RESUMO
Monoubiquitination of histones H2B-K120 (H2BK120ub) and H2A-K119 (H2AK119ub) play opposing roles in regulating transcription and chromatin compaction. H2BK120ub is a hallmark of actively transcribed euchromatin, while H2AK119ub is highly enriched in transcriptionally repressed heterochromatin. Whereas H2BK120ub is known to stimulate the binding or activity of various chromatin-modifying enzymes, this post-translational modification (PTM) also interferes with the binding of several proteins to the nucleosome H2A/H2B acidic patch via an unknown mechanism. Here, we report cryoEM structures of an H2BK120ub nucleosome showing that ubiquitin adopts discrete positions that occlude the acidic patch. Molecular dynamics simulations show that ubiquitin remains stably positioned over this nucleosome region. By contrast, our cryoEM structures of H2AK119ub nucleosomes show ubiquitin adopting discrete positions that minimally occlude the acidic patch. Consistent with these observations, H2BK120ub, but not H2AK119ub, abrogates nucleosome interactions with acidic patch-binding proteins RCC1 and LANA, and single-domain antibodies specific to this region. Our results suggest a mechanism by which H2BK120ub serves as a gatekeeper to the acidic patch and point to distinct roles for histone H2AK119 and H2BK120 ubiquitination in regulating protein binding to nucleosomes.
Assuntos
Microscopia Crioeletrônica , Histonas , Simulação de Dinâmica Molecular , Nucleossomos , Ubiquitina , Ubiquitinação , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Nucleossomos/química , Histonas/metabolismo , Histonas/química , Ubiquitina/metabolismo , Ubiquitina/química , Ubiquitina/genética , Humanos , Ligação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Phosphorylation of histone H3 threonine 3 (H3T3) by Haspin recruits the chromosomal passenger complex to the inner centromere and ensures proper cell cycle progression through mitosis. The mechanism by which Haspin binds to nucleosomes to phosphorylate H3T3 is not known. We report here cryo-EM structures of the Haspin kinase domain bound to a nucleosome. In contrast with previous structures of histone-modifying enzymes, Haspin solely contacts the nucleosomal DNA, inserting into a supergroove formed by apposing major grooves of two DNA gyres. This unique binding mode provides a plausible mechanism by which Haspin can bind to nucleosomes in a condensed chromatin environment to phosphorylate H3T3. We identify key basic residues in the Haspin kinase domain that are essential for phosphorylation of nucleosomal histone H3 and binding to mitotic chromatin. Our structure is the first of a kinase domain bound to a nucleosome and is the first example of a histone-modifying enzyme that binds to nucleosomes solely through DNA contacts.
RESUMO
The discovery of ubiquitin conjugation to lysines and the role of K48-linked polyubiquitin in targeting substrates for proteasomal degradation was followed by revelation of non-degradative roles of ubiquitination and, more recently, of non-canonical covalent ubiquitin linkages. Here we summarize findings of the ever-expanding array of ubiquitin signals and their biological roles.
Assuntos
Poliubiquitina , Ubiquitina , Ubiquitina/metabolismo , Proteólise , Ubiquitinação , Poliubiquitina/metabolismo , Lisina/metabolismoRESUMO
The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is a transcriptional co-activator that both acetylates and deubiquitinates histones. The histone acetyltransferase (HAT) subunit, Gcn5, is part of a subcomplex of SAGA called the HAT module. A minimal HAT module complex containing Gcn5 bound to Ada2 and Ada3 is required for full Gcn5 activity on nucleosomes. Deletion studies have suggested that the Ada2 SWIRM domain plays a role in tethering the HAT module to the remainder of SAGA. While recent cryo-EM studies have resolved the structure of the core of the SAGA complex, the HAT module subunits and molecular details of its interactions with the SAGA core could not be resolved. Here we show that the SWIRM domain is required for incorporation of the HAT module into the yeast SAGA complex, but not the ADA complex, a distinct six-protein acetyltransferase complex that includes the SAGA HAT module proteins. In the isolated Gcn5/Ada2/Ada3 HAT module, deletion of the SWIRM domain modestly increased activity but had negligible effect on nucleosome binding. Loss of the HAT module due to deletion of the SWIRM domain decreases the H2B deubiquitinating activity of SAGA, indicating a role for the HAT module in regulating SAGA DUB module activity. A model of the HAT module created with Alphafold Multimer provides insights into the structural basis for our biochemical data, as well as prior deletion studies.
Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/química , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Histona Acetiltransferases/metabolismoRESUMO
The human Mixed Lineage Leukemia-1 (MLL1) complex methylates histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variability in the docking of MLL1-WRAD on nucleosomes. In addition, portions of the Ash2L structure and the position of DPY30 remain ambiguous. We used an integrated approach combining cryoelectron microscopy (cryo-EM) and mass spectrometry cross-linking to determine a structure of the MLL1-WRAD complex bound to ubiquitinated nucleosomes. The resulting model contains the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We also resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The docking of subunits in all four states differs from structures of MLL1-WRAD bound to unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex.
Assuntos
Histona-Lisina N-Metiltransferase , Peptídeos e Proteínas de Sinalização Intracelular , Proteína de Leucina Linfoide-Mieloide , Nucleossomos , Ubiquitinação , Microscopia Crioeletrônica , Histona-Lisina N-Metiltransferase/química , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Nucleossomos/enzimologia , Ligação ProteicaRESUMO
Structure-based drug discovery efforts require knowledge of where drug-binding sites are located on target proteins. To address the challenge of finding druggable sites, we developed a machine-learning algorithm called TACTICS (trajectory-based analysis of conformations to identify cryptic sites), which uses an ensemble of molecular structures (such as molecular dynamics simulation data) as input. First, TACTICS uses k-means clustering to select a small number of conformations that represent the overall conformational heterogeneity of the data. Then, TACTICS uses a random forest model to identify potentially bindable residues in each selected conformation, based on protein motion and geometry. Lastly, residues in possible binding pockets are scored using fragment docking. As proof-of-principle, TACTICS was applied to the analysis of simulations of the SARS-CoV-2 main protease and methyltransferase and the Yersinia pestis aryl carrier protein. Our approach recapitulates known small-molecule binding sites and predicts the locations of sites not previously observed in experimentally determined structures. The TACTICS code is available at https://github.com/Albert-Lau-Lab/tactics_protein_analysis.
Assuntos
COVID-19 , SARS-CoV-2 , Sítios de Ligação , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica , ProteínasRESUMO
Lysine methylation can modify noncovalent interactions by altering lysine's hydrophobicity as well as its electronic structure. Although the ramifications of the former are documented, the effects of the latter remain largely unknown. Understanding the electronic structure is important for determining how biological methylation modulates protein-protein binding, and the impact of artificial methylation experiments in which methylated lysines are used as spectroscopic probes and protein crystallization facilitators. The benchmarked first-principles calculations undertaken here reveal that methyl-induced polarization weakens the electrostatic attraction of amines with protein functional groups - salt bridges, hydrogen bonds and cation-π interactions weaken by as much as 10.3, 7.9 and 3.5 kT, respectively. Multipole analysis shows that weakened electrostatics is due to the altered inductive effects, which overcome increased attraction from methyl-enhanced polarizability and dispersion. Due to their fundamental nature, these effects are expected to be present in many cases. A survey of methylated lysines in protein structures reveals several cases in which methyl-induced polarization is the primary driver of altered noncovalent interactions; in these cases, destabilizations are found to be in the 0.6-4.7 kT range. The clearest case of where methyl-induced polarization plays a dominant role in regulating biological function is that of the PHD1-PHD2 domain, which recognizes lysine-methylated states on histones. These results broaden our understanding of how methylation modulates noncovalent interactions.
Assuntos
Lisina , Proteínas , Ligação de Hidrogênio , Lisina/metabolismo , Ligação Proteica , Proteínas/metabolismo , Eletricidade EstáticaRESUMO
Lysine methylation is a key regulator of protein-protein binding. The amine group of lysine can accept up to three methyl groups, and experiments show that protein-protein binding free energies are sensitive to the extent of methylation. These sensitivities have been rationalized in terms of chemical and structural features present in the binding pockets of methyllysine binding domains. However, understanding their specific roles requires an energetic analysis. Here we propose a theoretical framework to combine quantum and molecular mechanics methods, and compute the effect of methylation on protein-protein binding free energies. The advantages of this approach are that it derives contributions from all local non-trivial effects of methylation on induction, polarizability and dispersion directly from self-consistent electron densities, and at the same time determines contributions from well-characterized hydration effects using a computationally efficient classical mean field method. Limitations of the approach are discussed, and we note that predicted free energies of fourteen out of the sixteen cases agree with experiment. Critical assessment of these cases leads to the following overarching principles that drive methylation-state recognition by protein domains. Methylation typically reduces the pairwise interaction between proteins. This biases binding toward lower methylated states. Simultaneously, however, methylation also makes it easier to partially dehydrate proteins and place them in protein-protein complexes. This latter effect biases binding in favor of higher methylated states. The overall effect of methylation on protein-protein binding depends ultimately on the balance between these two effects, which is observed to be tuned via several combinations of local features.
Assuntos
Proteínas de Transporte/química , Lisina/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas/química , Sítios de Ligação , Proteínas de Transporte/metabolismo , Ligação de Hidrogênio , Lisina/metabolismo , Metilação , Ligação Proteica , Proteínas/metabolismo , Solventes , Relação Estrutura-AtividadeRESUMO
Trk-A is a receptor tyrosine kinase (RTK) that plays an essential role in the development and functioning of the nervous system. Trk-A is expressed in neurons and signals in response to two ligands, NGF and neurotrophin-3 (NT-3), with very different functional consequences. Thus, NGF and NT-3 are "biased" ligands for Trk-A. Because it has been hypothesized that biased RTK ligands induce differential stabilization of RTK dimers, here, we seek to test this hypothesis for NGF and NT-3. In particular, we use Förster resonance energy transfer (FRET) and fluorescence intensity fluctuation spectroscopy to assess the strength of Trk-A interactions and Trk-A oligomer size in the presence of the two ligands. Although the difference in Trk-A behavior in response to the two ligands has been previously attributed to differences in their binding to Trk-A in the endosomes at low pH, here, we further show differences in the stabilities of the NGF- and NT-3-bound Trk-A dimers in the plasma membrane and at neutral pH. We discuss the biological significance of these new findings and their implications for the design of Trk-A ligands with novel functionalities.