RESUMEN
The Polycomb Group (PcG) complex PRC1 represses transcription, forms condensates in cells, and modifies chromatin architecture. These processes are connected through the essential, polymerizing Sterile Alpha Motif (SAM) present in the PRC1 subunit Polyhomeotic (Ph). In vitro, Ph SAM drives formation of short oligomers and phase separation with DNA or chromatin in the context of a Ph truncation ("mini-Ph"). Oligomer length is controlled by the long disordered linker (L) that connects the SAM to the rest of Ph--replacing Drosophila PhL with the evolutionarily diverged human PHC3L strongly increases oligomerization. How the linker controls SAM polymerization, and how polymerization and the linker affect condensate formation are not know. We analyzed PhL and PHC3L using biochemical assays and molecular dynamics (MD) simulations. PHC3L promotes mini-Ph phase separation and makes it relatively independent of DNA. In MD simulations, basic amino acids in PHC3L form contacts with acidic amino acids in the SAM. Engineering the SAM to make analogous charge-based contacts with PhL increased polymerization and phase separation, partially recapitulating the effects of the PHC3L. Ph to PHC3 linker swaps and SAM surface mutations alter Ph condensate formation in cells, and Ph function in Drosophila imaginal discs. Thus, SAM-driven phase separation and polymerization are conserved between flies and mammals, but the underlying mechanisms have diverged through changes to the disordered linker.
RESUMEN
Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) has been shown to play an important role in chromatin compaction and large-scale chromatin organization. Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand the underlying mechanism, here we analyze the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. We show that overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, SAM-induced phase separation, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Drosophila/química , Complejo Represivo Polycomb 1/química , Proteínas del Grupo Polycomb/metabolismo , Motivo alfa Estéril/fisiología , Animales , Compartimento Celular , Línea Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Polimerizacion , Motivo alfa Estéril/genética , UbiquitinaciónRESUMEN
Polycomb repression complex 1 (PRC1) is a multiprotein assembly that regulates transcription. The Polycomb group ring finger 1 protein (PCGF1) is central in the assembly of the noncanonical PRC1 variant called PRC1.1 through its direct interaction with BCOR (BCL-6-interacting corepressor) or its paralog, BCOR-like 1 (BCORL1). Previous structural studies revealed that the C-terminal PUFD domain of BCORL1 is necessary and sufficient to heterodimerize with the RAWUL domain of PCGF1 and, together, form a new protein-protein binding interface that associates with the histone demethylase KDM2B. Here, we show that the PUFD of BCOR and BCORL1 differ in their abilities to assemble with KDM2B. Unlike BCORL1, the PUFD of BCOR alone does not stably assemble with KDM2B. Rather, additional residues N-terminal to the BCOR PUFD are necessary for stable association. Nuclear magnetic resonance (NMR) structure determination and 15N T2 relaxation time measurements of the BCOR PUFD alone indicate that the termini of the BCOR PUFD, which are critical for binding PCGF1 and KDM2B, are disordered. This suggests a hierarchical mode of assembly whereby BCOR PUFD termini become structurally ordered upon binding PCGF1, which then allows stable association with KDM2B. Notably, BCOR internal tandem duplications (ITDs) leading to pediatric kidney and brain tumors map to the PUFD termini. Binding studies with the BCOR ITD indicate the ITD would disrupt PRC1.1 assembly, suggesting loss of the ability to assemble PRC1.1 is a critical molecular event driving tumorigenesis.
Asunto(s)
Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/química , Histona Demetilasas con Dominio de Jumonji/metabolismo , Modelos Moleculares , Complejo Represivo Polycomb 1/química , Unión Proteica , Dominios Proteicos , Mapas de Interacción de Proteínas , Multimerización de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Represoras/químicaRESUMEN
KDM2B recruits H2A-ubiquitinating activity of a non-canonical Polycomb Repression Complex 1 (PRC1.1) to CpG islands, facilitating gene repression. We investigated the molecular basis of recruitment using in vitro assembly assays to identify minimal components, subcomplexes, and domains required for recruitment. A minimal four-component PRC1.1 complex can be assembled by combining two separately isolated subcomplexes: the DNA-binding KDM2B/SKP1 heterodimer and the heterodimer of BCORL1 and PCGF1, a core component of PRC1.1. The crystal structure of the KDM2B/SKP1/BCORL1/PCGF1 complex illustrates the crucial role played by the PCGF1/BCORL1 heterodimer. The BCORL1 PUFD domain positions residues preceding the RAWUL domain of PCGF1 to create an extended interface for interaction with KDM2B, which is unique to the PCGF1-containing PRC1.1 complex. The structure also suggests how KDM2B might simultaneously function in PRC1.1 and an SCF ubiquitin ligase complex and the possible molecular consequences of BCOR PUFD internal tandem duplications found in pediatric kidney and brain tumors.
Asunto(s)
Histona Demetilasas con Dominio de Jumonji/metabolismo , Complejo Represivo Polycomb 1/química , Complejo Represivo Polycomb 1/metabolismo , Proteínas Represoras/metabolismo , Animales , Islas de CpG , Cristalografía por Rayos X , Regulación de la Expresión Génica , Humanos , Técnicas In Vitro , Histona Demetilasas con Dominio de Jumonji/química , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Proteínas Represoras/químicaRESUMEN
Polycomb Group (PcG) proteins assemble a chromatin state that maintains developmental gene repression. A new study combining structure and in vivo analysis details a molecular network from DNA recognition to PcG recruitment, highlighting the essential role of Sterile Alpha Motifs.
Asunto(s)
Cromatina , Proteínas de Drosophila/genética , Enfermedades del Desarrollo Óseo , Conducto Auditivo Externo/anomalías , Mandíbula/anomalías , Complejo Represivo Polycomb 1/genética , Proteínas del Grupo Polycomb/genética , Proteínas RepresorasRESUMEN
The self-association of sterile alpha motifs (SAMs) into a helical polymer architecture is a critical functional component of many different and diverse array of proteins. For the Drosophila Polycomb group (PcG) protein Polyhomeotic (Ph), its SAM polymerization serves as the structural foundation to cluster multiple PcG complexes, helping to maintain a silenced chromatin state. Ph SAM shares 64% sequence identity with its human ortholog, PHC3 SAM, and both SAMs polymerize. However, in the context of their larger protein regions, PHC3 SAM forms longer polymers compared with Ph SAM. Motivated to establish the precise structural basis for the differences, if any, between Ph and PHC3 SAM, we determined the crystal structure of the PHC3 SAM polymer. PHC3 SAM uses the same SAM-SAM interaction as the Ph SAM sixfold repeat polymer. Yet, PHC3 SAM polymerizes using just five SAMs per turn of the helical polymer rather than the typical six per turn observed for all SAM polymers reported to date. Structural analysis suggested that malleability of the PHC3 SAM would allow formation of not just the fivefold repeat structure but also possibly others. Indeed, a second PHC3 SAM polymer in a different crystal form forms a sixfold repeat polymer. These results suggest that the polymers formed by PHC3 SAM, and likely others, are dynamic. The functional consequence of the variable PHC3 SAM polymers may be to create different chromatin architectures.
Asunto(s)
Modelos Moleculares , Fragmentos de Péptidos/química , Complejo Represivo Polycomb 1/química , Ingeniería de Proteínas , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Proteínas de Unión al ADN/química , Bases de Datos de Proteínas , Proteínas de Drosophila/química , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Polimerizacion , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Aminoácido , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at very high frequency (â¼70%) in uterine leiomyomas. However, the influence of these mutations on Mediator function and the molecular basis for their tumorigenic potential remain unknown. To clarify the impact of these mutations, we used affinity-purification mass spectrometry to establish the global protein-protein interaction profiles for both wild-type and mutant MED12. We found that uterine leiomyoma-linked mutations in MED12 led to a highly specific decrease in its association with Cyclin C-CDK8/CDK19 and loss of Mediator-associated CDK activity. Mechanistically, this occurs through disruption of a MED12-Cyclin C binding interface that we also show is required for MED12-mediated stimulation of Cyclin C-dependent CDK8 kinase activity. These findings indicate that uterine leiomyoma-linked mutations in MED12 uncouple Cyclin C-CDK8/19 from core Mediator and further identify the MED12/Cyclin C interface as a prospective therapeutic target in CDK8-driven cancers.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Leiomioma/genética , Complejo Mediador/genética , Complejo Mediador/metabolismo , Neoplasias Uterinas/genética , Ciclina C/metabolismo , Femenino , Células HEK293 , Humanos , Leiomioma/metabolismo , Leiomioma/patología , Mutagénesis Sitio-Dirigida , Unión Proteica , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologíaRESUMEN
Polycomb-group RING finger homologs (PCGF1, PCGF2, PCGF3, PCGF4, PCGF5, and PCGF6) are critical components in the assembly of distinct Polycomb repression complex 1 (PRC1)-related complexes. Here, we identify a protein interaction domain in BCL6 corepressor, BCOR, which binds the RING finger- and WD40-associated ubiquitin-like (RAWUL) domain of PCGF1 (NSPC1) and PCGF3 but not of PCGF2 (MEL18) or PCGF4 (BMI1). Because of the selective binding, we have named this domain PCGF Ub-like fold discriminator (PUFD). The structure of BCOR PUFD bound to PCGF1 reveals that (1) PUFD binds to the same surfaces as observed for a different Polycomb group RAWUL domain and (2) the ability of PUFD to discriminate among RAWULs stems from the identity of specific residues within these interaction surfaces. These data show the molecular basis for determining the binding preference for a PCGF homolog, which ultimately helps determine the identity of the larger PRC1-like assembly.
Asunto(s)
Modelos Moleculares , Complejos Multiproteicos/química , Complejo Represivo Polycomb 1/química , Conformación Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Represoras/química , Secuencia de Aminoácidos , Cristalización , Humanos , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , UltracentrifugaciónRESUMEN
Sterile alpha motifs (SAMs) are frequently found in eukaryotic genomes. An intriguing property of many SAMs is their ability to self-associate, forming an open-ended polymer structure whose formation has been shown to be essential for the function of the protein. What remains largely unresolved is how polymerization is controlled. Previously, we had determined that the stretch of unstructured residues N-terminal to the SAM of a Drosophila protein called polyhomeotic (Ph), a member of the polycomb group (PcG) of gene silencers, plays a key role in controlling Ph SAM polymerization. Ph SAM with its native linker created shorter polymers compared to Ph SAM attached to either a random linker or no linker. Here, we show that the SAM linker for the human Ph ortholog, polyhomeotic homolog 3 (PHC3), also controls PHC3 SAM polymerization but does so in the opposite fashion. PHC3 SAM with its native linker allows longer polymers to form compared to when attached to a random linker. Attaching the PHC3 SAM linker to Ph SAM also resulted in extending Ph SAM polymerization. Moreover, in the context of full-length Ph protein, replacing the SAM linker with PHC3 SAM linker, intended to create longer polymers, resulted in greater repressive ability for the chimera compared to wild-type Ph. These findings show that polymeric SAM linkers evolved to modulate a wide dynamic range of SAM polymerization abilities and suggest that rationally manipulating the function of SAM containing proteins through controlling their SAM polymerization may be possible.
Asunto(s)
Complejo Represivo Polycomb 1/química , Multimerización de Proteína , Secuencia de Aminoácidos , Animales , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Complejo Represivo Polycomb 1/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Desplegamiento Proteico , Homología de Secuencia de AminoácidoRESUMEN
Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas de Unión al ADN/genética , Drosophila/química , Drosophila/genética , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Datos de Secuencia Molecular , Nucleoproteínas/genética , Complejo Represivo Polycomb 1 , Polimerizacion , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Polycomb group (PcG) proteins maintain the silent state of developmentally important genes. Recent evidence indicates that noncoding RNAs also play an important role in targeting PcG proteins to chromatin and PcG-mediated chromatin organization, although the molecular basis for how PcG and RNA function in concert remains unclear. The Phe-Cys-Ser (FCS) domain, named for three consecutive residues conserved in this domain, is a 30-40-residue Zn(2+) binding motif found in a number of PcG proteins. The FCS domain has been shown to bind RNA in a non-sequence specific manner, but how it does so is not known. Here, we present the three-dimensional structure of the FCS domain from human Polyhomeotic homologue 1 (HPH1, also known as PHC1) determined using multidimensional nuclear magnetic resonance methods. Chemical shift perturbations upon addition of RNA and DNA resulted in the identification of Lys 816 as a potentially important residue required for nucleic acid binding. The role played by this residue in Polyhomeotic function was demonstrated in a transcription assay conducted in Drosophila S2 cells. Mutation of the Arg residue to Ala in the Drosophila Polyhomeotic (Ph) protein, which is equivalent to Lys 816 in HPH1, was unable to repress transcription of a reporter gene to the level of wild-type Ph. These results suggest that direct interaction between the Ph FCS domain and nucleic acids is required for Ph-mediated repression.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Drosophila/química , Nucleoproteínas/química , Proteínas Represoras/química , Animales , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Cromatina/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Humanos , Resonancia Magnética Nuclear Biomolecular , Nucleoproteínas/metabolismo , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Estructura Terciaria de Proteína , Proteínas Represoras/metabolismoRESUMEN
Oncogenic transformation by adenovirus E1A and E1B-55K requires E1B-55K inhibition of p53 activity to prevent E1A-induced apoptosis. During viral infection, E1B-55K and E4orf6 substitute for the substrate-binding subunits of the host cell cullin 5 class of ubiquitin ligases, resulting in p53 polyubiquitinylation and proteasomal degradation. Here we show that E1B-55K alone also functions as an E3 SUMO1-p53 ligase. Fluorescence microscopy studies showed that E1B-55K alone, in the absence of other viral proteins, causes p53 to colocalize with E1B-55K in promyelocytic leukemia (PML) nuclear bodies, nuclear domains with a high concentration of sumoylated proteins. Photobleaching experiments with live cells revealed that E1B-55K tethering of p53 in PML nuclear bodies decreases the in vivo nuclear mobility of p53 nearly 2 orders of magnitude. E1B-55K-induced p53 sumoylation contributes to maximal inhibition of p53 function since mutation of the major p53 sumoylation site decreases E1B-55K-induced p53 sumoylation, tethering in PML nuclear bodies, and E1B-55K inhibition of p53 activity. Mutation of the E1B-55K sumoylation site greatly inhibits E1B-55K association with PML nuclear bodies and the p53 nuclear export to cytoplasmic aggresomes observed in E1A-E1B-transformed cells. Purified E1B-55K and p53 form high-molecular-weight complexes potentially through the formation of a network of E1B-55K dimers bound to the N termini of p53 tetramers. In support of this model, a p53 mutation that prevents tetramer formation greatly reduces E1B-55K-induced tethering in PML nuclear bodies and p53 nuclear export. These data indicate that E1B-55K's association with PML nuclear bodies inactivates p53 by first sequestering it in PML nuclear bodies and then greatly facilitating its nuclear export.
Asunto(s)
Proteínas E1B de Adenovirus/metabolismo , Transformación Celular Neoplásica/metabolismo , Cuerpos de Inclusión Intranucleares/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteína SUMO-1/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Proteínas E1B de Adenovirus/genética , Línea Celular Tumoral , Dimerización , Humanos , Microscopía Fluorescente , Modelos Biológicos , Mutación/genética , Fotoblanqueo , Proteína de la Leucemia Promielocítica , SumoilaciónRESUMEN
RING1B, a Polycomb Group (PcG) protein, binds methylated chromatin through its association with another PcG protein called Polycomb (Pc). However, RING1B can associate with nonmethylated chromatin suggesting an alternate mechanism for RING1B interaction with chromatin. Here, we demonstrate that two proteins with little sequence identity between them, the Pc cbox domain and RYBP, bind the same surface on the C-terminal domain of RING1B (C-RING1B). Pc cbox and RYBP each fold into a nearly identical, intermolecular beta sheet with C-RING1B and a loop structure which are completely different in the two proteins. Both the beta sheet and loop are required for stable binding and transcription repression. Further, a mutation engineered to disrupt binding on the Drosophila dRING1 protein prevents chromatin association and PcG function in vivo. These results suggest that PcG targeting to different chromatin locations relies, in part, on binding partners of C-RING1B that are diverse in sequence and structure.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Cromatina/metabolismo , Cristalografía por Rayos X , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Electroforesis en Gel de Poliacrilamida , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Datos de Secuencia Molecular , Mutación/genética , Resonancia Magnética Nuclear Biomolecular , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Represoras/química , Alineación de Secuencia , UltracentrifugaciónRESUMEN
We compare here the utility of sedimentation velocity (SV) to sedimentation equilibrium (SE) analysis for the characterization of reversible systems. Genetic algorithm optimization in UltraScan is used to optimize the model and to obtain solution properties of all components present in the system. We apply our method to synthetic and experimental data, and suggest limits for the accessible kinetic range. We conclude that equilibrium constants obtained from SV and SE analysis are equivalent, but that SV experiments provide better confidence for the K(d), can better account for the presence of contaminants and provide additional information including rate constants and shape parameters.
Asunto(s)
Algoritmos , Ultracentrifugación/métodos , Fraccionamiento Químico , Simulación por Computador , Proteínas de Unión al ADN/metabolismo , Humanos , Cinética , Peso Molecular , Complejo Represivo Polycomb 1 , Multimerización de Proteína , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Polycomb group (PcG) proteins are required for maintaining cell identity and stem cell self-renewal. RING1B and Polycomb (Pc) are two components of a multiprotein complex called polycomb repression complex 1 (PRC1) that is essential for establishing and maintaining long-term repressed gene states. Here we characterize the interaction between the C-terminal region of RING1B (C-RING1B) and the Pc cbox domain. The C-RING1B-cbox interaction displays a 1:1 stoichiometry with dissociation constants ranging from 9.2 to 180 nM for the different Pc orthologues. NMR analysis of C-RING1B alone reveals line broadening. However, when it is in complex with the cbox domain, there is a striking change to the NMR spectrum indicative of conformational tightening. This conformational change may arise from the organization of the C-RING1B subdomains. The C-terminal regions of all PcG RING1 proteins are composed of two stretches of conserved sequences separated by a variable linker sequence. While the entire C-RING1B region is required for cbox binding, the N- and C-terminal halves of C-RING1B can be separated and are able to interact, suggesting the presence of an intramolecular interaction within C-RING1B. The flexibility within the C-RING1B structure allowing transitions between the intramolecular bound and unbound states may cause the broadened peaks of the C-RING1B NMR spectrum. Binding the cbox domain stabilizes C-RING1B, whereby broadening is eliminated. The presence of flexible regions could allow C-RING1B to bind a variety of different factors, ultimately recruiting RING1B and its associated PcG proteins to different genomic loci.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas Represoras/química , Ubiquitina-Proteína Ligasas/química , Secuencia de Aminoácidos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Datos de Secuencia Molecular , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UltracentrifugaciónRESUMEN
The polycomb group proteins are required for the stable maintenance of gene repression patterns established during development. They function as part of large multiprotein complexes created via a multitude of protein-protein interaction domains. Here we examine the interaction between the SAM domains of the polycomb group proteins polyhomeotic (Ph) and Sex-comb-on-midleg (Scm). Previously we showed that Ph-SAM polymerizes as a helical structure. We find that Scm-SAM also polymerizes, and a crystal structure reveals an architecture similar to the Ph-SAM polymer. These results suggest that Ph-SAM and Scm-SAM form a copolymer. Binding affinity measurements between Scm-SAM and Ph-SAM subunits in different orientations indicate a preference for the formation of a single junction copolymer. To provide a model of the copolymer, we determined the structure of the Ph-SAM/Scm-SAM junction. Similar binding modes are observed in both homo- and heterocomplex formation with minimal change in helix axis direction at the polymer joint. The copolymer model suggests that polymeric Scm complexes could extend beyond the local domains of polymeric Ph complexes on chromatin, possibly playing a role in long range repression.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Nucleoproteínas/genética , Proteínas Represoras/genética , Secuencia de Aminoácidos , Animales , Cromatina/química , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Drosophila/química , Drosophila melanogaster , Glutatión Transferasa/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Mutación , Nucleoproteínas/química , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Polímeros/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Represoras/química , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , Factores de TiempoRESUMEN
Yan, an ETS family transcriptional repressor, is regulated by receptor tyrosine kinase signaling via the Ras/MAPK pathway. Phosphorylation and downregulation of Yan is facilitated by a protein called Mae. Yan and Mae interact through their SAM domains. We find that repression by Yan requires the formation of a higher order structure mediated by Yan-SAM polymerization. Moreover, a crystal structure of the Yan-SAM/Mae-SAM complex shows that Mae-SAM specifically recognizes a surface on Yan-SAM that is also required for Yan-SAM polymerization. Mae-SAM binds to Yan-SAM with approximately 1000-fold higher affinity than Yan-SAM binds to itself and can effectively depolymerize Yan-SAM. Mutations on Mae that specifically disrupt its SAM domain-dependent interactions with Yan disable the derepression function of Mae in vivo. Depolymerization of Yan by Mae represents a novel mechanism of transcriptional control that sensitizes Yan for regulation by receptor tyrosine kinases.
Asunto(s)
Proteínas Portadoras/química , Proteínas de Drosophila/química , Proteínas del Ojo/química , Genes Reguladores/fisiología , Péptidos y Proteínas de Señalización Intracelular , Proteínas Represoras/química , Secuencia de Aminoácidos/fisiología , Animales , Sitios de Unión/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Mutación/genética , Polímeros/química , Polímeros/metabolismo , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Sterile alpha motif (SAM) domains are known to exhibit diverse protein-protein interaction modes. They can form multiple self-association architectures and also bind to various non-SAM domain-containing proteins. Surprising new work adds a completely unanticipated function for some SAM domains - the ability to bind RNA. Such functional diversity within a homologous protein family presents a significant challenge for bioinformatic function assignment.
Asunto(s)
Proteínas/química , Proteínas/metabolismo , Sitios de Unión , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
SAM (sterile alpha motif) domains are protein-protein interaction modules found in a large number of regulatory proteins. Byr2 and Ste4 are two SAM domain-containing proteins in the mating pheromone response pathway of the fission yeast, Schizosaccharomyces pombe. Byr2 is a mitogen-activated protein kinase kinase kinase that is regulated by Ste4. Tu et al. (Tu, H., Barr, M., Dong, D. L., and Wigler, M. (1997) Mol. Cell. Biol. 17, 5876-5887) showed that the isolated SAM domain of Byr2 binds a fragment of Ste4 that contains both a leucine zipper (Ste4-LZ) domain as well as a SAM domain, suggesting that Byr2-SAM and Ste4-SAM may form a hetero-oligomer. Here, we show that the individual SAM domains of Ste4 and Byr2 are monomeric at low concentrations and bind to each other in a 1:1 stoichiometry with a relatively weak dissociation constant of 56 +/- 3 microm. Inclusion of the Ste4-LZ domain, which determines the oligomeric state of Ste4, has a dramatic effect on binding affinity, however. We find that the Ste4-LZ domain is trimeric and, when included with the Ste4-SAM domain, yields a 3:1 Ste4-LZ-SAM:Byr2-SAM complex with a tight dissociation constant of 19 +/- 4 nm. These results suggest that the Ste4-LZ-SAM protein may recognize multiple binding sites on Byr2-SAM, indicating a new mode of oligomeric organization for SAM domains. The fact that high affinity binding occurs only with the addition of an oligomerization domain suggests that it may be necessary to include ancillary oligomerization modules when searching for binding partners of SAM domains.
Asunto(s)
Proteínas Fúngicas/química , Subunidades beta de la Proteína de Unión al GTP , Proteínas de Unión al GTP Heterotriméricas/química , Quinasas Quinasa Quinasa PAM , Proteínas Quinasas Activadas por Mitógenos/química , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimología , Sitios de Unión , Cromatografía en Gel , Dimerización , Relación Dosis-Respuesta a Droga , Glutatión Transferasa/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
BACKGROUND: TEL is a transcriptional repressor containing a SAM domain that forms a helical polymer. In a number of hematologic malignancies, chromosomal translocations lead to aberrant fusions of TEL-SAM to a variety of other proteins, including many tyrosine kinases. TEL-SAM polymerization results in constitutive activation of the tyrosine kinase domains to which it becomes fused, leading to cell transformation. Thus, inhibitors of TEL-SAM self-association could abrogate transformation in these cells. In previous work, we determined the structure of a mutant TEL-SAM polymer bearing a Val to Glu substitution in center of the subunit interface. It remained unclear how much the mutation affected the architecture of the polymer, however. RESULTS: Here we determine the structure of the native polymer interface. To accomplish this goal, we introduced mutations that block polymer extension, producing a heterodimer with a wild-type interface. We find that the structure of the wild-type polymer interface is quite similar to the mutant structure determined previously. With the structure of the native interface, it is possible to evaluate the potential for developing therapeutic inhibitors of the interaction. We find that the interacting surfaces of the protein are relatively flat, containing no obvious pockets for the design of small molecule inhibitors. CONCLUSION: Our results confirm the architecture of the TEL-SAM polymer proposed previously based on a mutant structure. The fact that the interface contains no obvious potential binding pockets suggests that it may be difficult to find small molecule inhibitors to treat malignancies in this way.