K27-linked diubiquitin inhibits UCHL3 via an unusual kinetic trap

Functional analysis of lysine 27-linked ubiquitin chains ((K27)Ub) is difficult due to the inability to make them through enzymatic methods and due to a lack of model tools and substrates. Here we generate a series of ubiquitin (Ub) tools to study how the deubiquitinase UCHL3 responds to (K27)Ub chains in comparison to lysine 63-linked chains and mono-Ub. From a crystal structure of a complex between UCHL3 and synthetic (K27)Ub(2), we unexpectedly discover that free (K27)Ub(2) and (K27)Ub(2)-conjugated substrates are natural inhibitors of UCHL3. Using our Ub tools to profile UCHL3's activity, we generate a quantitative kinetic model of the inhibitory mechanism and we find that (K27)Ub(2) can inhibit UCHL3 covalently, by binding to its catalytic cysteine, and allosterically, by locking its catalytic loop tightly in place. Based on this inhibition mechanism, we propose that UCHL3 and (K27)Ub chains likely sense and regulate each other in cells.Chemical Immunolog

The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here, we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone

"4D Biology for health and disease" workshop report

The "4D Biology Workshop for Health and Disease", held on 16-17th ofMarch 2010 in Brussels, aimed at finding the best organising principlesfor large-scale proteomics, interactomics and structural genomics/biology initiatives, and setting the vision for future high-throughputresearch and large-scale data gathering in biological and medical science.Major conclusions of the workshop include the following. (i)Development of new technologies and approaches to data analysis iscrucial. Biophysical methods should be developed that span a broadrange of time/spatial resolution and characterise structures andkinetics of interactions. Mathematics, physics, computational andengineering tools need to be used more in biology and new tools needto be developed. (ii) Database efforts need to focus on improveddefinitions of ontologies and standards so that system-scale data andassociated metadata can be understood and shared efficiently. (iii)Research infrastructures should play a key role in fosteringmultidisciplinary research, maximising knowledge exchange betweendisciplines and facilitating access to diverse technologies. (iv)Understanding disease on a molecular level is crucial. Systemapproaches may represent a new paradigm in the search for biomarkersand new targets in human disease. (v) Appropriate education andtraining should be provided to help efficient exchange of knowledgebetween theoreticians, experimental biologists and clinicians. Theseconclusions provide a strong basis for creating major possibilities inadvancing research and clinical applications towards personalisedmedicine.Biophysical Structural Chemistr

Structuur een functie geven

Rede in verkorte vorm, uigesproken ter gelegenheid van het aanvaarden van het ambt van bijzonder hoogleraar met als leeropdracht 'Structuur en Functie van Eiwitten' aan het Erasmus MC, faculteit van de Erasmus Universiteit Rotterdam op 20 januari 200

Acetylcholine binding protein (AChBP): A secreted glial protein that provides a high-resolution model for the extracellular domain of pentameric ligand-gated ion channels

Acetylcholine binding protein (AChBP) has recently been identified from molluskan glial cells. Glial cells secrete it into cholinergic synapses, where it plays a role in modulating synaptic transmission. This novel mechanism resembles glia-dependent modulation of glutamate synapses, with several key differences. AChBP is a homolog of the ligand binding domain of the pentameric ligand-gated ion-channels. The crystal structure of AChBP provides the first high-resolution structure for this family of Cys-loop receptors. Nicotinic acetylcholine receptors and related ion-channels such as GAB

Insight in nAChR subtype selectivity from AChBP crystal structures

International audienceNicotinic acetylcholine receptors (nAChRs) display a broad variety of subtypes, which in turn present a complex subcellular and regional expression pattern in the brain, as well as a specific pharmacological profile. The association of these nAChRs with different types of brain disease has turned them into interesting drug targets for the treatment of Alzheimer's disease or schizophrenia, or for anti-smoking compounds among others. In the same way, muscle-type nAChRs present at neuromuscular junctions are also being targeted by muscle relaxants. However, to date no high-resolution structural data is available on functional pentameric forms of membrane bound nicotinic receptors. Therefore, characterization of the selectivity profiles of different nicotinic receptor subtypes, enabling efficient drug design, is a serious issue. Over the last eight years various high resolution structures of acetylcholine binding protein (AChBP), which is homologous to the extracellular ligand binding domain of the nicotinic acetylcholine receptor, have been obtained. AChBPs in complex with different ligands have provided detailed insight into the neurotransmitter binding site of nicotinic acetylcholine receptors. We present here the various efforts towards rationalizing subtype specificity in these receptors through the structural studies of acetylcholine binding protein-ligand complexes

E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6

In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for “error prone” DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation. This activity is prevented during PCNA ubiquitination by the interaction of Rad6 with E3 enzyme Rad18. Using NMR and X-ray crystallography we show that the R6BD of Rad18 inhibits this activity by competing with ubiquitin for a noncovalent “backside” binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process