Chemical Protein Synthesis via Direction-Switching One-Pot Peptide Ligation Enabled by Orthogonal Cysteinyl Protection.

A one-pot multiple peptide ligation strategy accelerates the demanding processes of chemical protein synthesis. Ligation intermediates generated during peptide ligation differ depending on the order of peptide segment assembly, affecting the aggregation properties of the ligation mixture. Therefore, expanding the scope of the ligation order in one-pot methods contributes to optimizing a ligation scheme for each target protein in chemical protein synthesis. Herein, we present the first example of direction-switching one-pot peptide ligation, utilizing the two mutually orthogonal cysteinyl protections compatible with native chemical ligation (NCL): thiazolidine (Thz) and a new allylic thiol protecting group, N-(2,2,2-trifluoroethyl)-allyloxycarbonylaminomethyl (TfeAllocam). By optimizing the structure and metal-mediated deprotection conditions for the allylic protecting group, we established efficient methods for consecutive NCLs and deprotections in two direction-switching schemes with the same peptide segments: one scheme from C-terminus-to-N-terminus (C-to-N) to N-terminus-to-C-terminus (N-to-C) and the other from N-to-C to C-to-N. Using this ligation approach, we accomplished the semisynthesis of dimonoubiquitinated histone H3 bearing trimethyl-lysine by condensing five peptide segments in a one-pot manner.

Journal of the American Chemical Society


Structural basis for E3 ubiquitin ligase UHRF1 binding to nucleosome core particle and histone H3 ubiquitination.

The maintenance of DNA methylation in differentiated cells is regulated by a ubiquitin signal generated by UHRF1 (ubiquitin-like with plant homeodomain and RING finger domain 1), which plays a pivotal role in recruitment of DNA methyltransferase DNMT1 to hemimethylated CpG sites. UHRF1 catalyzes multiple monoubiquitinations on histone H3 within nucleosomes. However, the structural mechanisms underlying the binding of UHRF1 to nucleosomes and the subsequent formation of the ubiquitin signal remain incompletely understood. Here, we report cryo-EM structures of UHRF1 bound to nucleosome core particle (NCP) harboring H3K9me3 and a single hemimethylated CpG site. The structures of the UHRF1-NCP complexes reveal an unanticipated interaction between the UHRF1 tandem Tudor domain and the acidic patch of the NCP. This interaction enhances histone H3 ubiquitination and stabilizes UHRF1 binding to the NCP in a manner that is dependent on the position of the hemimethylated CpG site. These findings provide mechanistic insights into the binding of UHRF1 to the NCP and the multiple monoubiquitination of histone H3 within the NCP.


Cryo-EM reveals evolutionarily conserved and distinct structural features of plant CG maintenance methyltransferase MET1.

DNA methylation is essential for genomic function and transposable element silencing. In plants, DNA methylation occurs in CG, CHG, and CHH contexts (where H = A, T, or C), with the maintenance of CG methylation mediated by the DNA methyltransferase MET1. The molecular mechanism by which MET1 maintains CG methylation, however, remains unclear. Here, we report cryogenic electron microscopy structures of Arabidopsis thaliana MET1. We find that the methyltransferase domain of MET1 specifically methylates hemimethylated DNA in vitro. The structure of MET1 bound to hemimethylated DNA reveals the activation mechanism of MET1 resembling that of mammalian DNMT1. Curiously, the structure of apo-MET1 shows an autoinhibitory state distinct from that of DNMT1, where the RFTS2 domain and the connecting linker inhibit DNA binding. The autoinhibition of MET1 is relieved upon binding of a potential activator, ubiquitinated histone H3. Taken together, our structural analysis demonstrates both conserved and distinct molecular mechanisms regulating CG maintenance methylation in plant and animal DNA methyltransferases.


Structures of USP7 in active and inactive states bound to DNMT1 revealed by cryo-EM.

The ubiquitin signal generated by UHRF1 is essential for DNA methylation maintenance by recruiting DNA methyltransferase 1 (DNMT1) to hemimethylated DNA through strong binding of its replication foci targeting sequence (RFTS) domain to ubiquitinated histone H3. The ubiquitin-specific protease 7 (USP7) forms a complex with DNMT1 and removes ubiquitin from H3. However, it remains unknown how USP7 deubiquitinates ubiquitinated H3 upon strong binding of the DNMT1 RFTS domain. Here, we show the activation mechanism of USP7 by combining biochemical and structural studies. USP7 is inactive toward ubiquitinated H3 in complex with the RFTS domain. However, when complexed with DNMT1, USP7 efficiently deubiquitinates ubiquitinated H3. Cryogenic electron microscopy (cryo-EM) single particle analysis revealed that USP7 bound to DNMT1 undergoes an open (inactive) and closed (active) conformational transition. Our findings provide mechanistic insights into the activation of USP7 upon binding to DNMT1 and contribute to a better understanding of the deubiquitination process in DNA methylation maintenance.


A comprehensive review of structural insights into DNA methylation maintenance.

DNA methylation is faithfully inherited during cell division, playing a crucial role in maintaining cellular identity. The process of DNA methylation maintenance relies on the DNA methyltransferase DNMT1 and the ubiquitin E3 ligase UHRF1. UHRF1 facilitates the ubiquitination of both the replication factor PAF15 and histone H3, with each ubiquitin signal regulating replication-coupled and -uncoupled DNA methylation maintenance, respectively. Over the past decades, advances in structural biology have significantly deepened our understanding of the molecular mechanisms governing DNA methylation maintenance. In particular, the emergence of cryo-electron microscopy-often referred to as the "resolution revolution"-has transformed many areas of biology, including epigenetics and chromatin biology. This review focuses on the structural mechanisms of DNA methylation maintenance, as revealed by the three-dimensional structures of key biomolecular complexes, and discusses the potential development of inhibitors targeting DNA methylation maintenance factors based on structural insights.


Characterization of the Swi6/HP1 binding motif in its partner protein reveals the basis for the functional divergence of the HP1 family proteins in fission yeast.

The heterochromatin protein 1 (HP1) family recognizes lysine 9-methylated histone H3 (H3K9me) and recruits other transacting factors to establish higher order chromatin structures. In the fission yeast Schizosaccharomyces pombe (S. pombe), two HP1 family proteins, Swi6 and Chp2, play distinct roles in recruiting transacting factors: Swi6 primarily recruits Epe1, a Jumonji C domain-containing protein involved in histone H3K9 demethylation, whereas Chp2 recruits Mit1, a component of the Snf2/Hdac Repressive Complex. However, detailed mechanisms of how multiple HP1 family proteins and their respective interactors work cooperatively or exclusively to form higher order chromatin structures remain elusive. In this study, we investigated the interactions between Swi6 and Epe1. We found that Swi6 interacts with Epe1 through its chromoshadow domain, and identified a unique motif, named the FVI motif, in Epe1 involved in this interaction through detailed mapping of the region. Enhanced green fluorescent protein (EGFP) tethering assays showed that the FVI motif is sufficient to recruit ectopically expressed EGFP to heterochromatic regions, and mutational analyses revealed that conserved hydrophobic residues in this motif are essential for proper targeting. Structural simulations further supported the importance of these residues in Swi6 binding. Interestingly, Mit1 containing the Epe1 FVI motif was recruited to the heterochromatic regions by Swi6 but not by Chp2. Cells expressing mutant Mit1 maintained heterochromatic silencing even in chp2 cells, suggesting that Chp2 is not required for heterochromatin formation when Mit1 is recruited by Swi6. These findings highlight distinct HP1-binding motifs in interactors, contributing to functional divergence among HP1 family proteins.


CDCA7 is an evolutionarily conserved hemimethylated DNA sensor in eukaryotes.

Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency, centromeric instability, and facial anomalies syndrome, characterized by DNA hypomethylation at heterochromatin. It remains unclear why CDCA7-HELLS is the sole nucleosome remodeling complex whose deficiency abrogates the maintenance of DNA methylation. We here identify the unique zinc-finger domain of CDCA7 as an evolutionarily conserved hemimethylation-sensing zinc finger (HMZF) domain. Cryo-electron microscopy structural analysis of the CDCA7-nucleosome complex reveals that the HMZF domain can recognize hemimethylated CpG in the outward-facing DNA major groove within the nucleosome core particle, whereas UHRF1, the critical activator of the maintenance methyltransferase DNMT1, cannot. CDCA7 recruits HELLS to hemimethylated chromatin and facilitates UHRF1-mediated H3 ubiquitylation associated with replication-uncoupled maintenance DNA methylation. We propose that the CDCA7-HELLS nucleosome remodeling complex assists the maintenance of DNA methylation on chromatin by sensing hemimethylated CpG that is otherwise inaccessible to UHRF1 and DNMT1.


Structure of human DPPA3 bound to the UHRF1 PHD finger reveals its functional and structural differences from mouse DPPA3.

DNA methylation maintenance is essential for cell fate inheritance. In differentiated cells, this involves orchestrated actions of DNMT1 and UHRF1. In mice, the high-affinity binding of DPPA3 to the UHRF1 PHD finger regulates UHRF1 chromatin dissociation and cytosolic localization, which is required for oocyte maturation and early embryo development. However, the human DPPA3 ortholog functions during these stages remain unclear. Here, we report the structural basis for human DPPA3 binding to the UHRF1 PHD finger. The conserved human DPPA3 85VRT87 motif binds to the acidic surface of UHRF1 PHD finger, whereas mouse DPPA3 binding additionally utilizes two unique α-helices. The binding affinity of human DPPA3 for the UHRF1 PHD finger was weaker than that of mouse DPPA3. Consequently, human DPPA3, unlike mouse DPPA3, failed to inhibit UHRF1 chromatin binding and DNA remethylation in Xenopus egg extracts effectively. Our data provide novel insights into the distinct function and structure of human DPPA3.


Cryo-electron microscopy reveals the impact of the nucleosome dynamics on transcription activity.

The structural biology of nucleosomes and their complexes with chromatin-associated factors contributes to our understanding of fundamental biological processes in the genome. With the advent of cryo-electron microscopy (cryo-EM), several structures are emerging with histone variants, various species and chromatin-associated proteins that bind to nucleosomes. Cryo-EM enables visualization of the dynamic states of nucleosomes, leading to the accumulation of knowledge on chromatin-templated biology. The cryo-EM structure of nucleosome in Komagataella pastoris, as studied by Fukushima et al., provided the insights into transcription ability of RNAPII with nucleosome dynamics. In this commentary, we review the recent advances in the structural biology of nucleosomes and their related biomolecules.


CDCA7 is a hemimethylated DNA adaptor for the nucleosome remodeler HELLS.

Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, characterized by hypomethylation at heterochromatin. The unique zinc-finger domain, zf-4CXXC_R1, of CDCA7 is widely conserved across eukaryotes but is absent from species that lack HELLS and DNA methyltransferases, implying its specialized relation with methylated DNA. Here we demonstrate that zf-4CXXC_R1 acts as a hemimethylated DNA sensor. The zf-4CXXC_R1 domain of CDCA7 selectively binds to DNA with a hemimethylated CpG, but not unmethylated or fully methylated CpG, and ICF disease mutations eliminated this binding. CDCA7 and HELLS interact via their N-terminal alpha helices, through which HELLS is recruited to hemimethylated DNA. While placement of a hemimethylated CpG within the nucleosome core particle can hinder its recognition by CDCA7, cryo-EM structure analysis of the CDCA7-nucleosome complex suggests that zf-4CXXC_R1 recognizes a hemimethylated CpG in the major groove at linker DNA. Our study provides insights into how the CDCA7-HELLS nucleosome remodeling complex uniquely assists maintenance DNA methylation.

bioRxiv - the preprint server for biology


Cooperative DNA-binding activities of Chp2 are critical for its function in heterochromatin assembly.

Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein that plays a critical role in heterochromatin assembly. HP1 proteins share a basic structure consisting of an N-terminal chromodomain (CD) and a C-terminal chromoshadow domain (CSD) linked by a disordered hinge region. The CD recognizes histone H3 lysine 9 methylation, a hallmark of heterochromatin, while the CSD forms a dimer to recruit other chromosomal proteins. HP1 proteins have been shown to bind DNA or RNA primarily through the hinge region. However, how DNA or RNA binding contributes to their function remains elusive. Here, we focus on Chp2, one of the two HP1 proteins in fission yeast, and investigate how Chp2's DNA-binding ability contributes to its function. Similar to other HP1 proteins, the Chp2 hinge exhibits clear DNA-binding activity. Interestingly, the Chp2 CSD also shows robust DNA-binding activity. Mutational analysis revealed that basic residues in the Chp2 hinge and at the N-terminus of the CSD are essential for DNA binding, and the combined amino acid substitutions of these residues alter Chp2 stability, impair Chp2 heterochromatin localization and lead to a silencing defect. These results demonstrate that the cooperative DNA-binding activities of Chp2 play an important role in heterochromatin assembly in fission yeast.


Novel compound heterozygous mutations in UHRF1 are associated with atypical immunodeficiency, centromeric instability and facial anomalies syndrome with distinctive genome-wide DNA hypomethylation.

Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is in most cases caused by mutations in either DNA methyltransferase (DNMT)3B, zinc finger and BTB domain containing 24, cell division cycle associated 7 or helicase lymphoid-specific. However, the causative genes of a few ICF patients remain unknown. We, herein, identified ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 (UHRF1) as a novel causative gene of one such patient with atypical symptoms. This patient is a compound heterozygote for two previously unreported mutations in UHRF1: c.886C > T (p.R296W) and c.1852C > T (p.R618X). The R618X mutation plausibly caused nonsense-mediated decay, while the R296W mutation changed the higher order structure of UHRF1, which is indispensable for the maintenance of CG methylation along with DNMT1. Genome-wide methylation analysis revealed that the patient had a centromeric/pericentromeric hypomethylation, which is the main ICF signature, but also had a distinctive hypomethylation pattern compared to patients with the other ICF syndrome subtypes. Structural and biochemical analyses revealed that the R296W mutation disrupted the protein conformation and strengthened the binding affinity of UHRF1 with its partner LIG1 and reduced ubiquitylation activity of UHRF1 towards its ubiquitylation substrates, histone H3 and proliferating cell nuclear antigen -associated factor 15 (PAF15). We confirmed that the R296W mutation causes hypomethylation at pericentromeric repeats by generating the HEK293 cell lines that mimic the patient's UHRF1 molecular context. Since proper interactions of the UHRF1 with LIG1, PAF15 and histone H3 are essential for the maintenance of CG methylation, the mutation could disturb the maintenance process. Evidence for the importance of the UHRF1 conformation for CG methylation in humans is, herein, provided for the first time and deepens our understanding of its role in regulation of CG methylation.


The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5.

UHRF1-dependent ubiquitin signaling plays an integral role in the regulation of maintenance DNA methylation. UHRF1 catalyzes transient dual mono-ubiquitylation of PAF15 (PAF15Ub2), which regulates the localization and activation of DNMT1 at DNA methylation sites during DNA replication. Although the initiation of UHRF1-mediated PAF15 ubiquitin signaling has been relatively well characterized, the mechanisms underlying its termination and how they are coordinated with the completion of maintenance DNA methylation have not yet been clarified. This study shows that deubiquitylation by USP7 and unloading by ATAD5 (ELG1 in yeast) are pivotal processes for the removal of PAF15 from chromatin. On replicating chromatin, USP7 specifically interacts with PAF15Ub2 in a complex with DNMT1. USP7 depletion or inhibition of the interaction between USP7 and PAF15 results in abnormal accumulation of PAF15Ub2 on chromatin. Furthermore, we also find that the non-ubiquitylated form of PAF15 (PAF15Ub0) is removed from chromatin in an ATAD5-dependent manner. PAF15Ub2 was retained at high levels on chromatin when the catalytic activity of DNMT1 was inhibited, suggesting that the completion of maintenance DNA methylation is essential for the termination of UHRF1-mediated ubiquitin signaling. This finding provides a molecular understanding of how the maintenance DNA methylation machinery is disassembled at the end of the S phase.


Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger.

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.


Structural basis for activation of DNMT1.

DNMT1 is an essential enzyme that maintains genomic DNA methylation, and its function is regulated by mechanisms that are not yet fully understood. Here, we report the cryo-EM structure of human DNMT1 bound to its two natural activators: hemimethylated DNA and ubiquitinated histone H3. We find that a hitherto unstudied linker, between the RFTS and CXXC domains, plays a key role for activation. It contains a conserved α-helix which engages a crucial "Toggle" pocket, displacing a previously described inhibitory linker, and allowing the DNA Recognition Helix to spring into the active conformation. This is accompanied by large-scale reorganization of the inhibitory RFTS and CXXC domains, allowing the enzyme to gain full activity. Our results therefore provide a mechanistic basis for the activation of DNMT1, with consequences for basic research and drug design.


Preparation of the ubiquitination-triggered active form of SETDB1 in Escherichia coli for biochemical and structural analyses.

Trimethylation of histone H3 at K9 by the lysine methyltransferase, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) plays a pivotal role in silencing tissue-specific genes and retrotransposable elements. In mammalian cells, SETDB1 undergoes monoubiquitination in the insertion region of the SET domain in an E3 ubiquitin ligase-independent manner. This ubiquitination has been shown to enhance the histone H3-K9 methyltransferase activity of SETDB1; however, the molecular mechanism underlying SETDB1 activation by ubiquitination is unknown. In this study, we developed an Escherichia coli ubiquitination plasmid for the preparation of ubiquitinated SETDB1. Western blotting and mutational analyses showed that co-expression of the SET domain of SETDB1 with the proteins encoded by the ubiquitination plasmid led to site-specific monoubiquitination of the SET domain at K867. An in vitro histone H3 methylation assay demonstrated that the ubiquitinated SET domain of SETDB1 acquired enzymatic activity. Taken together, these findings demonstrate successful preparation of the active form of SETDB1 with the E.coli ubiquitination system, which will aid biochemical and structural studies of ubiquitinated SETDB1. Graphical Abstract.


Structure-based screening combined with computational and biochemical analyses identified the inhibitor targeting the binding of DNA Ligase 1 to UHRF1.

The accumulation of epigenetic alterations is one of the major causes of tumorigenesis. Aberrant DNA methylation patterns cause genome instability and silencing of tumor suppressor genes in various types of tumors. Therefore, drugs that target DNA methylation-regulating factors have great potential for cancer therapy. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is an essential factor for DNA methylation maintenance. UHRF1 is overexpressed in various cancer cells and down-regulation of UHRF1 in these cells reactivates the expression of tumor suppressor genes, thus UHRF1 is a promising target for cancer therapy. We have previously shown that interaction between the tandem Tudor domain (TTD) of UHRF1 and DNA ligase 1 (LIG1) di/trimethylated on Lys126 plays a key role in the recruitment of UHRF1 to replication sites and replication-coupled DNA methylation maintenance. An arginine binding cavity (Arg-binding cavity) of the TTD is essential for LIG1 interaction, thus the development of inhibitors that target the Arg-binding cavity could potentially repress UHRF1 function in cancer cells. To develop such an inhibitor, we performed in silico screening using not only static but also dynamic metrics based on all-atom molecular dynamics simulations, resulting in efficient identification of 5-amino-2,4-dimethylpyridine (5A-DMP) as a novel TTD-binding compound. Crystal structure of the TTD in complex with 5A-DMP revealed that the compound stably bound to the Arg-binding cavity of the TTD. Furthermore, 5A-DMP inhibits the full-length UHRF1:LIG1 interaction in Xenopus egg extracts. Our study uncovers a UHRF1 inhibitor which can be the basis of future experiments for cancer therapy.


Structural dynamics of double-stranded DNA with epigenome modification.

Modification of cytosine plays an important role in epigenetic regulation of gene expression and genome stability. Cytosine is converted to 5-methylcytosine (5mC) by DNA methyltransferase; in turn, 5mC may be oxidized to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation enzyme. The structural flexibility of DNA is known to affect the binding of proteins to methylated DNA. Here, we have carried out a semi-quantitative analysis of the dynamics of double-stranded DNA (dsDNA) containing various epigenetic modifications by combining data from imino 1H exchange and imino 1H R1ρ relaxation dispersion NMR experiments in a complementary way. Using this approach, we characterized the base-opening (kopen) and base-closing (kclose) rates, facilitating a comparison of the base-opening and -closing process of dsDNA containing cytosine in different states of epigenetic modification. A particularly striking result is the increase in the kopen rate of hemi-methylated dsDNA 5mC/C relative to unmodified or fully methylated dsDNA, indicating that the Watson-Crick base pairs undergo selective destabilization in 5mC/C. Collectively, our findings imply that the epigenetic modulation of cytosine dynamics in dsDNA mediates destabilization of the GC Watson-Crick base pair to allow base-flipping in living cells.


Serine 298 Phosphorylation in Linker 2 of UHRF1 Regulates Ligand-Binding Property of Its Tandem Tudor Domain.

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an essential factor for the maintenance of mammalian DNA methylation and harbors several reader modules for recognizing epigenetic marks. The tandem Tudor domain (TTD) of UHRF1 has a peptide-binding groove that functions as a binding platform for intra- or intermolecular interactions. Besides the groove interacting with unphosphorylated linker 2 and spacer of UHRF1, it also interacts with di/tri-methylated histone H3 at Lys9 and DNA ligase 1 (LIG1) at Lys126. Here we focus on the phosphorylation of Ser298 in linker 2, which was implied to regulate the ligand-binding property of the TTD. Although the protein expression level of UHRF1 is unchanged throughout the cell cycle, Ser298 phosphorylated form of UHRF1 is notably increased in the G2/M phase, which is revealed by immunoprecipitation followed by Western blotting. Molecularly, while unphosphorylated linker 2 covers the peptide-binding groove to prevent access of other interactors, small-angle X-ray scattering, thermal stability assay and molecular dynamics simulation revealed that the phosphate group of Ser298 dissociates linker 2 from the peptide-binding groove of the TTD to permit the other interactors to access to the groove. Our data reveal a mechanism in which Ser298 phosphorylation in linker 2 triggers a change of the TTD's structure and may affect multiple functions of UHRF1 by facilitating associations with LIG1 at DNA replication sites and histone H3K9me2/me3 at heterochromatic regions.


Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation.

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.


Enhanced processivity of Dnmt1 by monoubiquitinated histone H3.

DNA methylation controls gene expression, and once established, DNA methylation patterns are faithfully copied during DNA replication by the maintenance DNA methyltransferase Dnmt1. In vivo, Dnmt1 interacts with Uhrf1, which recognizes hemimethylated CpGs. Recently, we reported that Uhrf1-catalyzed K18- and K23-ubiquitinated histone H3 binds to the N-terminal region (the replication focus targeting sequence, RFTS) of Dnmt1 to stimulate its methyltransferase activity. However, it is not yet fully understood how ubiquitinated histone H3 stimulates Dnmt1 activity. Here, we show that monoubiquitinated histone H3 stimulates Dnmt1 activity toward DNA with multiple hemimethylated CpGs but not toward DNA with only a single hemimethylated CpG, suggesting an influence of ubiquitination on the processivity of Dnmt1. The Dnmt1 activity stimulated by monoubiquitinated histone H3 was additively enhanced by the Uhrf1 SRA domain, which also binds to RFTS. Thus, Dnmt1 activity is regulated by catalysis (ubiquitination)-dependent and -independent functions of Uhrf1.


Structure of PCNA in complex with DNMT1 PIP box reveals the basis for the molecular mechanism of the interaction.

DNMT1 is a C5-DNA methyltransferase that plays a pivotal role in DNA methylation maintenance. During early and mid S-phase, DNMT1 accumulates at DNA replication sites by binding to proliferating cell nuclear antigen (PCNA), an essential factor for DNA replication, through a PIP box motif. However, the molecular mechanism by which the DNMT1 PIP box motif binds to PCNA remains unclear. Here, we report the crystal structure of PCNA bound to DNMT1 PIP box peptide. The structure reveals the detailed interaction between PCNA and DNMT1 PIP box; conserved glutamine and hydrophobic/aromatic residues in the PIP box are recognized by the Q- and hydrophobic pockets of PCNA, respectively. The structure also shows novel intramolecular interactions within the PIP box motif, which stabilize the helix conformation in the PIP box. Our data provide structural insight into the recruitment of DNMT1 to replication sites by PCNA.

Biochemical and Biophysical Research Communications


Structure of the UHRF1 Tandem Tudor Domain Bound to a Methylated Non-histone Protein, LIG1, Reveals Rules for Binding and Regulation.

The protein UHRF1 is crucial for DNA methylation maintenance. The tandem Tudor domain (TTD) of UHRF1 binds histone H3K9me2/3 with micromolar affinity, as well as unmethylated linker regions within UHRF1 itself, causing auto-inhibition. Recently, we showed that a methylated histone-like region of DNA ligase 1 (LIG1K126me2/me3) binds the UHRF1 TTD with nanomolar affinity, permitting UHRF1 recruitment to chromatin. Here we report the crystal structure of the UHRF1 TTD bound to a LIG1K126me3 peptide. The data explain the basis for the high TTD-binding affinity of LIG1K126me3 and reveal that the interaction may be regulated by phosphorylation. Binding of LIG1K126me3 switches the overall structure of UHRF1 from a closed to a flexible conformation, suggesting that auto-inhibition is relieved. Our results provide structural insight into how UHRF1 performs its key function in epigenetic maintenance.


Structure of the Dnmt1 Reader Module Complexed with a Unique Two-Mono-Ubiquitin Mark on Histone H3 Reveals the Basis for DNA Methylation Maintenance.

The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.


RFTS-dependent negative regulation of Dnmt1 by nucleosome structure and histone tails.

DNA methylation in promoter regions represses gene expression and is copied over mitotic divisions by Dnmt1. Dnmt1 activity is regulated by its replication foci targeting sequence (RFTS) domain which masks the catalytic pocket. It has been shown that Dnmt1 activity on unmethylated DNA is inhibited in nucleosome cores. In the present study, we aimed to assess the effect of nuclesome formation on maintenance methylation at single CpG resolution. We show that Dnmt1 fully methylates naked linker DNA in dinucleosomes, whereas maintenance methylation was repressed at all CpG sites in nucleosome core particles. Deletion of RFTS partly released obstruction of Dnmt1 activity in core particles. Histone H3 tail peptides inhibited Dnmt1 in an RFTS-dependent manner and repression was modulated by acetylation or methylation. We propose a novel function of RFTS to regulate Dnmt1 activity in nucleosomes.


Methylation of DNA Ligase 1 by G9a/GLP Recruits UHRF1 to Replicating DNA and Regulates DNA Methylation.

DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance.


Usp7-dependent histone H3 deubiquitylation regulates maintenance of DNA methylation.

Uhrf1-dependent histone H3 ubiquitylation plays a crucial role in the maintenance of DNA methylation via the recruitment of the DNA methyltransferase Dnmt1 to DNA methylation sites. However, the involvement of deubiquitylating enzymes (DUBs) targeting ubiquitylated histone H3 in the maintenance of DNA methylation is largely unknown. With the use of Xenopus egg extracts, we demonstrate here that Usp7, a ubiquitin carboxyl-terminal hydrolase, forms a stable complex with Dnmt1 and is recruited to DNA methylation sites during DNA replication. Usp7 deubiquitylates ubiquitylated histone H3 in vitro. Inhibition of Usp7 activity or its depletion in egg extracts results in enhanced and extended binding of Dnmt1 to chromatin, suppressing DNA methylation. Depletion of Usp7 in HeLa cells causes enhanced histone H3 ubiquitylation and enlargement of Dnmt1 nuclear foci during DNA replication. Our results thus suggest that Usp7 is a key factor that regulates maintenance of DNA methylation.


Analysis of the Fine-Tuning of Cyanobacterial Circadian Phase by Monochromatic Light and Long-Day Conditions.

The cyanobacterial circadian-related protein, Pex, accumulates in the dark period of the diurnal light-dark cycle. After the diurnal cycle, an approximately 3 h advance in the phase of the circadian bioluminescence rhythm is observed in pex-deficient mutants, as compared with the wild type. However, it is unclear what type of photosensing mechanism regulates the accumulation and the phase change. In monochromatic light irradiation experiments, Pex accumulation was strongly repressed under blue light conditions; however, only small reductions in Pex accumulation were observed under red or green light conditions. After the diurnal cycle of 12 h of white fluorescent light and 12 h of blue light, the phase advance was repressed more than that of the cycle of 12 h red (or green) light. The phase advance also occurred after 16 h light/8 h dark cycles (long-day cycles) but did not occur after 8 h light/16 h dark cycles (short-day cycles). While Pex is a unique winged helix transcription factor harboring secondary structures (α0 and α4 helices), the importance of the structures is not understood. In in vivo experiments with site-directed mutations in the α0 helix, the obtained mutants, in which Pex was missing the hydrophobic side chain at the 28th or 32nd amino acid residue, exhibited no phase delay after the light/dark cycle. In in vitro DNA binding assays, the mutant proteins showed no binding to the promoter region of the clock gene kaiA. From these results, we propose a molecular model which describes the phase delay in cyanobacteria.


The unexpected role of polyubiquitin chains in the formation of fibrillar aggregates.

Ubiquitin is known to be one of the most soluble and stably folded intracellular proteins, but it is often found in inclusion bodies associated with various diseases including neurodegenerative disorders and cancer. To gain insight into this contradictory behaviour, we have examined the physicochemical properties of ubiquitin and its polymeric chains that lead to aggregate formation. We find that the folding stability of ubiquitin chains unexpectedly decreases with increasing chain length, resulting in the formation of amyloid-like fibrils. Furthermore, when expressed in cells, polyubiquitin chains covalently linked to EGFP also form aggregates depending on chain length. Notably, these aggregates are selectively degraded by autophagy. We propose a novel model in which the physical and chemical instability of polyubiquitin chains drives the formation of fibrils, which then serve as an initiation signal for autophagy.


The structural basis for receptor recognition of human interleukin-18.

Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors' recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-18 activity.


Purification, crystallization and preliminary X-ray crystallographic analysis of human IL-18 and its extracellular complexes.

Interleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the interleukin-1 (IL-1) family, is involved in the pathogenesis of autoimmune/autoinflammatory and allergic diseases such as juvenile idiopathic arthritis and bronchial asthma. IL-18 forms a signalling complex with the IL-18 receptor α (IL-18Rα) and β (IL-18Rβ) chains; however, the detailed activation mechanism remains unclear. Here, the IL-18-IL-18Rα binary and IL-18-IL-18Rα-IL-18Rβ ternary complexes were purified and crystallized as well as IL-18 alone. An X-ray diffraction data set for IL-18 was collected to 2.33 Å resolution from a crystal belonging to space group P21, with unit-cell parameters a = 68.15, b = 79.51, c = 73.46 Å, β = 100.97°. Crystals of both the IL-18 binary and ternary complexes belonging to the orthorhombic space groups P21212 and P212121, respectively, diffracted to 3.10 Å resolution. Unit-cell parameters were determined as a = 135.49, b = 174.81, c = 183.40 Å for the binary complex and a = 72.56, b = 111.56, c = 134.57 Å for the ternary complex.

Acta Crystallographica. Section F, Structural Biology Communications


The DNA methyltransferase Dnmt1 directly interacts with the SET and RING finger-associated (SRA) domain of the multifunctional protein Uhrf1 to facilitate accession of the catalytic center to hemi-methylated DNA.

Dnmt1 is responsible for the maintenance DNA methylation during replication to propagate methylation patterns to the next generation. The replication foci targeting sequence (RFTS), which plugs the catalytic pocket, is necessary for recruitment of Dnmt1 to the replication site. In the present study we found that the DNA methylation activity of Dnmt1 was DNA length-dependent and scarcely methylated 12-bp short hemi-methylated DNA. Contrarily, the RFTS-deleted Dnmt1 and Dnmt1 mutants that destroyed the hydrogen bonds between the RFTS and catalytic domain showed significant DNA methylation activity even toward 12-bp hemi-methylated DNA. The DNA methylation activity of the RFTS-deleted Dnmt1 toward 12-bp hemi-methylated DNA was strongly inhibited on the addition of RFTS, but to a lesser extent by Dnmt1 harboring the mutations that impair the hydrogen bond formation. The SRA domain of Uhrf1, which is a prerequisite factor for maintenance methylation and selectively binds to hemi-methylated DNA, stimulated the DNA methylation activity of Dnmt1. The SRA to Dnmt1 concentration ratio was the determinant for the maximum stimulation. In addition, a mutant SRA, which had lost the DNA binding activity but was able to bind to Dnmt1, stimulated the DNA methylation activity of Dnmt1. The results indicate that the DNA methylation activity of Dnmt1 was stimulated on the direct interaction of the SRA and Dnmt1. The SRA facilitated acceptance of the 12-bp fluorocytosine-containing DNA by the catalytic center. We propose that the SRA removes the RFTS plug from the catalytic pocket to facilitate DNA acceptance by the catalytic center.


Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication.

Faithful propagation of DNA methylation patterns during DNA replication is critical for maintaining cellular phenotypes of individual differentiated cells. Although it is well established that Uhrf1 (ubiquitin-like with PHD and ring finger domains 1; also known as Np95 and ICBP90) specifically binds to hemi-methylated DNA through its SRA (SET and RING finger associated) domain and has an essential role in maintenance of DNA methylation by recruiting Dnmt1 to hemi-methylated DNA sites, the mechanism by which Uhrf1 coordinates the maintenance of DNA methylation and DNA replication is largely unknown. Here we show that Uhrf1-dependent histone H3 ubiquitylation has a prerequisite role in the maintenance DNA methylation. Using Xenopus egg extracts, we successfully reproduce maintenance DNA methylation in vitro. Dnmt1 depletion results in a marked accumulation of Uhrf1-dependent ubiquitylation of histone H3 at lysine 23. Dnmt1 preferentially associates with ubiquitylated H3 in vitro though a region previously identified as a replication foci targeting sequence. The RING finger mutant of Uhrf1 fails to recruit Dnmt1 to DNA replication sites and maintain DNA methylation in mammalian cultured cells. Our findings represent the first evidence, to our knowledge, of the mechanistic link between DNA methylation and DNA replication through histone H3 ubiquitylation.


Development of fluorogenic probes for quick no-wash live-cell imaging of intracellular proteins.

We developed novel fluorogenic probes for no-wash live-cell imaging of proteins fused to PYP-tag, which is a small protein tag recently reported by our group. Through the design of a new PYP-tag ligand, specific intracellular protein labeling with rapid kinetics and fluorogenic response was accomplished. The probes crossed the cell membrane, and cytosolic and nuclear localizations of PYP-tagged proteins without cell washing were visualized within a 6-min reaction time. The fluorogenic response was due to the environmental effect of fluorophore upon binding to PYP-tag. Furthermore, the PYP-tag-based method was applied to the imaging of methyl-CpG-binding domain localization. This rapid protein-labeling system combined with the small protein tag and designed fluorogenic probes offers a powerful method to study the localization, movement, and function of cellular proteins.

Journal of the American Chemical Society


Quantitative comparison of protein dynamics in live cells and in vitro by in-cell (19)F-NMR.

Here we describe how a (19)F-probe incorporated into an endogenous protein by a chemical biology method revealed protein dynamics. By explicit determination of ligand-bound and unbound structures with X-ray crystallography, the quantitative comparison of the protein's dynamics in live cells and in vitro is presented. These results clearly demonstrated the greater conformational fluctuations of the intracellular protein, partially due to macromolecular crowding effects.


Structural basis of the versatile DNA recognition ability of the methyl-CpG binding domain of methyl-CpG binding domain protein 4.

The methyl-CpG binding domain (MBD) protein MBD4 participates in DNA repair as a glycosylase that excises mismatched thymine bases in CpG sites and also functions in transcriptional repression. Unlike other MBD proteins, MBD4 recognizes not only methylated CpG dinucleotides ((5m)CG/(5m)CG) but also T/G mismatched sites generated by spontaneous deamination of 5-methylcytosine ((5m)CG/TG). The glycosylase activity of MBD4 is also implicated in active DNA demethylation initiated by the deaminase-catalyzed conversion of 5-methylcytosine to thymine. Here, we report the crystal structures of the MBD of MBD4 (MBDMBD4) complexed with (5m)CG/(5m)CG and (5m)CG/TG. The crystal structures show that the DNA interface of MBD4 has flexible structural features and harbors an extensive water network that supports its dual base specificities. Combined with the results of biochemical analyses, the crystal structure of MBD4 bound to 5-hydroxymethylcytosine further demonstrates that MBDMBD4 is able to recognize a wide range of 5-methylcytosine modifications through the unique water network. The versatile base recognition ability of MBDMBD4 implies multifunctional roles for MBD4 in the regulation of dynamic DNA methylation patterns coupled with deamination and/or oxidation of 5-methylcytosine.


Lysyl 5-hydroxylation, a novel histone modification, by Jumonji domain containing 6 (JMJD6).

JMJD6 is reported to hydroxylate lysyl residues of a splicing factor, U2AF65. In this study, we found that JMJD6 hydroxylates histone lysyl residues. In vitro experiments showed that JMJD6 has a binding affinity to histone proteins and hydroxylates multiple lysyl residues of histone H3 and H4 tails. Using JMJD6 knock-out mouse embryos, we revealed that JMJD6 hydroxylates lysyl residues of histones H2A/H2B and H3/H4 in vivo by amino acid composition analysis. 5-Hydroxylysine was detected at the highest level in histones purified from murine testis, which expressed JMJD6 at a significantly high level among various tissues examined, and JMJD6 overexpression increased the amount of 5-hydroxylysine in histones in human embryonic kidney 293 cells. These results indicate that histones are additional substrates of JMJD6 in vivo. Because 5-hydroxylation of lysyl residues inhibited N-acetylation and N-methylation by an acetyltransferase and a methyltransferase, respectively, in vitro, histone 5-hydroxylation may have important roles in epigenetic regulation of gene transcription or chromosomal rearrangement.


Recognition of modification status on a histone H3 tail by linked histone reader modules of the epigenetic regulator UHRF1.

Multiple covalent modifications on a histone tail are often recognized by linked histone reader modules. UHRF1 [ubiquitin-like, containing plant homeodomain (PHD) and really interesting new gene (RING) finger domains 1], an essential factor for maintenance of DNA methylation, contains linked two-histone reader modules, a tandem Tudor domain and a PHD finger, tethered by a 17-aa linker, and has been implicated to link histone modifications and DNA methylation. Here, we present the crystal structure of the linked histone reader modules of UHRF1 in complex with the amino-terminal tail of histone H3. Our structural and biochemical data provide the basis for combinatorial readout of unmodified Arg-2 (H3-R2) and methylated Lys-9 (H3-K9) by the tandem tudor domain and the PHD finger. The structure reveals that the intermodule linker plays an essential role in the formation of a histone H3-binding hole between the reader modules by making extended contacts with the tandem tudor domain. The histone H3 tail fits into the hole by adopting a compact fold harboring a central helix, which allows both of the reader modules to simultaneously recognize the modification states at H3-R2 and H3-K9. Our data also suggest that phosphorylation of a linker residue can modulate the relative position of the reader modules, thereby altering the histone H3-binding mode. This finding implies that the linker region plays a role as a functional switch of UHRF1 involved in multiple regulatory pathways such as maintenance of DNA methylation and transcriptional repression.

Proceedings of the National Academy of Sciences of the United States of America


Crystal structure of the ubiquitin-associated (UBA) domain of p62 and its interaction with ubiquitin.

p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62.


Structural basis for regulation of poly-SUMO chain by a SUMO-like domain of Nip45.

Post-translational modification by small ubiquitin-like modifier (SUMO) provides an important regulatory mechanism in diverse cellular processes. Modification of SUMO has been shown to target proteins involved in systems ranging from DNA repair pathways to the ubiquitin-proteasome degradation system by the action of SUMO-targeted ubiquitin ligases (STUbLs). STUbLs recognize target proteins modified with a poly-SUMO chain through their SUMO-interacting motifs (SIMs). STUbLs are also associated with RENi family proteins, which commonly have two SUMO-like domains (SLD1 and SLD2) at their C terminus. We have determined the crystal structures of SLD2 of mouse RENi protein, Nip45, in a free form and in complex with a mouse E2 sumoylation enzyme, Ubc9. While Nip45 SLD2 shares a beta-grasp fold with SUMO, the SIM interaction surface conserved in SUMO paralogues does not exist in SLD2. Biochemical data indicates that neither tandem SLDs or SLD2 of Nip45 bind to either tandem SIMs from either mouse STUbL, RNF4 or to those from SUMO-binding proteins, whose interactions with SUMO have been well characterized. On the other hand, Nip45 SLD2 binds to Ubc9 in an almost identical manner to that of SUMO and thereby inhibits elongation of poly-SUMO chains. This finding highlights a possible role of the RENi proteins in the modulation of Ubc9-mediated poly-SUMO formation.


Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX-DNMT3-DNMT3L domain.

DNMT3 proteins are de novo DNA methyltransferases that are responsible for the establishment of DNA methylation patterns in mammalian genomes. Here, we have determined the crystal structures of the ATRX-DNMT3-DNMT3L (ADD) domain of DNMT3A in an unliganded form and in a complex with the amino-terminal tail of histone H3. Combined with the results of biochemical analysis, the complex structure indicates that DNMT3A recognizes the unmethylated state of lysine 4 in histone H3. This finding indicates that the recruitment of DNMT3A onto chromatin, and thereby de novo DNA methylation, is mediated by recognition of the histone modification state by its ADD domain. Furthermore, our biochemical and nuclear magnetic resonance data show mutually exclusive binding of the ADD domain of DNMT3A and the chromodomain of heterochromatin protein 1alpha to the H3 tail. These results indicate that de novo DNA methylation by DNMT3A requires the alteration of chromatin structure.


Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism.

DNA methylation of CpG dinucleotides is an important epigenetic modification of mammalian genomes and is essential for the regulation of chromatin structure, of gene expression and of genome stability. Differences in DNA methylation patterns underlie a wide range of biological processes, such as genomic imprinting, inactivation of the X chromosome, embryogenesis, and carcinogenesis. Inheritance of the epigenetic methylation pattern is mediated by the enzyme DNA methyltransferase 1 (Dnmt1), which methylates newly synthesized CpG sequences during DNA replication, depending on the methylation status of the template strands. The protein UHRF1 (also known as Np95 and ICBP90) recognizes hemi-methylation sites via a SET and RING-associated (SRA) domain and directs Dnmt1 to these sites. Here we report the crystal structures of the SRA domain in free and hemi-methylated DNA-bound states. The SRA domain folds into a globular structure with a basic concave surface formed by highly conserved residues. Binding of DNA to the concave surface causes a loop and an amino-terminal tail of the SRA domain to fold into DNA interfaces at the major and minor grooves of the methylation site. In contrast to fully methylated CpG sites recognized by the methyl-CpG-binding domain, the methylcytosine base at the hemi-methylated site is flipped out of the DNA helix in the SRA-DNA complex and fits tightly into a protein pocket on the concave surface. The complex structure suggests that the successive flip out of the pre-existing methylated cytosine and the target cytosine to be methylated is associated with the coordinated transfer of the hemi-methylated CpG site from UHRF1 to Dnmt1.


Structural and biochemical characterization of a cyanobacterium circadian clock-modifier protein.

Circadian clocks are self-sustained biochemical oscillators. The oscillator of cyanobacteria comprises the products of three kai genes (kaiA, kaiB, and kaiC). The autophosphorylation cycle of KaiC oscillates robustly in the cell with a 24-h period and is essential for the basic timing of the cyanobacterial circadian clock. Recently, period extender (pex), mutants of which show a short period phenotype, was classified as a resetting-related gene. In fact, pex mRNA and the pex protein (Pex) increase during the dark period, and a pex mutant subjected to diurnal light-dark cycles shows a 3-h advance in rhythm phase. Here, we report the x-ray crystallographic analysis and biochemical characterization of Pex from cyanobacterium Synechococcus elongatus PCC 7942. The molecule has an (alpha+beta) structure with a winged-helix motif and is indicated to function as a dimer. The subunit arrangement in the dimer is unique and has not been seen in other winged-helix proteins. Electrophoresis mobility shift assay using a 25-base pair complementary oligonucleotide incorporating the kaiA upstream sequence demonstrates that Pex has an affinity for the double-stranded DNA. Furthermore, mutation analysis shows that Pex uses the wing region to recognize the DNA. The in vivo rhythm assay of Pex shows that the constitutive expression of the pex gene harboring the mutation that fails to bind to DNA lacks the period-prolongation activity in the pex-deficient Synechococcus, suggesting that Pex is a DNA-binding transcription factor.


Inhibitors and inactivators of protein arginine deiminase 4: functional and structural characterization.

Protein arginine deiminase 4 (PAD4) is a transcriptional coregulator that catalyzes the calcium-dependent conversion of specific arginine residues in proteins to citrulline. Recently, we reported the synthesis and characterization of F-amidine, a potent and bioavailable irreversible inactivator of PAD4. Herein, we report our efforts to identify the steric and leaving group requirements for F-amidine-induced PAD4 inactivation, the structure of the PAD4-F-amidine x calcium complex, and in vivo studies with N-alpha-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (Cl-amidine), a PAD4 inactivator with enhanced potency. The PAD4 inactivators described herein will be useful pharmacological probes in characterizing the incompletely defined physiological role(s) of this enzyme. In addition, they represent potential lead compounds for the treatment of rheumatoid arthritis because a growing body of evidence supports a role for PAD4 in the onset and progression of this chronic autoimmune disorder.


Structural basis for histone N-terminal recognition by human peptidylarginine deiminase 4.

Histone arginine methylation is a posttranslational modification linked to the regulation of gene transcription. Unlike other posttranslational modifications, methylation has generally been regarded as stable, and enzymes that demethylate histone arginine residues have not been identified. However, it has recently been shown that human peptidylarginine deiminase 4 (PAD4), a Ca(2+)-dependent enzyme previously known to convert arginine residues to citrulline in histones, can also convert monomethylated arginine residues to citrulline both in vivo and in vitro. Citrullination of histone arginine residues by the enzyme antagonizes methylation by histone arginine methyltransferases and is thus a novel posttranslational modification that regulates the level of histone arginine methylation and gene activity. Here we present the crystal structures of a Ca(2+)-bound PAD4 mutant in complex with three histone N-terminal peptides, each consisting of 10 amino acid residues that include one target arginine residue for the enzyme (H3/Arg-8, H3/Arg-17, and H4/Arg-3). To each histone N-terminal peptide, the enzyme induces a beta-turn-like bent conformation composed of five successive residues at the molecular surface near the active site cleft. The remaining five residues are highly disordered. The enzyme recognizes each peptide through backbone atoms of the peptide with a possible consensus recognition motif. The sequence specificity of the peptide recognized by this enzyme is thought to be fairly broad. These observations provide structural insights into target protein recognition by histone modification enzymes and illustrate how PAD4 can target multiple arginine sites in the histone N-terminal tails.


Structural characterization of calcineurin B homologous protein 1.

Calcineurin B homologous protein 1 (CHP1), also known as p22, is a calcium-binding EF-hand protein that plays a role in membrane trafficking. It binds to multiple effector proteins, including Na(+)/H(+) exchangers, a serine/threonine kinase, and calcineurin, potentially modulating their function. The crystal structure of calcium-bound CHP1 from rat has been determined at 2.2 Angstroms of resolution. The molecule has a compact alpha-helical structure containing four EF-hands. The overall folding topology of the protein is similar to that of the regulatory B subunit of calcineurin and to that of calcium- and integrin-binding protein. The calcium ion is coordinated in typical fashion in the third and fourth EF-hands, but the first and second EF-hands contain no calcium ion. The first EF-hand is maintained by internal interactions, and the second EF-hand is stabilized by hydrophobic interactions. CHP1 contains a hydrophobic pocket on the opposite side of the protein to the EF-hands that has been implicated in ligand binding.


Crystallization and preliminary X-ray crystallographic analysis of rat calcineurin B homologous protein 1.

Calcineurin B homologous protein 1 (CHP1), also known as p22, is a calcium-binding protein that plays a role in membrane trafficking and binds to multiple effector proteins, including Na+/H+ exchangers, serine/threonine protein kinase and calcineurin, potentially modulating their function. CHP1 has been crystallized at 277 K using polyethylene glycol as a precipitant. The crystal belongs to space group P2(1), with unit-cell parameters a = 55.5, b = 38.5, c = 90.0 A, beta = 90.7 degrees. A full set of diffraction data was collected to 2.2 A resolution at 100 K using the Photon Factory synchrotron-radiation source.

Acta Crystallographica. Section F, Structural Biology and Crystallization Communications


Structural basis for Ca(2+)-induced activation of human PAD4.

Peptidylarginine deiminase 4 (PAD4) is a Ca(2+)-dependent enzyme that catalyzes the conversion of protein arginine residues to citrulline. Its gene is a susceptibility locus for rheumatoid arthritis. Here we present the crystal structure of Ca(2+)-free wild-type PAD4, which shows that the polypeptide chain adopts an elongated fold in which the N-terminal domain forms two immunoglobulin-like subdomains, and the C-terminal domain forms an alpha/beta propeller structure. Five Ca(2+)-binding sites, none of which adopt an EF-hand motif, were identified in the structure of a Ca(2+)-bound inactive mutant with and without bound substrate. These structural data indicate that Ca(2+) binding induces conformational changes that generate the active site cleft. Our findings identify a novel mechanism for enzyme activation by Ca(2+) ions, and are important for understanding the mechanism of protein citrullination and for developing PAD-inhibiting drugs for the treatment of rheumatoid arthritis.


Crystallization and preliminary X-ray crystallographic analysis of human peptidylarginine deiminase V.

Human peptidylarginine deiminase V (PAD V) is a post-translational enzyme that catalyzes the conversion of arginine residues in protein into citrulline residues in the presence of calcium ion. Crystals of PAD V have been grown at 293 K using polyethylene glycol monomethylether as a precipitant. Crystals diffracted beyond 2.7 A resolution at 100 K at the SPring-8 synchrotron-radiation source. The crystal belongs to space group C2, with unit-cell parameters a = 144.6, b = 60.4, c = 113.4 A, beta = 123.6 degrees. The asymmetric unit contains one molecule, with a V(M) of 2.56 A(3) Da(-1) and a solvent content of 56.1%. A full set of X-ray diffraction data was collected to 2.8 A resolution with a completeness of 97.5%. Heavy-atom derivatives have been successfully prepared and structure analysis is in progress.

Acta Crystallographica. Section D, Biological Crystallography