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.epigenetics | multidomain structure | posttranslational modification | X-ray crystallography

The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T 37 /T 46 , followed by T 70 and S 65 . Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is a goal of interest for cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14-to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, 54 YXXXXLΦ 60 (motif 1) but lack known phosphorylation sites. We report here a 2.1-Å crystal structure of mouse eIF4E in complex with m 7 GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP1 50-84 ). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S 65 and T 70 ). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1-mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. The binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.translation initiation | phosphorylation | protein-protein interaction inhibitor | structure

Post-translational modification by small ubiquitin-like modifier (SUMO) proteins has been implicated in the regulation of a variety of cellular events. The functions of sumoylation are often mediated by downstream effector proteins harboring SUMOinteracting motifs (SIMs) that are composed of a hydrophobic core and a stretch of acidic residues. MBD1-containing chromatin-associated factor 1 (MCAF1), a transcription repressor, interacts with SUMO-2/3 and SUMO-1, with a preference for SUMO-2/3. We used NMR spectroscopy to solve the solution structure of the SIM of MCAF1 bound to SUMO-3. The hydrophobic core of the SIM forms a parallel ␤-sheet pairing with strand ␤2 of SUMO-3, whereas its C-terminal acidic stretch seems to mediate electrostatic interactions with a surface area formed by basic residues of SUMO-3. The significance of these electrostatic interactions was shown by mutations of both SUMO-3 and MCAF1. The present structural and biochemical data suggest that the acidic stretch of the SIM of MCAF1 plays an important role in the binding to SUMO-3. Small ubiquitin-like modifier (SUMO)2 proteins conjugate post-translationally with target proteins through a series of enzymatic reactions that resemble ubiquitination (1-5). In contrast to ubiquitination, which is largely involved in regulating the degradation of target proteins by proteasomes or lysosomes (6), sumoylation appears to regulate a wide variety of cellular events, such as nuclear transport, subnuclear localization, transcriptional regulation, DNA repair, and chromosome segregation (3). In particular, sumoylation of transcription factors represses transcription through a variety of different mechanisms, such as recruitment of histone deacetylases and regulation of nuclear body components (7). It has been suggested that sumoylation generally regulates the functions of target proteins by modulating their proteinprotein or protein-DNA interactions.In mammals, four SUMO paralogues, SUMO-1 through SUMO-4, have been identified, of which SUMO-1 to -3 can serve as protein modifiers (8). Whereas SUMO-2 and SUMO-3 share 97% amino acid identity, they have only 48 and 46% identity with SUMO-1, respectively. This indicates that SUMO-2 and SUMO-3 constitute a subgroup that is distinct from SUMO-1. Although these SUMO paralogues largely share common cellular functions, they also show paralogue-specific properties in regard to cellular localization and substrate specificity (9, 10). For example, SUMO-1 is preferentially found within nucleoli, nuclear envelopes, and cytoplasmic foci (8), whereas SUMO-2/3 accrue on chromosomes early in the nuclear reformation process (11). With respect to substrate specificity, RanGAP1 is preferentially modified by SUMO-1, whereas SUMO-2/3 show preference for topoisomerase 2 during mitosis. However, promyelocytic leukemia protein conjugates to all three SUMO paralogues (12). SUMO-1 is most abundant in the protein-conjugated form, whereas SUMO-2/3 isomers are more abundant in a free pool and are available to conjugate with target proteins...

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.