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bioRxiv2018

Structural and biochemical characterization on the cognate and heterologous interactions of the MazEF-mt9 TA system

The toxin-antitoxin (TA) modules widely exist in bacteria, and their activities are associated with the persister phenotype of the pathogen Mycobacterium tuberculosis (M. tb). M. tb causes Tuberculosis, a contagious and severe airborne disease. There are ten MazEF TA systems in M. tb, which play important roles in stress adaptation. How the antitoxins antagonize toxins in M. tb or how the ten TA systems crosstalk to each other are of interests, but the detailed molecular mechanisms are largely unclear. MazEF-mt9 is a unique member among the MazEF families due to its tRNase activity, which is usually carried out by the VapC family toxins. Here we present the cocrystal structure of the MazEF-mt9 complex at 2.7 [A]. By characterizing the association mode between the TA pairs through various characterization techniques, we found that MazF-mt9 not only bound its cognate antitoxin, but also the non-cognate antitoxin MazE-mt1, a phenomenon that could be also observed in vivo. Based on our structural and biochemical work, we proposed that the cognate and heterologous interactions among different TA systems work together to relieve MazF-mt9s toxicity to M. tb cells, which may facilitate their adaptation to the stressful conditions encountered during host infection.\n\nIMPORTANCETuberculosis (TB) is one of the most severe contagious diseases. Caused by Mycobacterium tuberculosis (M. tb), it poses a serious threat to human health. Additionally, TB is difficult to cure because of the multipledrug-resistant (MDR) and extensively drug-resistant (XDR) M. tb strains. Toxin-antitoxin (TA) systems have been discovered to widely exist in prokaryotic organisms with diverse roles, normally composed of a pair of molecules that antagonize each other. M. tb has ten MazEF systems, and some of them have been proved to be directly associated with the genesis of persisters and drug-resistance of M. tb. We here report the MazEF-mt9 complex structure, and thoroughly characterized the interactions between MazF-mt9 with MazEs within or outside the MazEF-mt9 family. Our study not only revealed the crosstalks between TA families and its significance to M. tb survival but also offers insights into potential anti-TB drug design.

biochemistry↗

bioRxiv2018

RNA polymerase I transcription fidelity, speed and processivity depend on the interplay of its lobe binding subunits

Eukaryotic RNA polymerases I and III (Pol I and III) consist of core subunits, which are conserved in RNA polymerase II (Pol II). Additionally, Pol I and III have specific subunits, associating with the so-called lobe structure first described within Pol II. In Pol I of the yeast S. cerevisiae, these are Rpa34.5, and the N-terminal domains of Rpa49 and Rpa12.2, here referred to as the lobe-binding module (lb-module). We analyzed functions of the lb-module in a defined in vitro transcription system. Cooperation between lb-module components influenced transcription fidelity, elongation speed, and release of stalled Pol I complexes to continue elongation. Interestingly, lb-module containing Pol I and III, but not Pol II, were able to transcribe nucleosomal templates. Our data suggest, how the Pol I specific subunits may contribute to accurate and processive transcription of ribosomal RNA genes.

biochemistry↗

bioRxiv2018

CSNAP, the smallest CSN subunit, modulates proteostasis through cullin-RING ubiquitin ligases

The cullin-RING ubiquitin E3 ligase (CRL) family consists of ~250 complexes that catalyze ubiquitylation of proteins to achieve cellular regulation. All CRLs are inhibited by the COP9 signalosome complex (CSN) through both enzymatic (deneddylation) and non-enzymatic (steric) mechanisms. The relative contribution of these two mechanisms is unclear. Here, we decouple the mechanisms using CSNAP, the recently discovered ninth subunit of the CSN. We find that CSNAP reduces the affinity of CSN toward CRL complexes. Removing CSNAP does not affect deneddylation, but leads to global effects on the CRL, causing altered reproductive capacity, suppressed DNA damage response, decreased viability, and delayed cell cycle progression. Thus, although CSNAP is only 2% of the CSN mass, it plays a critical role in the steric regulation of CRLs by the CSN.

biochemistry↗

bioRxiv2018

Probing the flavin transfer mechanism in alkanesulfonate monooxygenase system

Bacteria acquire sulfur through the sulfur assimilation pathway, but under sulfur limiting conditions bacteria must acquire sulfur from alternative sources. The alkanesulfonate monooxygenase enzymes are expressed under sulfur-limiting conditions, and catalyze the desulfonation of wide-range of alkanesulfonate substrates. The SsuE enzyme is an NADPH-dependent FMN reductase that provides reduced flavin to the SsuD monooxygenase. The mechanism for the transfer of reduced flavin in flavin dependent two-component systems occurs either by free-diffusion or channeling. Previous studies have shown the presence of protein-protein interactions between SsuE and SsuD, but the identification of putative interaction sights have not been investigated. Current studies utilized HDX-MS to identify protective sites on SsuE and SsuD. A conserved -helix on SsuD showed a decrease in percent deuteration when SsuE was included in the reaction. This suggests the role of -helix in promoting protein-protein interactions. Specific SsuD variants were generated in order to investigate the role of these residues in protein-protein interactions and catalysis. Variant containing substitutions at the charged residues showed a six-fold decrease in the activity, while a deletion variant of SsuD lacking the -helix showed no activity when compared to wild-type SsuD. In addition, there was no protein-protein interactions identified between SsuE and his-tagged SsuD variants in pull-down assays, which correlated with an increase in the Kd value. The -helix is located right next to a dynamic loop region, positioned at the entrance of the active site. The putative interaction site and dynamic loop region located so close to the active site of SsuD suggests the importance of this region in the SsuD catalysis. Stopped-flow studies were performed to analyze the lag-phase which signifies the stabilization and transfer of reduced flavin from SsuE to SsuD. The SsuD variants showed a decrease in lag-phase, which could be because of a downturn in flavin transfer. A competitive assay was devised to evaluate the mechanism of flavin transfer in the alkanesulfonate monooxygenase system. A variant of SsuE was generated which interacted with SsuD, but was not able to reduce FMN. Assays that included varying concentrations of Y118A SsuE and wild-type SsuE in the coupled assays showed a decrease in the desulfonation activity of SsuD. The decrease in activity could be by virtue of Y118A SsuE competing with the wild-type SsuE for the putative docking site on SsuD. These studies define the importance of protein-protein interactions for the efficient transfer of reduced flavin from SsuE to SsuD leading to the desulfonation of alkanesulfonates.

biochemistry↗

bioRxiv2018

Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity.

Inositol polyphosphate multikinase (IPMK) is a member of the IPK-superfamily of kinases, catalyzing phosphorylation of several soluble inositols and the signaling phospholipid PI(4,5)P2 (PIP2). IPMK also has critical non-catalytic roles in p53, mTOR/Raptor, TRAF6 and AMPK signaling mediated partly by two disordered domains. Although IPMK non-catalytic functions are well established, it is less clear if the disordered domains are important for IPMK kinase activity or ATP binding. Here, kinetic and structural analyses of an engineered human IPMK lacking all disordered domains ({Delta}IPMK) are presented. Although the KM for PIP2 is identical between {Delta}IPMK and wild type, {Delta}IPMK has a 1.8-fold increase in kcat for PIP2, indicating the native IPMK disordered domains decrease IPMK activity in vitro. The 2.5 [A] crystal structure of {Delta}IPMK is reported, confirming the conserved ATP-grasp fold. A comparison with other IPK-superfamily structures revealed a putative \"ATP-clamp\" in the disordered N-terminus, we predicted would stabilize ATP binding. Consistent with this observation, removal of the ATP clamp sequence increases the KM for ATP 4.9-fold, indicating the N-terminus enhances ATP binding to IPMK. Together, these structural and kinetic studies suggest in addition to mediating protein-protein interactions, the disordered domains of IPMK impart modulatory capacity to IPMK kinase activity through multiple kinetic mechanisms.

biochemistry↗

bioRxiv2018

An anti-Gn glycoprotein antibody from a convalescent patient potently inhibits the infection of severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease localized to China, Japan, and Korea that is characterized by severe hemorrhage and a high fatality rate. Currently, no specific vaccine or treatment has been approved for this disease. To develop a therapeutic agent for SFTS, we isolated antibodies from a phage-displayed antibody library that was constructed from a patient who recovered from SFTS virus (SFTSV) infection. One antibody, designated as Ab10, was reactive to the Gn envelope glycoprotein of SFTSV and protected host cells and A129 mice from infection in both in vitro and in vivo experiments. Notably, Ab10 protected 80% of mice, even when injected 5 days after inoculation with a lethal dose of SFTSV. Using cross-linker assisted mass spectrometry and alanine scanning, we located the non-linear epitope of Ab10 on the Gn glycoprotein domain II and an unstructured stem region, suggesting that Ab10 may inhibit a conformational alteration that is critical for cell membrane fusion between the virus and host cell. Ab10 reacted to recombinant Gn glycoprotein in Gangwon/Korea/2012, HB28, and SD4 strains. Additionally, based on its epitope, we predict that Ab10 binds the Gn glycoprotein in 247 of 272 reported SFTSV isolates previously reported. Together, these data suggest that Ab10 has potential to be developed into a therapeutic agent that could protect against more than 90% of reported SFTSV isolates.\n\nAuthor summarySevere fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease localized to China, Japan, and Korea. This tick-borne virus has infected more than 5,000 humans with a 6.4% to 20.9% fatality rate. Currently, there are no prophylactic or therapeutic measures against this virus. Historically, antibodies from patients who recovered from viral infection have been used to treat new patients. Until now, one recombinant monoclonal antibody was approved for the prophylaxis of respiratory syntial virus infection. We selected 10 antibodies from a patient who recovered from SFTS and found that one antibody potently inhibited SFTS viral infection in both test tube and animal studies. We determined the binding site of this antibody to SFTS virus, which allowed us to predict that this antibody could bind 247 out of 272 SFTS virus isolates reported up to now. We anticipate that this antibody could be developed into a therapeutic measure against SFTS.

biochemistry↗

bioRxiv2018

Snapshots of single particles from single cells using electron microscopy

Cryo-electron microscopy has become an indispensable tool for structural studies of biological macromolecules. There are two predominant methods for studying the architectures of multi-protein complexes: (1) single particle analysis of purified samples and (2) tomography of whole cells or cell sections. The former can produce high-resolution structures but is limited to highly purified samples, while the latter can capture proteins in their native state but is hindered by a low signal-to-noise ratio and results in lower-resolution structures. Here, we present a method combining microfluidic single cell extraction with single particle analysis by electron microscopy to characterize protein complexes from individual C. elegans embryos. Using this approach, we uncover three-dimensional structures of ribosomes directly from single embryo extracts. In addition, we investigate structural dynamics during development by counting the number of ribosomes per polysome in early and late embyros. This approach has significant potential applications for counting protein complexes and studying protein architectures from single cells in developmental, evolutionary and disease contexts.

biochemistry↗

bioRxiv2018

Telomere DNA G-quadruplex folding within actively extending human telomerase

Telomerase maintains telomere length by reverse transcribing short G-rich DNA repeat sequences from its internal RNA template. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using K+ versus Li+ salts yield changes in DNA product profiles consistent with formation of GQ structure within the telomerase-DNA complex. Single-molecule FRET experiments reveal complex DNA structural dynamics during real-time catalysis, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we fit telomerase time series data to a global kinetic model that converges to a unique set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis and provide a detailed characterization of GQ modulation of polymerase function.\n\nSIGNIFICANCETelomeres protect the ends of linear chromosomes from illicit DNA processing events that can threaten genome stability. Telomere structure is built upon repetitive G-rich DNA repeat sequences that have the ability to fold into stable secondary structures called G-quadruplexes (GQs). In rapidly dividing cells, including the majority of human cancers, telomeres are maintained by the specialized telomerase enzyme. Thus, telomerase and its telomere DNA substrates represent important targets for developing novel cancer drugs. In this work, we provide evidence for GQ folding within the newly synthesized DNA product of an actively extending telomerase enzyme. Our results highlight the delicate interplay between the structural properties of telomere DNA and telomerase function.

biochemistry↗

bioRxiv2018

Dynamic G-quadruplexes on the surface of the human ribosome

Profound similarities and critical differences mark ribosomes across phylogeny. The ribosomal core, approximated by the prokaryotic ribosome, is universal, yet mammalian ribosomes are nearly twice as large as those of prokaryotes. Differences in size are due in part to rRNA expansion segments. Here we show rRNA tentacles of Expansion Segment 7 (ES7) of Homo sapiens can form G-quadruplexes in vitro. G-quadruplex-forming regions are located on the most surface-exposed regions of the ribosome, near the termini of rRNA tentacles. We characterized rRNA of the large ribosomal subunit by computation, circular dichroism, gel mobility, fluorescent probes, nuclease accessibility, electrophoretic mobility shifts and blotting. We investigated ES7 and oligomers derived from ES7, intact 28S rRNA, and 80S ribosomes and polysomes. We used mass spectrometry to identify proteins that bind to rRNA G-quadruplexes in cell lysates. Proteins that associate with rRNA G-quadruplexes include helicases (DDX3, CNBP, DDX21, DDX17) and heterogeneous nuclear ribonucleoproteins (hnRNPs). And finally, by multiple sequence alignments, we observed that G-quadruplex-forming sequences appear to be a general feature LSU rRNA of the phylum Chordata but not in other phyla. It is known that G-quadruplexes form in telomeres, promoters, and untranslated regions of mRNA but, to our knowledge, they have not been reported previously in ribosomes.

biochemistry↗

bioRxiv2018

Dynamics and Selective Remodeling of the DNA Binding Domains of RPA

Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-strand DNA (ssDNA) intermediates, activating the DNA damage response, and handing off ssDNA to appropriate downstream players. Six DNA binding domains (DBDs) in RPA promote high affinity binding to ssDNA, but also allow RPA displacement by lower affinity proteins. We have made fluorescent versions of RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA, while RPA as a whole remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. This demonstrates how RPA interacting proteins, with lower ssDNA binding affinity, can access the occluded ssDNA and remodel individual DBDs to replace RPA.\n\nOne Sentence SummaryThe choreography of binding and rearrangement of the individual domains of RPA during homologous recombination is revealed.

biochemistry↗

bioRxiv2018

Human telomere repeat binding factor TRF1 replaces TRF2 bound to shelterin core hub TIN2 when TPP1 is absent

Human telomeric repeat binding factors TRF1, TRF2 along with TIN2 form a core of shelterin complex that protects chromosome ends against unwanted end-joining and DNA repair. We applied a single-molecule approach to assess TRF1-TIN2-TRF2 complex formation in solution at physiological conditions. Fluorescence Cross-Correlation Spectroscopy (FCCS) was used to describe the complex formation by analyzing how coincident fluctuations of differently labeled TRF1 and TRF2 correlate when they move together through the confocal volume of the microscope. We observed, at the single-molecule level, that TRF1 effectively substituted TRF2 on TIN2. We assessed the effect of another telomeric factor TPP1 that recruits telomerase to telomeres. We found that TPP1 upon binding to TIN2 induces allosteric changes that expand TIN2 binding capacity, such that TIN2 can accommodate both TRF1 and TRF2 simultaneously. We suggest a molecular model that explains why TPP1 is essential for the stable formation of TRF1-TIN2-TRF2 core complex.

biochemistry↗

bioRxiv2018

Molecular basis for high affinity agonist binding in GPCRs

A characteristic of GPCRs in the G protein-coupled state is that the affinity of the agonist often increases significantly, but the molecular basis for this is unclear. We have determined six active-state structures of the {beta}1-adrenoceptor ({beta}1AR) bound to conformation-specific nanobodies in the presence of agonists of varying efficacy. A direct comparison with structures of {beta}1AR in inactive states bound to the identical ligands showed a 24-42% reduction in the volume of the orthosteric binding site. Potential hydrogen bonds were also shorter, and there was up to a 30% increase in the number of atomic contacts between the receptor and ligand. GPCRs are highly conserved, so these factors will likely be essential in increasing the affinity of a wide range of structurally distinct agonists.\n\nOne Sentence SummaryHigh affinity agonist binding to G protein-coupled GPCRs results from an increase in the number and strength of protein-ligand interactions.

biochemistry↗

bioRxiv2018

Targeting translation activity at the ribosome interface with UV-active small molecules

Puromycin is a well-known antibiotic that is used to study the mechanism of protein synthesis and to monitor translation efficiency due to its incorporation into nascent peptide chains. However, puromycin effects outside the ribo-some catalytic core remain unexplored. Here, we developed two puromycin analogues (3PB and 3PC) that can efficiently interact with several proteins involved in translation, ribosome function and RNA processing. We biochemically characterized the binding of these analogues and globally mapped the direct small molecule-protein interactions in living cells using clickable and photoreactive puromycin-like probes in combination with in-depth mass spectrometry. We identified a list of proteins that interact with ribosomes during translation (e.g. eEF1A, ENO1 and GRP78) and we addressed possible uses of the probes to sense the activity of protein synthesis and to capture associated RNA. By coupling genome-wide RNA sequencing methods with these molecules, the characterization of unexplored translational control mechanisms will be feasible.

biochemistry↗

bioRxiv2018

Reporter Ion Data Analysis Reduction (R.I.D.A.R) for isobaric proteomics quantification studies

Isobaric labeling-based relative quantification techniques such as iTRAQ and TMT were introduced 15 years ago and are now nearly ubiquitous in shotgun proteomics labs around the world. The methods for data processing in these experiments has changed little since inception, with peptide database searching of all MS/MS spectra occurring concurrent or asynchronous to the quantification of the reporter fragment regions. In this study we present an alternative method for data processing whereby the reporter ion region of all MS/MS spectra are first examined and spectra that are not quantitatively interesting to the end user are discarded. The remaining MS/MS spectra that are retained can then be more rapidly searched for computationally expensive database alterations such as post-translational modifications and single amino acid variations in more practical time. We have termed this method Reporter Ion Data Analysis Reduction (RIDAR). To demonstrate the application of RIDAR, we reprocess a recent CPTAC 2 study containing approximately 7.8 million MS/MS spectra. Post RIDAR processing we can search this public dataset versus a human canonical FASTA database and a compiled proteogenomic database of over 875,000 known cancer mutations in a single day on a standard desktop computer, a time reduction of 85% compared to the conventional workflow. With the rapidly increasing size and density of shotgun proteomics data files, RIDAR facilitates rapid analysis of large proteomics datasets for researchers without access to high performance computational resources.\n\nAbstract Graphic\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC=\"FIGDIR/small/437210_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (14K):\norg.highwire.dtl.DTLVardef@1bcf00eorg.highwire.dtl.DTLVardef@134510eorg.highwire.dtl.DTLVardef@1b594b4org.highwire.dtl.DTLVardef@35e268_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2018

Structure of transcribing RNA polymerase II-nucleosome complex

Transcription of eukaryotic protein-coding genes requires passage of RNA polymerase II (Pol II) through chromatin. Pol II passage is impaired by nucleosomes and requires elongation factors that help Pol II to efficiently overcome the nucleosomal barrier1-4. How the Pol II machinery transcribes through a nucleosome remains unclear because structural studies have been limited to Pol II elongation complexes formed on DNA templates lacking nucleosomes5. Here we report the cryo-electron microscopy (cryo-EM) structure of transcribing Pol II from the yeast Saccharomyces cerevisiae engaged with a downstream nucleosome core particle (NCP) at an overall resolution of 4.4 [A] with resolutions ranging from 4-6 [A] in Pol II and 6-8 [A] in the NCP. Pol II and the NCP adopt a defined orientation that could not be predicted from modelling. Pol II contacts DNA of the incoming NCP on both sides of the nucleosomal dyad with its domains clamp head and lobe. Comparison of the Pol II-NCP structure to known structures of Pol II complexes reveals that the elongation factors TFIIS, DSIF, NELF, PAF1 complex, and SPT6 can be accommodated on the Pol II surface in the presence of the oriented nucleosome. Further structural comparisons show that the chromatin remodelling enzyme Chd1, which is also required for efficient Pol II passage6,7, could bind the oriented nucleosome with its motor domain. The DNA-binding region of Chd1 must however be released from DNA when Pol II approaches the nucleosome, and based on published data8,9 this is predicted to stimulate Chd1 activity and to facilitate Pol II passage. Our results provide a starting point for a mechanistic analysis of chromatin transcription.

biochemistry↗

bioRxiv2018

The ataxin-1 interactome reveals direct connection with multiple disrupted nuclear transport pathways

The expanded polyglutamine (polyQ) tract form of ataxin-1 drives disease progression in spinocerebellar ataxia type 1 (SCA1). Although polyQ-ataxin-1 is known to form distinctive intranuclear bodies, the cellular pathways and functions it influences remain poorly understood. Here, we identify direct and proximal partners constituting the interactome of ataxin-1[85Q] in Neuro-2a cells. Pathways analyses indicate a significant enrichment of essential nuclear transporters in the interactome, pointing to disruptions in nuclear transport processes in the presence of polyQ-ataxin-1. Our direct assessments of nuclear transporters and their cargoes reinforce these observations, revealing disrupted trafficking often with relocalisation of transporters and/or cargoes to ataxin-1[85Q] nuclear bodies. Strikingly, the nucleoporin Nup98, dependent on its GLFG repeats, is recruited into polyQ-ataxin-1 nuclear bodies. Our results highlight a disruption of multiple essential nuclear protein trafficking pathways by polyQataxin-1, a key contribution to furthering understanding of pathogenic mechanisms initiated by polyQ tract proteins.

biochemistry↗

bioRxiv2018

Association of the types of alcoholic beverages and blood lipids in a local population in Jharkhand, India

Although light-to-moderate alcohol consumption is considered beneficial, alcohol in binge doses or high cumulative lifetime consumption leads to cardiovascular diseases, metabolic syndrome and structural damage to various organs. Alcohol is known to alter blood lipid concentrations; however, the association of the types of alcohol on the lipid profile has not been investigated extensively. A cross-sectional study involving male participants (n = 86) aged 20 to 60 from the Ranchi and Dhanbad zone of Jharkhand, India, was carried out to investigate the effects of cumulative lifetime consumption of Haria, a local rice-based fermented alcohol, Indian made foreign liquor (IMFL), and a combination of the two on the blood lipid profiles. Demographic characteristics, dietary intake and medical history were obtained from the participants by questionnaire, and lipid levels were determined by analysis of blood samples. The effect of Haria alone on the blood lipids was also investigated on the local female population (n = 31). After adjusting for demographic and dietary factors, IMFL and combination of IMFL and Haria consumption was associated with increased serum total cholesterol, triglyceride, and low density lipoprotein (LDL) cholesterol levels (P < 0.05) and decreased high density lipoprotein (HDL) cholesterol levels (P < 0.05). None of the blood lipids changed significantly in Haria consumers in both male and female groups. This study suggests that Haria, a popular alcoholic beverage of West Bengal and east-central India, is a relatively safe local alcoholic beverage and does not alter the lipid profile in consumers.

biochemistry↗

bioRxiv2018

Covalent Ligand Screening Uncovers a RNF4 E3 Ligase Recruiter for Targeted Protein Degradation Applications

Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome- and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.

biochemistry↗