Consecutive Alkylation, "Click", and "Clip" Reactions for the Traceless Methionine-Based Conjugation and Release of Methionine-Containing Peptides.

"Click" reactions have revolutionized research in many areas of science. However, a disadvantage of the high stability of the Click product is that identifying simple treatments for cleanly dissociating the latter under the same guiding principles, i.e., a "Clip" reaction, remains a challenge. This study demonstrates that electron-deficient alkynes, conveniently installed on methionine residues, can participate in well-known Click (nucleophilic thiol-allene addition) and subsequent Clip reactions (radical thiol-ene addition). To illustrate this concept, a variety of bioconjugates (peptide-peptide; peptide-fluorophore; peptide-polymer; and peptide-protein) were prepared. Interestingly, the Clip reaction of these bioconjugates releases the original peptides concurrent with regeneration of their unmodified methionine residue, in minutes. Moreover, the conjugates demonstrate substantial stability toward endogenous levels of reactive species in bacteria, illustrating the potential for this chemistry in the biosciences. The reaction conditions employed in the Click and Clip steps are compatible with the preservation of the integrity of biomolecules/fluorophores and involve readily accessible reagents and the natural functional groups on peptides/proteins.

The CRL4DCAF1 cullin-RING ubiquitin ligase is activated following a switch in oligomerization state.

The cullin-4-based RING-type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the ˜29 CRL4 substrate receptors reported, the DDB1- and CUL4-associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4DCAF1 ligase. In the 8.4 Å cryo-EM map of CRL4DCAF1 , four CUL4-RBX1-DDB1-DCAF1 protomers are organized into two dimeric sub-assemblies. In this arrangement, the WD40 domain of DCAF1 mediates binding with the cullin C-terminal domain (CTD) and the RBX1 subunit of a neighboring CRL4DCAF1 protomer. This renders RBX1, the catalytic subunit of the ligase, inaccessible to the E2 ubiquitin-conjugating enzymes. Upon CRL4DCAF1 activation by neddylation, the interaction between the cullin CTD and the neighboring DCAF1 protomer is broken, and the complex assumes an active dimeric conformation. Accordingly, a tetramerization-deficient CRL4DCAF1 mutant has higher ubiquitin ligase activity compared to the wild-type. This study identifies a novel mechanism by which unneddylated and substrate-free CUL4 ligases can be maintained in an inactive state.

Publisher Correction: DNA damage detection in nucleosomes involves DNA register shifting.

In this Article, in Fig. 1a, the 5' and 3' labels were reversed in the DNA sequence, and Fig. 4 was missing panel labels a-e. These errors have been corrected online.

DNA damage detection in nucleosomes involves DNA register shifting.

Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and DNA repair. In humans, the UV-damaged DNA-binding protein (UV-DDB) complex detects UV-light-induced pyrimidine dimers throughout the genome; however, it remains unknown how these lesions are recognized in chromatin, in which nucleosomes restrict access to DNA. Here we report cryo-electron microscopy structures of UV-DDB bound to nucleosomes bearing a 6-4 pyrimidine-pyrimidone dimer or a DNA-damage mimic in various positions. We find that UV-DDB binds UV-damaged nucleosomes at lesions located in the solvent-facing minor groove without affecting the overall nucleosome architecture. In the case of buried lesions that face the histone core, UV-DDB changes the predominant translational register of the nucleosome and selectively binds the lesion in an accessible, exposed position. Our findings explain how UV-DDB detects occluded lesions in strongly positioned nucleosomes, and identify slide-assisted site exposure as a mechanism by which high-affinity DNA-binding proteins can access otherwise occluded sites in nucleosomal DNA.

Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome.

The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.

Self-assembled metallic nanowires on a dielectric support: Pd on rutile TiO2(110).

Palladium nanoparticles supported on rutile TiO(2)(110)-1 x 1 have been studied using the complementary techniques of scanning tunneling microscopy and X-ray photoemission electron microscopy. Two distinct types of palladium nanoparticles are observed, namely long nanowires up to 1000 nm long, and smaller dotlike features with diameters ranging from 80-160 nm. X-ray photoemission electron microscopy reveals that the nanoparticles are composed of metallic palladium, separated by the bare TiO(2)(110) surface.