Homo-BacPROTAC-induced degradation of ClpC1 as a strategy against drug-resistant mycobacteria.

Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance.

Clp-targeting BacPROTACs impair mycobacterial proteostasis and survival.

The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics.

Structural basis for regulation of apoptosis and autophagy by the BIRC6/SMAC complex.

Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. The atypical ubiquitin ligase BIRC6 is the only essential IAP, additionally functioning as a suppressor of autophagy. We performed a structure-function analysis of BIRC6 in complex with caspase-9, HTRA2, SMAC, and LC3B, which are critical apoptosis and autophagy proteins. Cryo-electron microscopy structures showed that BIRC6 forms a megadalton crescent shape that arcs around a spacious cavity containing receptor sites for client proteins. Multivalent binding of SMAC obstructs client binding, impeding ubiquitination of both autophagy and apoptotic substrates. On the basis of these data, we discuss how the BIRC6/SMAC complex can act as a stress-induced hub to regulate apoptosis and autophagy drivers.

A molecular network of conserved factors keeps ribosomes dormant in the egg.

Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed1-4. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4-eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.

BacPROTACs mediate targeted protein degradation in bacteria.

Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis-targeting chimeras. Despite their great promise for medical chemistry, so far, it has not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, drug susceptibility and degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, allowing selective targeting of endogenous proteins via fusion to an established degron. In addition to guiding antibiotic discovery, the BacPROTAC technology presents a versatile research tool enabling the inducible degradation of bacterial proteins.

Cryo-EM structure of the plant 26S proteasome.

Targeted proteolysis is a hallmark of life. It is especially important in long-lived cells that can be found in higher eukaryotes, like plants. This task is mainly fulfilled by the ubiquitin-proteasome system. Thus, proteolysis by the 26S proteasome is vital to development, immunity, and cell division. Although the yeast and animal proteasomes are well characterized, there is only limited information on the plant proteasome. We determined the first plant 26S proteasome structure from Spinacia oleracea by single-particle electron cryogenic microscopy at an overall resolution of 3.3 Å. We found an almost identical overall architecture of the spinach proteasome compared with the known structures from mammals and yeast. Nevertheless, we noticed a structural difference in the proteolytic active ß1 subunit. Furthermore, we uncovered an unseen compression state by characterizing the proteasome's conformational landscape. We suspect that this new conformation of the 20S core protease, in correlation with a partial opening of the unoccupied gate, may contribute to peptide release after proteolysis. Our data provide a structural basis for the plant proteasome, which is crucial for further studies.

HUWE1 employs a giant substrate-binding ring to feed and regulate its HECT E3 domain.

HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin-proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure, where the C-terminal HECT domain heads an extended alpha-solenoid body that coils in on itself and houses various protein-protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins.

McsB forms a gated kinase chamber to mark aberrant bacterial proteins for degradation.

In Gram-positive bacteria, the McsB protein arginine kinase is central to protein quality control, labeling aberrant molecules for degradation by the ClpCP protease. Despite its importance for stress response and pathogenicity, it is still elusive how the bacterial degradation labeling is regulated. Here, we delineate the mechanism how McsB targets aberrant proteins during stress conditions. Structural data reveal a self-compartmentalized kinase, in which the active sites are sequestered in a molecular cage. The 'closed' octamer interconverts with other oligomers in a phosphorylation-dependent manner and, unlike these 'open' forms, preferentially labels unfolded proteins. In vivo data show that heat-shock triggers accumulation of higher order oligomers, of which the octameric McsB is essential for surviving stress situations. The interconversion of open and closed oligomers represents a distinct regulatory mechanism of a degradation labeler, allowing the McsB kinase to adapt its potentially dangerous enzyme function to the needs of the bacterial cell.

The oxidoreductase PYROXD1 uses NAD(P)+ as an antioxidant to sustain tRNA ligase activity in pre-tRNA splicing and unfolded protein response.

The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism.

Native Mass Spectrometry Can Effectively Predict PROTAC Efficacy.

Protein degraders, also known as proteolysis targeting chimeras (PROTACs), are bifunctional small molecules that promote cellular degradation of a protein of interest (POI). PROTACs act as molecular mediators, bringing an E3 ligase and a POI into proximity, thus promoting ubiquitination and degradation of the targeted POI. Despite their great promise as next-generation pharmaceutical drugs, the development of new PROTACs is challenged by the complexity of the system, which involves binary and ternary interactions between components. Here, we demonstrate the strength of native mass spectrometry (nMS), a label-free technique, to provide novel insight into PROTAC-mediated protein interactions. We show that nMS can monitor the formation of ternary E3-PROTAC-POI complexes and detect various intermediate species in a single experiment. A unique benefit of the method is its ability to reveal preferentially formed E3-PROTAC-POI combinations in competition experiments with multiple substrate proteins, thereby positioning it as an ideal high-throughput screening strategy during the development of new PROTACs.

Moyamoya disease factor RNF213 is a giant E3 ligase with a dynein-like core and a distinct ubiquitin-transfer mechanism.

RNF213 is the major susceptibility factor for Moyamoya disease, a progressive cerebrovascular disorder that often leads to brain stroke in adults and children. Characterization of disease-associated mutations has been complicated by the enormous size of RNF213. Here, we present the cryo-EM structure of mouse RNF213. The structure reveals the intricate fold of the 584 kDa protein, comprising an N-terminal stalk, a dynein-like core with six ATPase units, and a multidomain E3 module. Collaboration with UbcH7, a cysteine-reactive E2, points to an unexplored ubiquitin-transfer mechanism that proceeds in a RING-independent manner. Moreover, we show that pathologic MMD mutations cluster in the composite E3 domain, likely interfering with substrate ubiquitination. In conclusion, the structure of RNF213 uncovers a distinct type of an E3 enzyme, highlighting the growing mechanistic diversity in ubiquitination cascades. Our results also provide the molecular framework for investigating the emerging role of RNF213 in lipid metabolism, hypoxia, and angiogenesis.

Moyamoya disease is a genetic disorder affecting both adults and children. It is characterized by narrowing of the blood vessels in the brain, which can lead to strokes. Moyamoya patients often have mutations in the gene for a protein called RNF213. This protein is linked to multiple processes in the body, including the development of blood vessels. Despite this, its role in Moyamoya disease is still something of a mystery. RNF213 is known to fall into two protein 'classes'. First, it is an E3 enzyme. This type of protein tags unwanted or defective proteins for disposal by the cell. Second, it is a motor protein. Motor proteins contain tiny molecular 'engines', called ATPases, that normally convert chemical energy to movement. No other human protein combines these two activities, making RNF213 unique. RNF213 is also an extremely large protein, which means it is difficult to manipulate in the laboratory and thus hard to study. Scientists still need more detailed information on RNF213's structure and chemical activity before we can understand what the mutant protein might be doing in Moyamoya disease. Ahel et al. therefore set out to make the RNF213 protein and 'dissect' it in a test tube. Electron microscopy experiments using the mouse-version of RNF213 revealed that it consisted of a single, giant molecule, folded up to form three regions with distinct structures. These were a long 'arm' at one end, a ring-shaped part in the middle, containing the ATPase 'motor', and the E3 enzyme module at the other end. Further chemical analysis showed that RNF213's ATPase and E3 modules worked in unexpected ways. Although the ATPase did resemble another well-known motor protein, in RNF213 it did not generate movement but rather appeared to act like an intricate molecular 'switch'. The E3 module of RNF213 'tagged' other molecules as expected but did not contain an additional structure that all other known E3 enzymes need to work properly. This suggests that RNF213 represents a distinct class of E3 enzymes. Biochemical tests of the mutation most commonly found in Moyamoya patients revealed that it left RNF213's overall structure, ATPase motor and E3 module intact. That is, the disease-causing mutation appeared to hinder interactions with other partner proteins, rather than disrupting RNF213 itself. By providing the first detailed molecular description of the architecture of RNF213, Ahel et al. hope that these findings will help future investigations of both this giant protein's biological role in the cell and its contribution to Moyamoya disease.

Molecular features of the UNC-45 chaperone critical for binding and folding muscle myosin.

Myosin is a motor protein that is essential for a variety of processes ranging from intracellular transport to muscle contraction. Folding and assembly of myosin relies on a specific chaperone, UNC-45. To address its substrate-targeting mechanism, we reconstitute the interplay between Caenorhabditis elegans UNC-45 and muscle myosin MHC-B in insect cells. In addition to providing a cellular chaperone assay, the established system enabled us to produce large amounts of functional muscle myosin, as evidenced by a biochemical and structural characterization, and to directly monitor substrate binding to UNC-45. Data from in vitro and cellular chaperone assays, together with crystal structures of binding-deficient UNC-45 mutants, highlight the importance of utilizing a flexible myosin-binding domain. This so-called UCS domain can adopt discrete conformations to efficiently bind and fold substrate. Moreover, our data uncover the molecular basis of temperature-sensitive UNC-45 mutations underlying one of the most prominent motility defects in C. elegans.

Structure of McsB, a protein kinase for regulated arginine phosphorylation.

Protein phosphorylation regulates key processes in all organisms. In Gram-positive bacteria, protein arginine phosphorylation plays a central role in protein quality control by regulating transcription factors and marking aberrant proteins for degradation. Here, we report structural, biochemical, and in vivo data of the responsible kinase, McsB, the founding member of an arginine-specific class of protein kinases. McsB differs in structure and mechanism from protein kinases that act on serine, threonine, and tyrosine residues and instead has a catalytic domain related to that of phosphagen kinases (PhKs), metabolic enzymes that phosphorylate small guanidino compounds. In McsB, the PhK-like phosphotransferase domain is structurally adapted to target protein substrates and is accompanied by a novel phosphoarginine (pArg)-binding domain that allosterically controls protein kinase activity. The identification of distinct pArg reader domains in this study points to a remarkably complex signaling system, thus challenging simplistic views of bacterial protein phosphorylation.

Capsid protein structure, self-assembly, and processing reveal morphogenesis of the marine virophage mavirus.

Virophages have the unique property of parasitizing giant viruses within unicellular hosts. Little is understood about how they form infectious virions in this tripartite interplay. We provide mechanistic insights into assembly and maturation of mavirus, a marine virophage, by combining structural and stability studies on capsomers, virus-like particles (VLPs), and native virions. We found that the mavirus protease processes the double jelly-roll (DJR) major capsid protein (MCP) at multiple C-terminal sites and that these sites are conserved among virophages. Mavirus MCP assembled in Escherichia coli in the absence and presence of penton protein, forming VLPs with defined size and shape. While quantifying VLPs in E. coli lysates, we found that full-length rather than processed MCP is the competent state for capsid assembly. Full-length MCP was thermally more labile than truncated MCP, and crystal structures of both states indicate that full-length MCP has an expanded DJR core. Thus, we propose that the MCP C-terminal domain serves as a scaffolding domain by adding strain on MCP to confer assembly competence. Mavirus protease processed MCP more efficiently after capsid assembly, which provides a regulation mechanism for timing capsid maturation. By analogy to Sputnik and adenovirus, we propose that MCP processing renders mavirus particles infection competent by loosening interactions between genome and capsid shell and destabilizing pentons for genome release into host cells. The high structural similarity of mavirus and Sputnik capsid proteins together with conservation of protease and MCP processing suggest that assembly and maturation mechanisms described here are universal for virophages.

RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination.

Class switch recombination (CSR) at the immunoglobulin heavy-chain (IgH) locus is associated with the formation of R-loop structures over switch (S) regions. While these often occur co-transcriptionally between nascent RNA and template DNA, we now show that they also form as part of a post-transcriptional mechanism targeting AID to IgH S-regions. This depends on the RNA helicase DDX1 that is also required for CSR in vivo. DDX1 binds to G-quadruplex (G4) structures present in intronic switch transcripts and converts them into S-region R-loops. This in turn targets the cytidine deaminase enzyme AID to S-regions so promoting CSR. Notably R-loop levels over S-regions are diminished by chemical stabilization of G4 RNA or by the expression of a DDX1 ATPase-deficient mutant that acts as a dominant-negative protein to reduce CSR efficiency. In effect, we provide evidence for how S-region transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH locus.

The ng_ζ1 toxin of the gonococcal epsilon/zeta toxin/antitoxin system drains precursors for cell wall synthesis.

Bacterial toxin-antitoxin complexes are emerging as key players modulating bacterial physiology as activation of toxins induces stasis or programmed cell death by interference with vital cellular processes. Zeta toxins, which are prevalent in many bacterial genomes, were shown to interfere with cell wall formation by perturbing peptidoglycan synthesis in Gram-positive bacteria. Here, we characterize the epsilon/zeta toxin-antitoxin (TA) homologue from the Gram-negative pathogen Neisseria gonorrhoeae termed ng_ɛ1 / ng_ζ1. Contrary to previously studied streptococcal epsilon/zeta TA systems, ng_ɛ1 has an epsilon-unrelated fold and ng_ζ1 displays broader substrate specificity and phosphorylates multiple UDP-activated sugars that are precursors of peptidoglycan and lipopolysaccharide synthesis. Moreover, the phosphorylation site is different from the streptococcal zeta toxins, resulting in a different interference with cell wall synthesis. This difference most likely reflects adaptation to the individual cell wall composition of Gram-negative and Gram-positive organisms but also the distinct involvement of cell wall components in virulence.

Photoactivation Mechanism of a Bacterial Light-Regulated Adenylyl Cyclase.

Light-regulated enzymes enable organisms to quickly respond to changing light conditions. We characterize a photoactivatable adenylyl cyclase (AC) from Beggiatoa sp. (bPAC) that translates a blue light signal into the production of the second messenger cyclic AMP. bPAC contains a BLUF photoreceptor domain that senses blue light using a flavin chromophore, linked to an AC domain. We present a dark state crystal structure of bPAC that closely resembles the recently published structure of the homologous OaPAC from Oscillatoria acuminata. To elucidate the structural mechanism of light-dependent AC activation by the BLUF domain, we determined the crystal structures of illuminated bPAC and of a pseudo-lit state variant. We use hydrogen-deuterium exchange measurements of secondary structure dynamics and hypothesis-driven point mutations to trace the activation pathway from the chromophore in the BLUF domain to the active site of the cyclase. The structural changes are relayed from the residues interacting with the excited chromophore through a conserved kink of the BLUF ß-sheet to a tongue-like extrusion of the AC domain that regulates active site opening and repositions catalytic residues. Our findings not only show the specific molecular pathway of photoactivation in BLUF-regulated ACs but also have implications for the general understanding of signaling in BLUF domains and of the activation of ACs.

3'-NADP and 3'-NAADP, Two Metabolites Formed by the Bacterial Type III Effector AvrRxo1.

An arsenal of effector proteins is injected by bacterial pathogens into the host cell or its vicinity to increase virulence. The commonly used top-down approaches inferring the toxic mechanism of individual effector proteins from the host's phenotype are often impeded by multiple targets of different effectors as well as by their pleiotropic effects. Here we describe our bottom-up approach, showing that the bacterial type III effector AvrRxo1 of plant pathogens is an authentic phosphotransferase that produces two novel metabolites by phosphorylating nicotinamide/nicotinic acid adenine dinucleotide at the adenosine 3'-hydroxyl group. Both products of AvrRxo1, 3'-NADP and 3'-nicotinic acid adenine dinucleotide phosphate (3'-NAADP), are substantially different from the ubiquitous co-enzyme 2'-NADP and the calcium mobilizer 2'-NAADP. Interestingly, 3'-NADP and 3'-NAADP have previously been used as inhibitors or signaling molecules but were regarded as "artificial" compounds so far. Our findings now necessitate a shift in thinking about the biological importance of 3'-phosphorylated NAD derivatives.

Protein structure determination by single-wavelength anomalous diffraction phasing of X-ray free-electron laser data.

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems that are prone to radiation damage. However, phasing XFEL data de novo is complicated by the inherent inaccuracy of SFX data, and only a few successful examples, mostly based on exceedingly strong anomalous or isomorphous difference signals, have been reported. Here, it is shown that SFX data from thaumatin microcrystals can be successfully phased using only the weak anomalous scattering from the endogenous S atoms. Moreover, a step-by-step investigation is presented of the particular problems of SAD phasing of SFX data, analysing data from a derivative with a strong anomalous signal as well as the weak signal from endogenous S atoms.

Type II toxin: antitoxin systems. More than small selfish entities?

Toxin-antitoxin (TA) modules regulate metabolism and viability of bacteria and archaea. In type II TA systems these functions are generally thought to be performed by two small proteins. However, evidence is increasing that the toxins are much more diverse and can form multi-domain proteins. Recently, we published a novel type II TA system in which toxin and antitoxin are covalently linked into a single polypeptide chain. In this review we summarize the current knowledge on these elongated toxin homologs and provide perspectives for future study.