP thus provides a means of selectively and rapidly modulating cellular InsP(7) levels, providing a new and versatile tool to study the biological function and metabolic relationships of inositol pyrophosphates.
Asunto(s)
Difosfatos/metabolismo , Inhibidores Enzimáticos , Fosfatos de Inositol/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Purinas , Secuencia de Aminoácidos , Animales , Línea Celular , Difosfatos/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Fosfatos de Inositol/química , Insulina/metabolismo , Datos de Secuencia Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Purinas/química , Purinas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Tapsigargina/metabolismo , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
Eukaryotes possess numerous inositol phosphate (IP) and diphosphoinositol phosphate (PP-IPs or inositol pyrophosphates) species that act as chemical codes important for intracellular signaling pathways. Production of IP and PP-IP molecules occurs through several classes of evolutionarily conserved inositol phosphate kinases. Here we report the characterization of a human inositol hexakisphosphate (IP6) and diphosphoinositol pentakisphosphate (PP-IP5 or IP7) kinase with similarity to the yeast enzyme Vip1, a recently identified IP6/IP7 kinase (Mulugu, S., Bai, W., Fridy, P. C., Bastidas, R. J., Otto, J. C., Dollins, D. E., Haystead, T. A., Ribeiro, A. A., and York, J. D. (2007) Science 316, 106-109). Recombinant human VIP1 exhibits in vitro IP6 and IP7 kinase activities and restores IP7 synthesis when expressed in mutant yeast. Expression of human VIP1 in HEK293T cells engineered to produce high levels of IP7 results in dramatic increases in bisdiphosphoinositol tetrakisphosphate (PP2-IP4 or IP8). Northern blot analysis indicates that human VIP1 is expressed in a variety of tissues and is enriched in skeletal muscle, heart, and brain. The subcellular distribution of tagged human VIP1 is indicative of a cytoplasmic non-membrane localization pattern. We also characterized human and mouse VIP2, an additional gene product with nearly 90% similarity to VIP1 in the kinase domain, and observed both IP6 and IP7 kinase activities. Our data demonstrate that human VIP1 and VIP2 function as IP6 and IP7 kinases that act along with the IP6K/Kcs1-class of kinases to convert IP6 to IP8 in mammalian cells, a process that has been found to occur in response to various stimuli and signaling events.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Fosfatos de Inositol/biosíntesis , Fosfotransferasas (Aceptor del Grupo Fosfato)/biosíntesis , Proteínas Recombinantes/biosíntesis , Transducción de Señal/fisiología , Animales , Clonación Molecular , Citosol/enzimología , Humanos , Ratones , Especificidad de Órganos/fisiología , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Homología de Secuencia de AminoácidoRESUMEN
GRP94, an essential endoplasmic reticulum chaperone, is required for the conformational maturation of proteins destined for cell-surface display or export. The extent to which GRP94 and its cytosolic paralog, Hsp90, share a common mechanism remains controversial. GRP94 has not been shown conclusively to hydrolyze ATP or bind cochaperones, and both activities, by contrast, result in conformational changes and N-terminal dimerization in Hsp90 that are critical for its function. Here, we report the 2.4 A crystal structure of mammalian GRP94 in complex with AMPPNP and ADP. The chaperone is conformationally insensitive to the identity of the bound nucleotide, adopting a "twisted V" conformation that precludes N-terminal domain dimerization. We also present conclusive evidence that GRP94 possesses ATPase activity. Our observations provide a structural explanation for GRP94's observed rate of ATP hydrolysis and suggest a model for the role of ATP binding and hydrolysis in the GRP94 chaperone cycle.
Asunto(s)
Proteínas HSP70 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/química , Proteínas de la Membrana/química , Chaperonas Moleculares/química , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Animales , Cristalografía por Rayos X , Dimerización , Perros , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia MolecularRESUMEN
Inositol pyrophosphates are a diverse group of high-energy signaling molecules whose cellular roles remain an active area of study. We report a previously uncharacterized class of inositol pyrophosphate synthase and find it is identical to yeast Vip1 and Asp1 proteins, regulators of actin-related protein-2/3 (ARP 2/3) complexes. Vip1 and Asp1 acted as enzymes that encode inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase activities. Alterations in kinase activity led to defects in cell growth, morphology, and interactions with ARP complex members. The functionality of Asp1 and Vip1 may provide cells with increased signaling capacity through metabolism of IP6.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Ácido Fítico/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Alineación de Secuencia , Proteína 3 Relacionada con la Actina/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Secuencia Conservada , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/aislamiento & purificación , Humanos , Fosfatos de Inositol/metabolismo , Datos de Secuencia Molecular , Enzimas Multifuncionales , Fosforilación , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/aislamiento & purificación , Estructura Terciaria de Proteína , Pirofosfatasas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/aislamiento & purificación , Especificidad por Sustrato , TemperaturaRESUMEN
GRP94, the endoplasmic reticulum paralog of Hsp90, is regulated by adenosine nucleotides that bind to its N-terminal regulatory domain. Because of its weak affinity for nucleotides, the functionally relevant transition in GRP94 is likely to be between the unliganded and nucleotide-bound states. We have determined the structure of the unliganded GRP94 N-domain. The helix 1-4-5 subdomain of the unliganded protein adopts the closed conformation seen in the structure of the protein in complex with inhibitors. This conformation is distinct from the open conformation of the subdomain seen when the protein is bound to ATP or ADP. ADP soaked into crystals of the unliganded protein reveals an intermediate conformation midway between the open and closed states and demonstrates that in GRP94 the conversion between the open and closed states is driven by ligand binding. The direction of the observed movement in GRP94 shows that nucleotides act to open the subdomain elements rather than close them, which is contrary to the motion proposed for Hsp90. These observations support a model where ATP binding dictates the conformation of the N-domain and regulates its ability to form quaternary structural interactions.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de la Membrana/química , Adenosina Difosfato/química , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Secuencia Conservada , Cristalografía por Rayos X , Electrones , Escherichia coli/metabolismo , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Steroid receptors bind as dimers to a degenerate set of response elements containing inverted repeats of a hexameric half-site separated by 3 bp of spacer (IR3). Naturally occurring selective androgen response elements have recently been identified that resemble direct repeats of the hexameric half-site (ADR3). The 3D crystal structure of the androgen receptor (AR) DNA-binding domain bound to a selective ADR3 reveals an unexpected head-to-head arrangement of the two protomers rather than the expected head-to-tail arrangement seen in nuclear receptors bound to response elements of similar geometry. Compared with the glucocorticoid receptor, the DNA-binding domain dimer interface of the AR has additional interactions that stabilize the AR dimer and increase the affinity for nonconsensus response elements. This increased interfacial stability compared with the other steroid receptors may account for the selective binding of AR to ADR3 response elements.
Asunto(s)
Andrógenos/metabolismo , Receptores Androgénicos/metabolismo , Animales , Secuencia de Bases , ADN , Dimerización , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Ratas , Receptores Androgénicos/química , Receptores Androgénicos/genéticaRESUMEN
GRP94 is the endoplasmic reticulum paralog of cytoplasmic Hsp90. Models of Hsp90 action posit an ATP-dependent conformational switch in the N-terminal ligand regulatory domain of the chaperone. However, crystal structures of the isolated N-domain of Hsp90 in complex with a variety of ligands have yet to demonstrate such a conformational change. We have determined the structure of the N-domain of GRP94 in complex with ATP, ADP, and AMP. Compared with the N-ethylcarboxamidoadenosine and radicicol-bound forms, these structures reveal a large conformational rearrangement in the protein. The nucleotide-bound form exposes new surfaces that interact to form a biochemically plausible dimer that is reminiscent of those seen in structures of MutL and DNA gyrase. Weak ATP binding and a conformational change in response to ligand identity are distinctive mechanistic features of GRP94 and suggest a model for how GRP94 functions in the absence of co-chaperones and ATP hydrolysis.