Epigenetic inheritance in mammals is characterized by high-fidelity replication of CpG methylation patterns during development. UHRF1 (also known as ICBP90 in humans and Np95 in mouse) is an E3 ligase important for the maintenance of global and local DNA methylation in vivo. The preferential affinity of UHRF1 for hemi-methylated DNA over symmetrically methylated DNA by means of its SET and RING-associated (SRA) domain and its association with the maintenance DNA methyltransferase 1 (DNMT1) suggests a role in replication of the epigenetic code. Here we report the 1.7 A crystal structure of the apo SRA domain of human UHRF1 and a 2.2 A structure of its complex with hemi-methylated DNA, revealing a previously unknown reading mechanism for methylated CpG sites (mCpG). The SRA-DNA complex has several notable structural features including a binding pocket that accommodates the 5-methylcytosine that is flipped out of the duplex DNA. Two specialized loops reach through the resulting gap in the DNA from both the major and the minor grooves to read the other three bases of the CpG duplex. The major groove loop confers both specificity for the CpG dinucleotide and discrimination against methylation of deoxycytidine of the complementary strand. The structure, along with mutagenesis data, suggests how UHRF1 acts as a key factor for DNMT1 maintenance methylation through recognition of a fundamental unit of epigenetic inheritance, mCpG.

The ubiquitin system regulates virtually all aspects of cellular function. We report a method to target the myriad enzymes that govern ubiquitination of protein substrates. We used massively diverse combinatorial libraries of ubiquitin variants to develop inhibitors of four deubiquitinases (DUBs) and analyzed the DUB-inhibitor complexes with crystallography. We extended the selection strategy to the ubiquitin conjugating (E2) and ubiquitin ligase (E3) enzymes and found that ubiquitin variants can also enhance enzyme activity. Last, we showed that ubiquitin variants can bind selectively to ubiquitin-binding domains. Ubiquitin variants exhibit selective function in cells and thus enable orthogonal modulation of specific enzymatic steps in the ubiquitin system.

Histone modifications and DNA methylation represent two layers of heritable epigenetic information that regulate eukaryotic chromatin structure and gene activity. UHRF1 is a unique factor that bridges these two layers; it is required for maintenance DNA methylation at hemimethylated CpG sites, which are specifically recognized through its SRA domain and also interacts with histone H3 trimethylated on lysine 9 (H3K9me3) in an unspecified manner. Here we show that UHRF1 contains a tandem Tudor domain (TTD) that recognizes H3 tail peptides with the heterochromatin-associated modification state of trimethylated lysine 9 and unmodified lysine 4 (H3K4me0/K9me3). Solution NMR and crystallographic data reveal the TTD simultaneously recognizes H3K9me3 through a conserved aromatic cage in the first Tudor subdomain and unmodified H3K4 within a groove between the tandem subdomains. The subdomains undergo a conformational adjustment upon peptide binding, distinct from previously reported mechanisms for dual histone mark recognition. Mutant UHRF1 protein deficient for H3K4me0/K9me3 binding shows altered localization to heterochromatic chromocenters and fails to reduce expression of a target gene, p16INK4A , when overexpressed. Our results demonstrate a novel recognition mechanism for the combinatorial readout of histone modification states associated with gene silencing and add to the growing evidence for coordination of, and cross-talk between, the modification states of H3K4 and H3K9 in regulation of gene expression.Histone modifications and DNA methylation represent two layers of heritable epigenetic information that regulate chromatin structure and gene activity in eukaryotic organisms. Methylated DNA sequences are generally associated with long term transcriptional silencing through the recruitment of repressor complexes, including methyl-binding proteins, histone deacetylases, and chromatin remodeling machinery (1, 2). Likewise, specific histone methylation states can recruit multivalent adaptor proteins, which lead to chromatin condensation, further inhibiting gene expression. Accumulating evidence shows that these two methylation systems act cooperatively to establish the epigenetic state of the cell (3-5); however, the mechanisms of this cooperation remain vague.During replication, CpG methylation patterns are maintained in mammals by the DNA methyltransferase 1 with hemimethylated CpG dinucleotides serving as a substrate. This enzyme is aided by UHRF1 (ubiquitin-like, PHD and RING finger containing 1, also known as ICBP90 in humans and NP95 in mouse), which interacts with DNA methyltransferase 1 and specifically recognizes hemimethylated CpG dinucleotides through its SET-and RING-associated domain (SRA) 4 (6, 7). 4 The abbreviations used are: SRA, SET-and RING-associated domain; UHRF1, ubiquitin-like, PHD and RING finger containing 1; mUHRF1, murine UHRF1; TTD, tandem tudor domain; TTD N , N-terminal tudor subdomain; TTD C , C-terminal tudor subdomain; NMR, nuclear magnetic resonance; RDC, residual dipolar coupli...

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

Ubiquitin-specific protease 8 (USP8) hydrolyzes mono and polyubiquitylated targets such as epidermal growth factor receptors and is involved in clathrin-mediated internalization. In 1182 residues, USP8 contains multiple domains, including coiled-coil, rhodanese, and catalytic domains. We report the first high-resolution crystal structures of these domains and discuss their implications for USP8 function. The amino-terminal domain is a homodimer with a novel fold. It is composed of two five-helix bundles, where the first helices are swapped, and carboxyl-terminal helices are extended in an antiparallel fashion. The structure of the rhodanese domain, determined in complex with the E3 ligase NRDP1, reveals the canonical rhodanese fold but with a distorted primordial active site. The USP8 recognition domain of NRDP1 has a novel protein fold that interacts with a conserved peptide loop of the rhodanese domain. A consensus sequence of this loop is found in other NRDP1 targets, suggesting a common mode of interaction. The structure of the carboxyl-terminal catalytic domain of USP8 exhibits the conserved tripartite architecture but shows unique traits. Notably, the active site, including the ubiquitin binding pocket, is in a closed conformation, incompatible with substrate binding. The presence of a zinc ribbon subdomain near the ubiquitin binding site further suggests a polyubiquitin-specific binding site and a mechanism for substrate induced conformational changes.The post-translational ubiquitylation system is composed of a cascade of E1, 2 E2, and E3 enzymes that activate and transfer ubiquitin or ubiquitin homologs to target proteins, including those targets that are membrane-anchored (1, 2). Modification by ubiquitin or ubiquitin-like molecules typically affects localization of the protein within the cell. For example, Lys-48-polyubiquitylated targets are recognized by the proteasome, which catalyzes complete target proteolysis (3). By affecting the abundance and/or half-life of signaling molecules, the ubiquitylation system can have dramatic effects on signal transduction pathways and play major roles in cellular biology. Degradation of integral membrane proteins can also be initiated by the ubiquitylation system but subsequent steps differ (4). Ubiquitylated receptors for example are rounded up by multivalent adaptors (e.g. AP2, epsin), which in turn recruit clathrin molecules that induce internalization through membrane budding. Dissociation of clathrin coats releases endosomes, which are either recycled back to the plasma membrane or fused with lysosomes, where membrane proteins undergo proteolytic degradation. Budding, uncoating, trafficking, and fusion events are influenced by the ubiquitylation state of the various intermediates, which is regulated not just by E3 ligases but by deubiquitylases as well.The human genome contains over 90 deubiquitylases, the specific biological roles of which are mostly unknown. Some deubiquitylases have been implicated in ubiquitin recycling, which typically can occur at the ...