RESUMEN
The C-terminal transactivation domain (TAD) of BMAL1 (brain and muscle ARNT-like 1) is a regulatory hub for transcriptional coactivators and repressors that compete for binding and, consequently, contributes to period determination of the mammalian circadian clock. Here, we report the discovery of two distinct conformational states that slowly exchange within the dynamic TAD to control timing. This binary switch results from cis/trans isomerization about a highly conserved Trp-Pro imide bond in a region of the TAD that is required for normal circadian timekeeping. Both cis and trans isomers interact with transcriptional regulators, suggesting that isomerization could serve a role in assembling regulatory complexes in vivo. Toward this end, we show that locking the switch into the trans isomer leads to shortened circadian periods. Furthermore, isomerization is regulated by the cyclophilin family of peptidyl-prolyl isomerases, highlighting the potential for regulation of BMAL1 protein dynamics in period determination.
Asunto(s)
Factores de Transcripción ARNTL/metabolismo , Relojes Circadianos , Ritmo Circadiano , Factores de Transcripción ARNTL/química , Factores de Transcripción ARNTL/genética , Animales , Línea Celular Tumoral , Ciclofilinas/genética , Ciclofilinas/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Humanos , Isomerismo , Ratones , Mutación , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Filogenia , Prolina , Dominios Proteicos , Transducción de Señal , Relación Estructura-Actividad , Factores de Tiempo , Transfección , TriptófanoRESUMEN
The effort to modulate challenging protein targets has stimulated interest in ligands that are larger and more complex than typical small-molecule drugs. While combinatorial techniques such as mRNA display routinely produce high-affinity macrocyclic peptides against classically undruggable targets, poor membrane permeability has limited their use toward primarily extracellular targets. Understanding the passive membrane permeability of macrocyclic peptides would, in principle, improve our ability to design libraries whose leads can be more readily optimized against intracellular targets. Here, we investigate the permeabilities of over 200 macrocyclic 10-mers using the thioether cyclization motif commonly found in mRNA display macrocycle libraries. We identified the optimal lipophilicity range for achieving permeability in thioether-cyclized 10-mer cyclic peptide-peptoid hybrid scaffolds and showed that permeability could be maintained upon extensive permutation in the backbone. In one case, changing a single amino acid from d-Pro to d-NMe-Ala, representing the loss of a single methylene group in the side chain, resulted in a highly permeable scaffold in which the low-dielectric conformation shifted from the canonical cross-beta geometry of the parent compounds into a novel saddle-shaped fold in which all four backbone NH groups were sequestered from the solvent. This work provides an example by which pre-existing physicochemical knowledge of a scaffold can benefit the design of macrocyclic peptide mRNA display libraries, pointing toward an approach for biasing libraries toward permeability by design. Moreover, the compounds described herein are a further demonstration that geometrically diverse, highly permeable scaffolds exist well beyond conventional drug-like chemical space.
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Péptidos Cíclicos , Péptidos , Péptidos/química , Péptidos Cíclicos/química , Biblioteca de Péptidos , Permeabilidad , ARN Mensajero , SulfurosRESUMEN
B-Myb is a highly conserved member of the vertebrate Myb family of transcription factors that plays a critical role in cell-cycle progression and proliferation. Myb proteins activate Myb-dependent promoters by interacting specifically with Myb-binding site (MBS) sequences using their DNA-binding domain (DBD). Transactivation of MBS promoters by B-Myb is repressed by its negative regulatory domain (NRD), and phosphorylation of the NRD by Cdk2-CyclinA relieves the repression to activate B-Myb-dependent promoters. However, the structural mechanisms underlying autoinhibition and activation of B-Myb-mediated transcription have been poorly characterized. Here, we determined that a region in the B-Myb NRD (residues 510-600) directly associates with the DBD and inhibits binding of the DBD to the MBS DNA sequence. We demonstrate using biophysical assays that phosphorylation of the NRD at T515, T518, and T520 is sufficient to disrupt the interaction between the NRD and the DBD, which results in increased affinity for MBS DNA and increased B-Myb-dependent promoter activation in cell assays. Our biochemical characterization of B-Myb autoregulation and the activating effects of phosphorylation provide insight into how B-Myb functions as a site-specific transcription factor.
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Proteínas de Ciclo Celular , Quinasa 2 Dependiente de la Ciclina , ADN , Transactivadores , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , ADN/metabolismo , Humanos , Fosforilación , Dominios Proteicos , Transactivadores/química , Transactivadores/metabolismo , Activación TranscripcionalRESUMEN
Circadian rhythms are generated by interlocked transcription-translation feedback loops that establish cell-autonomous biological timing of â¼24 h. Mutations in core clock genes that alter their stability or affinity for one another lead to changes in circadian period. The human CRY1Δ11 mutant lengthens circadian period to cause delayed sleep phase disorder (DSPD), characterized by a very late onset of sleep. CRY1 is a repressor that binds to the transcription factor CLOCK:BMAL1 to inhibit its activity and close the core feedback loop. We previously showed how the PHR (photolyase homology region) domain of CRY1 interacts with distinct sites on CLOCK and BMAL1 to sequester the transactivation domain from coactivators. However, the Δ11 variant alters an intrinsically disordered tail in CRY1 downstream of the PHR. We show here that the CRY1 tail, and in particular the region encoded by exon 11, modulates the affinity of the PHR domain for CLOCK:BMAL1. The PHR-binding epitope in exon 11 is necessary and sufficient to disrupt the interaction between CRY1 and the subunit CLOCK. Moreover, PHR-tail interactions are conserved in the paralog CRY2 and reduced when either CRY is bound to the circadian corepressor PERIOD2. Discovery of this autoregulatory role for the mammalian CRY1 tail and conservation of PHR-tail interactions in both mammalian cryptochromes highlights functional conservation with plant and insect cryptochromes, which also utilize PHR-tail interactions to reversibly control their activity.
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Factores de Transcripción ARNTL/metabolismo , Proteínas CLOCK/metabolismo , Ritmo Circadiano/fisiología , Criptocromos/metabolismo , Animales , Relojes Circadianos , Humanos , Ratones , Proteínas Circadianas Period/metabolismo , Dominios Proteicos , Transcripción GenéticaRESUMEN
Boron-containing compounds are commonly used in synthetic chemistry and are known to play important roles in biology. Despite the widespread relevance of boronated compounds, there have been limited methods to discover, characterize, and study them. Here, we describe the use of 11B NMR, including 1H-11B HMBC, for the isolation and characterization of the boron-containing natural product diadenosine borate. Utilizing synthetic standards, we optimized coupling parameters for 1H-11B HMBC experiments to allow for the analysis of small quantities (â¼1 mg) of boron-containing compounds. This work can facilitate the broader application of 11B NMR to the study of boron in a range of applications, from synthetic chemistry to the role of boron in naturally occurring systems.
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Adenosina , Productos Biológicos , Boratos , Espectroscopía de Resonancia Magnética , Boratos/química , Adenosina/química , Productos Biológicos/químicaRESUMEN
The chameleonic behavior of cyclosporin A (CsA) was investigated through conformational ensembles employing multicanonical molecular dynamics simulations that could sample the cis and trans isomers of N-methylated amino acids; these assessments were conducted in explicit water, dimethyl sulfoxide, acetonitrile, methanol, chloroform, cyclohexane (CHX), and n-hexane (HEX) using AMBER ff03, AMBER10:EHT, AMBER12:EHT, and AMBER14:EHT force fields. The conformational details were discussed employing the free-energy landscapes (FELs) at T = 300 K; it was observed that the experimentally determined structures of CsA were only a part of the conformational space. Comparing the ROESY measurements in CHX-d12 and HEX-d14, the major conformations in those apolar solvents were essentially the same as that in CDCl3 except for the observation of some sidechain rotamers. The effects of the metal ions on the conformations, including the cis/trans isomerization, were also investigated. Based on the analysis of FELs, it was concluded that the AMBER ff03 force field best described the experimentally derived conformations, indicating that CsA intrinsically formed membrane-permeable conformations and that the metal ions might be the key to the cis/trans isomerization of N-methylated amino acids before binding a partner protein.
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Ciclosporina , Simulación de Dinámica Molecular , Conformación Molecular , Conformación Proteica , Solventes , AguaRESUMEN
Human epithelial 15-lipoxygenase-2 (h15-LOX-2, ALOX15B) is expressed in many tissues and has been implicated in atherosclerosis, cystic fibrosis and ferroptosis. However, there are few reported potent/selective inhibitors that are active ex vivo. In the current work, we report newly discovered molecules that are more potent and structurally distinct from our previous inhibitors, MLS000545091 and MLS000536924 (Jameson et al, PLoS One, 2014, 9, e104094), in that they contain a central imidazole ring, which is substituted at the 1-position with a phenyl moiety and with a benzylthio moiety at the 2-position. The initial three molecules were mixed-type, non-reductive inhibitors, with IC50 values of 0.34⯱â¯0.05⯵M for MLS000327069, 0.53⯱â¯0.04⯵M for MLS000327186 and 0.87⯱â¯0.06⯵M for MLS000327206 and greater than 50-fold selectivity versus h5-LOX, h12-LOX, h15-LOX-1, COX-1 and COX-2. A small set of focused analogs was synthesized to demonstrate the validity of the hits. In addition, a binding model was developed for the three imidazole inhibitors based on computational docking and a co-structure of h15-LOX-2 with MLS000536924. Hydrogen/deuterium exchange (HDX) results indicate a similar binding mode between MLS000536924 and MLS000327069, however, the latter restricts protein motion of helix-α2 more, consistent with its greater potency. Given these results, we designed, docked, and synthesized novel inhibitors of the imidazole scaffold and confirmed our binding mode hypothesis. Importantly, four of the five inhibitors mentioned above are active in an h15-LOX-2/HEK293 cell assay and thus they could be important tool compounds in gaining a better understanding of h15-LOX-2's role in human biology. As such, a suite of similar pharmacophores that target h15-LOX-2 both in vitro and ex vivo are presented in the hope of developing them as therapeutic agents.
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Araquidonato 15-Lipooxigenasa/metabolismo , Inhibidores de la Lipooxigenasa/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Inhibidores de la Lipooxigenasa/síntesis química , Inhibidores de la Lipooxigenasa/química , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Methionine (Met) oxidation is an important biological redox node, with hundreds if not thousands of protein targets. The process yields methionine oxide (MetO). It renders the sulfur chiral, producing two distinct, diastereomerically related products. Despite the biological significance of Met oxidation, a reliable protocol to separate the resultant MetO diastereomers is currently lacking. This hampers our ability to make peptides and proteins that contain stereochemically defined MetO to then study their structural and functional properties. We have developed a facile method that uses supercritical CO2 chromatography and allows obtaining both diastereomers in purities exceeding 99 %. 1 Hâ NMR spectra were correlated with X-ray structural information. The stereochemical interconversion barrier at sulfur was calculated as 45.2â kcal mol-1 , highlighting the remarkable stereochemical stability of MetO sulfur chirality. Our protocol should open the road to synthesis and study of a wide variety of stereochemically defined MetO-containing proteins and peptides.
RESUMEN
Amyloidogenic peptides and proteins aggregate into fibrillary structures that are usually deposited in tissues and organs and are often involved in the development of diseases. In contrast to native structured proteins, amyloids do not follow a defined energy landscape toward the fibrillary state and often generate a vast population of aggregation intermediates that are transient and exceedingly difficult to study. Here, we employ chiral editing as a tool to study the aggregation mechanism of the Amyloid ß (Aß) 42 peptide, whose aggregation intermediates are thought to be one of the main driving forces in Alzheimer's disease (AD). Through the design of a focused chiral mutant library (FCML) of 16 chiral Aß42 variants, we identified several point D-substitutions that allowed us to modulate the aggregation propensity and the biological activity of the peptide. Surprisingly, the reduced propensity toward aggregation and the stabilization of oligomeric intermediates did not always correlate with an increase in toxicity. In the present study, we show how chiral editing can be a powerful tool to trap and stabilize Aß42 conformers that might otherwise be too transient and dynamic to study, and we identify sites within the Aß42 sequence that could be potential targets for therapeutic intervention.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Humanos , Fragmentos de Péptidos , Electricidad EstáticaRESUMEN
The cellular prion protein (PrPC) is a zinc-binding protein that contributes to the regulation of Zn2+ and other divalent species of the central nervous system. Zn2+ coordinates to the flexible, N-terminal repeat region of PrPC and drives a tertiary contact between this repeat region and a well-defined cleft of the C-terminal domain. The tertiary structure promoted by Zn2+ is thought to regulate inherent PrPC toxicity. Despite the emerging consensus regarding the interaction between Zn2+ and PrPC, there is little direct spectroscopic confirmation of the metal ion's coordination details. Here, we address this conceptual gap by using Cd2+ as a surrogate for Zn2+. NMR finds that Cd2+ binds exclusively to the His imidazole side chains of the repeat segment, with a dissociation constant of â¼1.2 mM, and promotes an N-terminal-C-terminal cis interaction very similar to that observed with Zn2+. Analysis of 113Cd NMR spectra of PrPC, along with relevant control proteins and peptides, suggests that coordination of Cd2+ in the full-length protein is consistent with a three- or four-His geometry. Examination of the mutation E199K in mouse PrPC (E200K in humans), responsible for inherited Creutzfeldt-Jakob disease, finds that the mutation lowers metal ion affinity and weakens the cis interaction. These findings not only provide deeper insight into PrPC metal ion coordination but they also suggest new perspectives on the role of familial mutations in prion disease.
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Resonancia Magnética Nuclear Biomolecular , Proteínas Priónicas/química , Proteínas Priónicas/metabolismo , Zinc/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cadmio/química , Histidina/química , Imidazoles/química , Modelos Moleculares , Mutación , Proteínas Priónicas/genética , Unión ProteicaRESUMEN
Covering: up to 2018With contributions from the global natural product (NP) research community, and continuing the Raw Data Initiative, this review collects a comprehensive demonstration of the immense scientific value of disseminating raw nuclear magnetic resonance (NMR) data, independently of, and in parallel with, classical publishing outlets. A comprehensive compilation of historic to present-day cases as well as contemporary and future applications show that addressing the urgent need for a repository of publicly accessible raw NMR data has the potential to transform natural products (NPs) and associated fields of chemical and biomedical research. The call for advancing open sharing mechanisms for raw data is intended to enhance the transparency of experimental protocols, augment the reproducibility of reported outcomes, including biological studies, become a regular component of responsible research, and thereby enrich the integrity of NP research and related fields.
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Productos Biológicos/química , Espectroscopía de Resonancia Magnética/métodos , Conformación Molecular , Reproducibilidad de los ResultadosRESUMEN
Correction for 'The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research' by James B. McAlpine et al., Nat. Prod. Rep., 2018, DOI: .
RESUMEN
Syndecans potently modulate agouti-related peptide (AgRP) signaling in the central melanocortin system. Through heparan sulfate moieties, syndecans are thought to anchor AgRP near its receptor, enhancing its orexigenic effects. Original work proposed that the N-terminal domain of AgRP facilitates this interaction. However, this is not compatible with evidence that this domain is posttranslationally cleaved. Addressing this long-standing incongruity, we used calorimetry and magnetic resonance to probe interactions of AgRP peptides with glycosaminoglycans, including heparan sulfate. We show that mature, cleaved, C-terminal AgRP, not the N-terminal domain, binds heparan sulfate. NMR shows that the binding site consists of regions distinct from the melanocortin receptor-binding site. Using a library of designed AgRP variants, we find that the strength of the syndecan interaction perfectly tracks orexigenic action. Our data provide compelling evidence that AgRP is a heparan sulfate-binding protein and localizes critical regions in the AgRP structure required for this interaction.
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Proteína Relacionada con Agouti/química , Heparitina Sulfato/química , Péptidos/química , Proteína Relacionada con Agouti/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Péptidos/metabolismo , Unión Proteica , Dominios ProteicosRESUMEN
Organically functionalized silicene quantum dots (SiQDs) were synthesized by chemical exfoliation of calcium silicide and stabilized by hydrosilylation with olefin/acetylene derivatives forming Si-CH2-CH2- or Si-CHâCH- interfacial bonds. Transmission electron microscopy and atomic force microscopy measurements showed that the resultant SiQDs were ca. 2 nm in diameter and consisted of ca. four atomic layers of silicon. The structure was further characterized by 1H and 29Si NMR and X-ray photoelectron spectroscopic measurements. In photoluminescence measurements, the SiQDs exhibited a strong emission at 385 nm and the intensity varied with the interfacial linkage. In electrochemical measurements, both ethynylferrocene- and vinylferrocene-functionalized SiQDs exhibited a pair of well-defined voltammetric peaks at +0.15 V (vs Fc+/Fc) in the dark for the redox reaction of the ferrocene/ferrocenium couple; yet under UV photoirradiation, an additional pair of voltammetric peaks appeared at -0.41 V, most likely because of the redox reaction of ferrocene anions formed by photoinduced electron transfer from the SiQD to the ferrocene metal centers.
RESUMEN
Oligomeric proteins are important targets for structure determination in solution. While in most cases the fold of individual subunits can be determined experimentally, or predicted by homology-based methods, protein-protein interfaces are challenging to determine de novo using conventional NMR structure determination protocols. Here we focus on a member of the bet-V1 superfamily, Aha1 from Colwellia psychrerythraea. This family displays a broad range of crystallographic interfaces none of which can be reconciled with the NMR and SAXS data collected for Aha1. Unlike conventional methods relying on a dense network of experimental restraints, the sparse data are used to limit conformational search during optimization of a physically realistic energy function. This work highlights a new approach for studying minor conformational changes due to structural plasticity within a single dimeric interface in solution.
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Proteínas Bacterianas/química , Alteromonadaceae/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Soluciones , Difracción de Rayos XRESUMEN
The second round of the community-wide initiative Critical Assessment of automated Structure Determination of Proteins by NMR (CASD-NMR-2013) comprised ten blind target datasets, consisting of unprocessed spectral data, assigned chemical shift lists and unassigned NOESY peak and RDC lists, that were made available in both curated (i.e. manually refined) or un-curated (i.e. automatically generated) form. Ten structure calculation programs, using fully automated protocols only, generated a total of 164 three-dimensional structures (entries) for the ten targets, sometimes using both curated and un-curated lists to generate multiple entries for a single target. The accuracy of the entries could be established by comparing them to the corresponding manually solved structure of each target, which was not available at the time the data were provided. Across the entire data set, 71 % of all entries submitted achieved an accuracy relative to the reference NMR structure better than 1.5 Å. Methods based on NOESY peak lists achieved even better results with up to 100% of the entries within the 1.5 Å threshold for some programs. However, some methods did not converge for some targets using un-curated NOESY peak lists. Over 90% of the entries achieved an accuracy better than the more relaxed threshold of 2.5 Å that was used in the previous CASD-NMR-2010 round. Comparisons between entries generated with un-curated versus curated peaks show only marginal improvements for the latter in those cases where both calculations converged.
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Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , Proteínas/química , Espectroscopía de Resonancia Magnética con Carbono-13 , Conjuntos de Datos como Asunto , Espectroscopía de Protones por Resonancia Magnética , Reproducibilidad de los ResultadosRESUMEN
Single-stranded DNA (ssDNA) binding proteins are important in basal metabolic pathways for gene transcription, recombination, DNA repair and replication in all domains of life. Their main cellular role is to stabilize melted duplex DNA and protect genomic DNA from degradation. We have uncovered the molecular function of protein domain family domain of unknown function DUF2128 (PF09901) as a novel ssDNA binding domain. This bacterial domain strongly associates into a dimer and presents a highly positively charged surface that is consistent with its function in non-specific ssDNA binding. Lactococcus lactis YdbC is a representative of DUF2128. The solution NMR structures of the 20 kDa apo-YdbC dimer and YdbC:dT(19)G(1) complex were determined. The ssDNA-binding energetics to YdbC were characterized by isothermal titration calorimetry. YdbC shows comparable nanomolar affinities for pyrimidine and mixed oligonucleotides, and the affinity is sufficiently strong to disrupt duplex DNA. In addition, YdbC binds with lower affinity to ssRNA, making it a versatile nucleic acid-binding domain. The DUF2128 family is related to the eukaryotic nuclear protein positive cofactor 4 (PC4) family and to the PUR family both by fold similarity and molecular function.
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Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Lactococcus lactis , Secuencia de Aminoácidos , Apoproteínas/química , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/metabolismo , ADN/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/clasificación , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteoma , ARN/metabolismo , Alineación de SecuenciaRESUMEN
We have developed an approach for determining NMR structures of proteins over 20 kDa that utilizes sparse distance restraints obtained using transverse relaxation optimized spectroscopy experiments on perdeuterated samples to guide RASREC Rosetta NMR structure calculations. The method was tested on 11 proteins ranging from 15 to 40 kDa, seven of which were previously unsolved. The RASREC Rosetta models were in good agreement with models obtained using traditional NMR methods with larger restraint sets. In five cases X-ray structures were determined or were available, allowing comparison of the accuracy of the Rosetta models and conventional NMR models. In all five cases, the Rosetta models were more similar to the X-ray structures over both the backbone and side-chain conformations than the "best effort" structures determined by conventional methods. The incorporation of sparse distance restraints into RASREC Rosetta allows routine determination of high-quality solution NMR structures for proteins up to 40 kDa, and should be broadly useful in structural biology.
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Medición de Intercambio de Deuterio/métodos , Genómica/métodos , Proteínas de Unión a Maltosa/química , Resonancia Magnética Nuclear Biomolecular/métodos , Rodopsinas Sensoriales/química , Soluciones/química , Algoritmos , Animales , Cristalografía por Rayos X , Humanos , Proteínas de Unión a Maltosa/genética , Peso Molecular , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Rodopsinas Sensoriales/genéticaRESUMEN
Casein kinase 1 δ (CK1δ) controls essential biological processes including circadian rhythms and Wnt signaling, but how its activity is regulated is not well understood. CK1δ is inhibited by autophosphorylation of its intrinsically disordered C-terminal tail. Two CK1 splice variants, δ 1 and δ 2 , are known to have very different effects on circadian rhythms. These variants differ only in the last 16 residues of the tail, referred to as the extreme C-termini (XCT), but with marked changes in potential phosphorylation sites. Here we test if the XCT of these variants have different effects in autoinhibition of the kinase. Using NMR and HDX-MS, we show that the δ 1 XCT is preferentially phosphorylated by the kinase and the δ 1 tail makes more extensive interactions across the kinase domain. Mutation of δ1 -specific XCT phosphorylation sites increases kinase activity both in vitro and in cells and leads to changes in circadian period, similar to what is reported in vivo. Mechanistically, loss of the phosphorylation sites in XCT disrupts tail interaction with the kinase domain. δ1 autoinhibition relies on conserved anion binding sites around the CK1 active site, demonstrating a common mode of product inhibition of CK1δ . These findings demonstrate how a phosphorylation cycle controls the activity of this essential kinase.
RESUMEN
A high-quality structure of the 68-residue protein CD1104B from Clostridium difficile strain 630 exhibits a distinct all α-helical fold. The structure presented here is the first representative of bacterial protein domain family PF14203 (currently 180 members) of unknown function (DUF4319) and reveals that the side-chains of the only two strictly conserved residues (Glu 8 and Lys 48) form a salt bridge. Moreover, these two residues are located in the vicinity of the largest surface cleft which is predicted to contribute to a surface area involved in protein-protein interactions. This, along with its coding in transposon CTn4, suggests that CD1104B (and very likely all members of Pfam 14203) functions by interacting with other proteins required for the transfer of transposons between different bacterial species.