RESUMEN
The circadian clock is a biological timekeeper that operates through transcription-translation feedback loops in mammals. Cryptochrome 1 (CRY1) and Cryptochrome 2 (CRY2) are highly conserved core clock components having redundant and distinct functions. We recently identified the CRY1- and CRY2-selective compounds KL101 and TH301, respectively, which provide useful tools for the exploration of isoform-selective CRY regulation. However, intrinsic differences in the compound-binding FAD (flavin adenine dinucleotide) pockets between CRY1 and CRY2 are not well understood, partly because of nonoptimal properties of previously reported apo form structures in this particular region constituted by almost identical sequences. Here, we show unliganded CRY1 and CRY2 crystal structures with well-defined electron densities that are largely free of crystal contacts at the FAD pocket and nearby lid loop. We revealed conformational isomerism in key residues. In particular, CRY1 W399 and corresponding CRY2 W417 in the FAD pocket had distinct conformations ("out" for CRY1 and "in" for CRY2) by interacting with the lid loop residues CRY1 Q407 and CRY2 F424, respectively, resulting in different overall lid loop structures. Molecular dynamics simulations supported that these conformations were energetically favorable to each isoform. Isoform-selective compounds KL101 and TH301 preferred intrinsic "out" and "in" conformations of the tryptophan residue in CRY1 and CRY2, respectively, while the nonselective compound KL001 fit to both conformations. Mutations of lid loop residues designed to perturb their isoform-specific interaction with the tryptophan resulted in reversed responses of CRY1 and CRY2 to KL101 and TH301. We propose that these intrinsic structural differences of CRY1 and CRY2 can be targeted for isoform-selective regulation.
Asunto(s)
Criptocromos/química , Criptocromos/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Mamíferos/metabolismo , Animales , Sitios de Unión , Criptocromos/genética , Cristalografía por Rayos X , Simulación de Dinámica Molecular , Mutación/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Cryptochrome 1 (CRY1) and CRY2 are core regulators of the circadian clock, and the development of isoform-selective modulators is important for the elucidation of their redundant and distinct functions. Here, we report the identification and functional characterization of a small-molecule modulator of the mammalian circadian clock that selectively controls CRY1. Cell-based circadian chemical screening identified a thienopyrimidine derivative KL201 that lengthened the period of circadian rhythms in cells and tissues. Functional assays revealed stabilization of CRY1 but not CRY2 by KL201. A structure-activity relationship study of KL201 derivatives in combination with X-ray crystallography of the CRY1-KL201 complex uncovered critical sites and interactions required for CRY1 regulation. KL201 bound to CRY1 in overlap with FBXL3, a subunit of ubiquitin ligase complex, and the effect of KL201 was blunted by knockdown of FBXL3. KL201 will facilitate isoform-selective regulation of CRY1 to accelerate chronobiology research and therapeutics against clock-related diseases.
Asunto(s)
Carbazoles/metabolismo , Ritmo Circadiano , Criptocromos/metabolismo , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Sitios de Unión , Carbazoles/química , Carbazoles/farmacología , Línea Celular Tumoral , Ritmo Circadiano/efectos de los fármacos , Criptocromos/química , Criptocromos/genética , Cristalografía por Rayos X , Proteínas F-Box/metabolismo , Genes Reporteros , Humanos , Simulación del Acoplamiento Molecular , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Unión Proteica , Relación Estructura-Actividad , UbiquitinaciónRESUMEN
CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY-compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure.
Asunto(s)
Criptocromos/química , Isoformas de Proteínas/química , Animales , Sitios de Unión , Relojes Circadianos , Criptocromos/genética , Fibroblastos/metabolismo , Células HEK293 , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Ratones Noqueados , Modelos Moleculares , Unión Proteica , Conformación Proteica , Isoformas de Proteínas/genética , TermodinámicaRESUMEN
Compounds targeting the circadian clock have been identified as potential treatments for clock-related diseases, including cancer. Our cell-based phenotypic screen revealed uncharacterized clock-modulating compounds. Through affinity-based target deconvolution, we identified GO289, which strongly lengthened circadian period, as a potent and selective inhibitor of CK2. Phosphoproteomics identified multiple phosphorylation sites inhibited by GO289 on clock proteins, including PER2 S693. Furthermore, GO289 exhibited cell type-dependent inhibition of cancer cell growth that correlated with cellular clock function. The x-ray crystal structure of the CK2α-GO289 complex revealed critical interactions between GO289 and CK2-specific residues and no direct interaction of GO289 with the hinge region that is highly conserved among kinases. The discovery of GO289 provides a direct link between the circadian clock and cancer regulation and reveals unique design principles underlying kinase selectivity.
Asunto(s)
Carcinoma de Células Renales/metabolismo , Proliferación Celular/efectos de los fármacos , Relojes Circadianos/efectos de los fármacos , Ritmo Circadiano/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Neoplasias Renales/metabolismo , Animales , Proteínas CLOCK/metabolismo , Carcinoma de Células Renales/patología , Quinasa de la Caseína II/antagonistas & inhibidores , Línea Celular Tumoral , Cristalografía por Rayos X , Células HEK293 , Humanos , Neoplasias Renales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación/efectos de los fármacosRESUMEN
Coenzyme A (CoA) plays pivotal roles in a variety of metabolic pathways in all organisms. The biosynthetic pathway of CoA is strictly regulated by feedback inhibition. In the hyperthermophilic archaeon Thermococcus kodakarensis, ketopantoate reductase (KPR), which catalyzes the NAD(P)H-dependent reduction of 2-oxopantoate, is a target of feedback inhibition by CoA. The crystal structure of KPR from T. kodakarensis (Tk-KPR) complexed with CoA and 2-oxopantoate has previously been reported. The structure provided an explanation for the competitive inhibition mechanism. Here, further biochemical analyses of Tk-KPR and the crystal structure of Tk-KPR in complex with NADP(+) are reported. A mutational analysis implies that the residues in the binding pocket cooperatively contribute to the recognition of CoA. The structure reveals the same dimer architecture as the Tk-KPR-CoA-2-oxopantoate complex. Moreover, the positions of the residues involved in the dimer interaction are not changed by the binding of CoA and 2-oxopantoate, suggesting individual conformational changes of Tk-KPR monomers.
Asunto(s)
Oxidorreductasas de Alcohol/química , NADP/química , Thermococcus/enzimología , Oxidorreductasas de Alcohol/genética , Coenzima A/química , Cristalografía por Rayos X , Dimerización , Estabilidad de Enzimas , Mutación , Conformación ProteicaRESUMEN
Coenzyme A (CoA) plays essential roles in a variety of metabolic pathways in all three domains of life. The biosynthesis pathway of CoA is strictly regulated by feedback inhibition. In bacteria and eukaryotes, pantothenate kinase is the target of feedback inhibition by CoA. Recent biochemical studies have identified ketopantoate reductase (KPR), which catalyzes the NAD(P)H-dependent reduction of 2-oxopantoate to pantoate, as a target of the feedback inhibition by CoA in archaea. However, the mechanism for recognition of CoA by KPR is still unknown. Here we report the crystal structure of KPR from Thermococcus kodakarensis in complex with CoA and 2-oxopantoate. CoA occupies the binding site of NAD(P)H, explaining the competitive inhibition by CoA. Our structure reveals a disulfide bond between CoA and Cys84 that indicates an irreversible inhibition upon binding of CoA. The structure also suggests the cooperative binding of CoA and 2-oxopantoate that triggers a conformational closure and seems to facilitate the disulfide bond formation. Our findings provide novel insights into the mechanism that regulates biosynthesis of CoA in archaea.
Asunto(s)
Oxidorreductasas de Alcohol/química , Proteínas Arqueales/química , Coenzima A/química , Hidroxibutiratos/química , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Retroalimentación Fisiológica , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Especificidad por SustratoRESUMEN
Proper protein folding is an essential process for all organisms. Prefoldin (PFD) is a molecular chaperone that assists protein folding by delivering non-native proteins to group II chaperonin. A heterohexamer of eukaryotic PFD has been shown to specifically recognize and deliver non-native actin and tubulin to chaperonin-containing TCP-1 (CCT), but the mechanism of specific recognition is still unclear. To determine its crystal structure, recombinant human PFD was reconstituted, purified and crystallized. X-ray diffraction data were collected to 4.7â Å resolution. The crystals belonged to space group P21212, with unit-cell parameters a = 123.2, b = 152.4, c = 105.9â Å.
Asunto(s)
Chaperonas Moleculares/química , Chaperonas Moleculares/aislamiento & purificación , Secuencia de Aminoácidos , Cristalización , Humanos , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Difracción de Rayos XRESUMEN
Small heat shock proteins (sHsps) play a role in preventing the fatal aggregation of denatured proteins in the presence of stresses. The sHsps exist as monodisperse oligomers in their resting state. Because the hydrophobic N-terminal regions of sHsps are possible interaction sites for denatured proteins, the manner of assembly of the oligomer is critical for the activation and inactivation mechanisms. Here, we report the oligomer architecture of SpHsp16.0 from Schizosaccharomyces pombe determined with X-ray crystallography and small angle X-ray scattering. Both results indicate that eight dimers of SpHsp16.0 form an elongated sphere with 422 symmetry. The monomers show nonequivalence in the interaction with neighboring monomers and conformations of the N- and C-terminal regions. Variants for the N-terminal phenylalanine residues indicate that the oligomer formation ability is highly correlated with chaperone activity. Structural and biophysical results are discussed in terms of their possible relevance to the activation mechanism of SpHsp16.0.