Mind the metal:a fragment library-derived zinc impurity binds the E2 ubiquitin-conjugating enzyme Ube2T and induces structural rearrangements

Efforts to develop inhibitors, activators, and effectors of biological reactions using small molecule libraries are often hampered by interference compounds, artifacts, and false positives that permeate the pool of initial hits. Here, we report the discovery of a promising initial hit compound targeting the Fanconi anemia ubiquitin-conjugating enzyme Ube2T and describe its biophysical and biochemical characterization. Analysis of the co-crystal structure led to the identification of a contaminating zinc ion as solely responsible for the observed effects. Zinc binding to the active site cysteine induces a domain swap in Ube2T that leads to cyclic trimerization organized in an open-ended linear assembly. Our study serves as a cautionary tale for screening small molecule libraries and provides insights into the structural plasticity of ubiquitin-conjugating enzymes

Allosteric targeting of the Fanconi anemia ubiquitin-conjugating enzyme Ube2T by fragment screening

Ube2T is the E2 ubiquitin-conjugating enzyme of the Fanconi anemia DNA repair pathway and it is overexpressed in several cancers, representing an attractive target for the development of inhibitors. Despite the extensive efforts in targeting the ubiquitin system, very few E2 binders have currently been discovered. Herein we report the identification of a new allosteric pocket on Ube2T through a fragment screening using biophysical methods. Several fragments binding to this site inhibit ubiquitin conjugation in vitro

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosi

Structural characterization of Mycobacterium tuberculosis RNA polymerase binding protein A (RbpA) and its interactions with sigma factors

The RNA polymerase binding protein A (RbpA) is a 13 kDa protein, encoded by the gene Rv2050, that was shown to be essential for the growth and survival of the important human pathogen Mycobacterium tuberculosis. Although is not clear yet why RbpA is essential in M. tuberculosis, significant progress has been made in the characterization of the protein. For instance, it was shown that RbpA binds to the β-subunit of the RNA polymerase (RNAP) and activates transcription. Interestingly, it was reported that RbpA can enhance the transcription activity of the RNAP containing the primary σ-subunit σ[superscript A] but does not have any detectable effect if the RNAP is associated with the alternative σ-subunit σ[superscript F]. Moreover, it was also shown that RbpA might influence the response of M. tuberculosis to the current frontline anti-tuberculosis drug rifampicin. The research project described in this thesis contributes to the ongoing efforts to characterize RbpA by providing the structure of the protein and identifying the principle σ-subunit σ[superscript A], and the principle-like σ-subunit σ[superscript B], as interaction partners. The solution structure of RbpA reveals the presence of a central structured region and highly dynamic N- and C- termini. Both termini are involved in the formation of a tight complex with the σ-subunit but only the C-terminal region appears to be essential for this interaction. The finding that RbpA also binds to the RNAP σ-subunit suggests new possibilities for the mechanism of action used by RbpA to activate transcription. Furthermore, preliminary data obtained using a ΔRv2050 conditional mutant strain of M. tuberculosis suggest that the interaction with the σ-subunit is essential for the functionality of RbpA

Structural characterization of Mycobacterium tuberculosis RNA polymerase binding protein A (RbpA) and its interactions with sigma factors

The RNA polymerase binding protein A (RbpA) is a 13 kDa protein, encoded by the gene Rv2050, that was shown to be essential for the growth and survival of the important human pathogen Mycobacterium tuberculosis. Although is not clear yet why RbpA is essential in M. tuberculosis, significant progress has been made in the characterization of the protein. For instance, it was shown that RbpA binds to the β-subunit of the RNA polymerase (RNAP) and activates transcription. Interestingly, it was reported that RbpA can enhance the transcription activity of the RNAP containing the primary σ-subunit σ[superscript A] but does not have any detectable effect if the RNAP is associated with the alternative σ-subunit σ[superscript F]. Moreover, it was also shown that RbpA might influence the response of M. tuberculosis to the current frontline anti-tuberculosis drug rifampicin. The research project described in this thesis contributes to the ongoing efforts to characterize RbpA by providing the structure of the protein and identifying the principle σ-subunit σ[superscript A], and the principle-like σ-subunit σ[superscript B], as interaction partners. The solution structure of RbpA reveals the presence of a central structured region and highly dynamic N- and C- termini. Both termini are involved in the formation of a tight complex with the σ-subunit but only the C-terminal region appears to be essential for this interaction. The finding that RbpA also binds to the RNAP σ-subunit suggests new possibilities for the mechanism of action used by RbpA to activate transcription. Furthermore, preliminary data obtained using a ΔRv2050 conditional mutant strain of M. tuberculosis suggest that the interaction with the σ-subunit is essential for the functionality of RbpA.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Dimethylcarbonate-Assisted Ring-Opening of Biobased Valerolactones with Methanol

The methanolysis reaction of renewable gamma-valerolactone and alpha-methyl-gamma-valerolactone in the presence of dimethylcarbonate and under acid conditions can be tuned to yield selectively each of three acyclic biobased products: 4-hydroxy-methylpentanoate I, 4-methoxy-methylpentanoate 2, and methyl-pent-3-enoate 3. The reaction was studied in batch and in continuous flow, and a reaction mechanism based on experimental and computational evidence was proposed. The protocol is based on a set of greener chemical technologies and was implemented in continuous flow

Targeting Ligandable Pockets on Plant Homeodomain (PHD) Zinc Finger Domains by a Fragment-Based Approach

Plant homeodomain (PHD) zinc fingers are histone reader domains that are often associated with human diseases. Despite this, they constitute a poorly targeted class of readers, suggesting low ligandability. Here, we describe a successful fragment-based campaign targeting PHD fingers from the proteins BAZ2A and BAZ2B as model systems. We validated a pool of <i>in silico</i> fragments both biophysically and structurally and solved the first crystal structures of PHD zinc fingers in complex with fragments bound to an anchoring pocket at the histone binding site. The best-validated hits were found to displace a histone H3 tail peptide in competition assays. This work identifies new chemical scaffolds that provide suitable starting points for future ligand optimization using structure-guided approaches. The demonstrated ligandability of the PHD reader domains could pave the way for the development of chemical probes to drug this family of epigenetic readers

Impaired glycosylation blocks DPP10 cell surface expression and alters the electrophysiology of i to channel complex

DPP10 is a transmembrane glycosylated protein belonging to the family of dipeptidyl aminopeptidase-like proteins (DPPLs). DPPLs are auxiliary subunits involved in the regulation of voltage-gated Kv4 channels, key determinants of cardiac and neuronal excitability. Although it is known that DPPLs are needed to generate native-like currents in heterologous expression systems, the molecular basis of this involvement are still poorly defined. In this study, we investigated the functional relevance of DPP10 glycosylation in modulating Kv4.3 channel activities. Using transfected Chinese hamster ovary (CHO) cells to reconstitute Kv4 complex, we show that the pharmacological inhibition of DPP10 glycosylation by tunicamycin and neuraminidase affects transient outward potassium current (I (to)) kinetics. Tunicamycin completely blocked DPP10 glycosylation and reduced DPP10 cell surface expression. The accelerating effects of DPP10 on Kv4.3 current kinetics, i.e. on inactivation and recovery from inactivation, were abolished. Neuraminidase produced different effects on current kinetics than tunicamycin, i.e., shifted the voltage dependence to more negative potentials. The effects of tunicamycin on the native I (to) currents of human atrial myocytes expressing DPP10 were similar to those of the KV4.3/KChIP2/DPP10 complex in CHO cells. Our results suggest that N-linked glycosylation of DPP10 plays an important role in modulating Kv4 channel activities