ABSTRACT
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.
ABSTRACT
Monoubiquitination of histone H2B-K120/123 plays several roles in regulating transcription, DNA replication and the DNA damage response. The structure of a nucleosome in complex with the dimeric RING E3 ligase Bre1 reveals that one RING domain binds to the nucleosome acidic patch, where it can position the E2 ubiquitin conjugating enzyme Rad6, while the other RING domain contacts the DNA. Comparisons with H2A-specific E3 ligases suggest a general mechanism of tuning histone specificity via the non-E2-binding RING domain.
Subject(s)
Histones , Saccharomyces cerevisiae Proteins , Histones/metabolism , Nucleosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Ubiquitin/metabolism , Ubiquitination , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
Post-translational modification of histones plays a central role in regulating transcription. Methylation of histone H3 at lysines 4 (H3K4) and 79 (H3K79) play roles in activating transcription whereas methylation of H3K27 is a repressive mark. These modifications, in turn, depend upon prior monoubiquitination of specific histone residues in a phenomenon known as histone crosstalk. Earlier work had provided insights into the mechanism by which monoubiquitination histone H2BK120 stimulates H3K4 methylation by COMPASS/MLL1 and H3K79 methylation by DOT1L, and monoubiquitinated H2AK119 stimulates methylation of H3K27 by the PRC2 complex. Recent studies have shed new light on the role of individual subunits and paralogs in regulating the activity of PRC2 and how additional post-translational modifications regulate yeast Dot1 and human DOT1L, as well as provided new insights into the regulation of MLL1 by H2BK120ub.
Subject(s)
Histones , Protein Processing, Post-Translational , Humans , Histones/metabolism , Ubiquitination , Methylation , Saccharomyces cerevisiae/metabolismABSTRACT
Monoubiquitination of histone H2BK120/123 plays multiple roles in regulating transcription, DNA replication and the DNA damage response. The structure of a nucleosome in complex with the dimeric RING E3 ligase, Bre1, reveals that one RING domain binds to the nucleosome acidic patch, where it can position the Rad6 E2, while the other RING domain contacts the DNA. Comparisons with H2A-specific E3 ligases suggests a general mechanism of tuning histone specificity via the non-E2-binding RING domain.
ABSTRACT
The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR), in most organisms, catalyzes the four-electron reduction of the thioester (S)-HMG-CoA to the primary alcohol (R)-mevalonate, utilizing NADPH as the hydride donor. In some organisms, including the opportunistic lung pathogen Burkholderia cenocepacia, it catalyzes the reverse reaction, utilizing NAD+ as a hydride acceptor in the oxidation of mevalonate. B. cenocepacia HMGR has been previously shown to exist as an ensemble of multiple non-additive oligomeric states, each with different levels of enzymatic activity, suggesting that the enzyme exhibits characteristics of the morpheein model of allostery. We have characterized a number of factors, including pH, substrate concentration, and enzyme concentration, that modulate the structural transitions that influence the interconversion among the multiple oligomers. We have also determined the crystal structure of B. cenocepacia HMGR in the hexameric state bound to coenzyme A and ADP. This hexameric assembly provides important clues about how the transition among oligomers might occur, and why B. cenocepacia HMGR, unique among characterized HMGRs, exhibits morpheein-like behavior.
Subject(s)
Bacterial Proteins/metabolism , Burkholderia cenocepacia/enzymology , Hydroxymethylglutaryl CoA Reductases/metabolism , Protein Structure, Quaternary , Adenosine Triphosphate/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Coenzyme A/chemistry , Crystallography, X-Ray , Enzyme Assays , Hydroxymethylglutaryl CoA Reductases/chemistry , Hydroxymethylglutaryl CoA Reductases/isolation & purification , Molecular Dynamics Simulation , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is conserved from yeast to humans. We report here cryo-EM structures of Dot1L bound to ubiquitinated nucleosome that show how H2B-Ub stimulates Dot1L activity and reveal a role for the histone H4 tail in positioning Dot1L. We find that contacts mediated by Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 that reorients K79 from an inaccessible position, thus enabling this side chain to insert into the active site in a position primed for catalysis. Our study provides a comprehensive mechanism of cross-talk between histone ubiquitination and methylation and reveals structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.
