The chromatophores of the cercozoan amoeba Paulinella are photosynthetic organelles that evolved from a cyanobacterial endosymbiont. Many nucleus-encoded chromatophore-targeted proteins carry unusual N-terminal targeting signals termed crTPs, which are bipartite. crTPpart1 likely mediates trafficking through the secretory pathway and is cleaved off during import, but crTPpart2 remains attached to its cargo protein and its function is unknown. To unravel the functional role of crTPpart2, here we elucidated the structures of crTPpart2 from two different chromatophore-targeted proteins by X-ray crystallography at ∼2.3 Å resolution. Interestingly, the crTPpart2 of both proteins adopts a structural fold. Both structures share a conserved structured core and a flexible N-terminal arm. The structured core resembles proteins of the γ-glutamyl cyclotransferase superfamily within which crTPpart2 structures form a protein (sub)-family. The proposed catalytic center typical for proteins with cyclotransferase activity is not conserved in crTPpart2. A Cys pair that is conserved in crTPpart2 of many chromatophore-targeted proteins has been captured as a disulfide bridge. Together, our data suggest that chromatophore-targeted proteins are imported in their folded state and that the fold adopted by crTPpart2 plays a functional role during import. The characterization of its structure and flexibility provides important steps toward elucidating this protein translocation mechanism.

Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Å resolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-β-thia and β-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.

Polyethylene terephthalate (PET) is a widely used synthetic polymer and known to contaminate marine and terrestrial ecosystems. Only few PET‐active microorganisms and enzymes (PETases) are currently known, and it is debated whether degradation activity for PET originates from promiscuous enzymes with broad substrate spectra that primarily act on natural polymers or other bulky substrates, or whether microorganisms evolved their genetic makeup to accepting PET as a carbon source. Here, we present a predicted diene lactone hydrolase designated PET40, which acts on a broad spectrum of substrates, including PET. It is the first esterase with activity on PET from a GC‐rich Gram‐positive Amycolatopsis species belonging to the Pseudonocardiaceae (Actinobacteria). It is highly conserved within the genera Amycolatopsis and Streptomyces. PET40 was identified by sequence‐based metagenome search using a PETase‐specific hidden Markov model. Besides acting on PET, PET40 has a versatile substrate spectrum, hydrolyzing δ‐lactones, β‐lactam antibiotics, the polyester‐polyurethane Impranil® DLN, and various para ‐nitrophenyl ester substrates. Molecular docking suggests that the PET degradative activity is likely a result of the promiscuity of PET40, as potential binding modes were found for substrates encompassing mono(2‐hydroxyethyl) terephthalate, bis(2‐hydroxyethyl) terephthalate, and a PET trimer. We also solved the crystal structure of the inactive PET40 variant S178A to 1.60 Å resolution. PET40 is active throughout a wide pH (pH 4–10) and temperature range (4–65 °C) and remarkably stable in the presence of 5% SDS, making it a promising enzyme as a starting point for further investigations and optimization approaches.

To enter epithelial cells, the obligate intracellular pathogen Chlamydia pneumoniae secretes early effector proteins, which bind to and modulate the host-cell's plasma membrane and recruit several pivotal endocytic host proteins. Here, we present the high-resolution structure of an entry-related chlamydial effector protein, SemD. Co-crystallisation of SemD with its host binding partners demonstrates that SemD co-opts the Cdc42 binding site to activate the actin cytoskeleton regulator N-WASP, making active, GTP-bound Cdc42 superfluous. While SemD binds N-WASP much more strongly than Cdc42 does, it does not bind the Cdc42 effector protein FMNL2, indicating effector protein specificity. Furthermore, by identifying flexible and structured domains, we show that SemD can simultaneously interact with the membrane, the endocytic protein SNX9, and N-WASP. Here, we show at the structural level how a single effector protein can hijack central components of the host's endocytic system for efficient internalization.

The chromatophores in Paulinella are evolutionary-early-stage photosynthetic organelles. Biological processes in chromatophores depend on a combination of chromatophore and nucleus-encoded proteins. Interestingly, besides proteins carrying chromatophore-targeting signals, a large arsenal of short chromatophore-targeted proteins (sCTPs; <90 amino acids) without recognizable targeting signals were found in chromatophores. This situation resembles endosymbionts in plants and insects that are manipulated by host-derived antimicrobial peptides. Previously, we identified an expanded family of sCTPs of unknown function, named here "DNA-binding (DB)-sCTPs". DB-sCTPs contain a ~45 amino acid motif that is conserved in some bacterial proteins with predicted functions in DNA processing. Here, we explored antimicrobial activity, DNA-binding capacity, and structures of three purified recombinant DB-sCTPs. All three proteins exhibited antimicrobial activity against bacteria involving membrane permeabilization, and bound to bacterial lipids in vitro. A combination of in vitro assays demonstrated binding of recombinant DB-sCTPs to chromatophore-derived genomic DNA sequences with an affinity in the low nM range. Additionally, we report the 1.2 Å crystal structure of one DB-sCTP. In silico docking studies suggest that helix α2 inserts into the DNA major grove and the exposed residues, that are highly variable between different DB-sCTPs, confer interaction with the DNA bases. Identification of photosystem II subunit CP43 as a potential interaction partner of one DB-sCTP, suggests DB-sCTPs to be involved in more complex regulatory mechanisms. We hypothesize that membrane binding of DB-sCTPs is related to their import into chromatophores. Once inside, they interact with the chromatophore genome potentially providing nuclear control over genetic information processing.

Polyethylene terephthalate (PET) is a commodity polymer known to globally contaminate marine and terrestrial environments. Today, around 80 bacterial and fungal PET-active enzymes (PETases) are known, originating from four bacterial and two fungal phyla. In contrast, no archaeal enzyme had been identified to degrade PET. Here we report on the structural and biochemical characterization of PET46 (RLI42440.1), an archaeal promiscuous feruloyl esterase exhibiting degradation activity on semi-crystalline PET powder comparable to IsPETase and LCC (wildtypes), and higher activity on bis-, and mono-(2-hydroxyethyl) terephthalate (BHET and MHET). The enzyme, found by a sequence-based metagenome search, is derived from a non-cultivated, deep-sea Candidatus Bathyarchaeota archaeon. Biochemical characterization demonstrated that PET46 is a promiscuous, heat-adapted hydrolase. Its crystal structure was solved at a resolution of 1.71 Å. It shares the core alpha/beta-hydrolase fold with bacterial PETases, but contains a unique lid common in feruloyl esterases, which is involved in substrate binding. Thus, our study widens the currently known diversity of PET-hydrolyzing enzymes, by demonstrating PET depolymerization by a plant cell wall-degrading esterase.

Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation.

ABSTRACT Polyethylene terephthalate (PET) is a commodity polymer known to globally contaminate marine and terrestrial environments. Today, around 40 bacterial and fungal PET-active enzymes (PETases) are known, originating from four bacterial and two fungal phyla. In contrast, no archaeal enzyme has been identified to degrade PET. Here we report on the structural and biochemical characterization of PET46, an archaeal promiscuous feruloyl esterase exhibiting degradation activitiy on PET, bis-, and mono-(2-hydroxyethyl) terephthalate (BHET and MHET). The enzyme, found by a sequence-based metagenome search, was derived from a non-cultivated, deep-sea Candidatus Bathyarchaeota archaeon. Biochemical characterization demonstrated that PET46 is a promiscuous, heat-adapted hydrolase. Its crystal structure was solved at a resolution of 1.71 Å. It shares the core alpha/beta-hydrolase fold with bacterial PETases, but contains a unique lid common in feruloyl esterases, which is involved in substrate binding. Thus, our study significantly widens the currently known diversity of PET-hydrolyzing enzymes, by demonstrating PET depolymerization by a lignin-degrading esterase.

CK2α and CK2α' are paralogous catalytic subunits of CK2, which belongs to the eukaryotic protein kinases. CK2 promotes tumorigenesis and the spread of pathogenic viruses like SARS-CoV-2 and is thus an attractive drug target. Efforts to develop selective CK2 inhibitors binding offside the ATP site had disclosed the αD pocket in CK2α; its occupation requires large conformational adaptations of the helix αD. As shown here, the αD pocket is accessible also in CK2α', where the necessary structural plasticity can be triggered with suitable ligands even in the crystalline state. A CK2α' structure with an ATP site and an αD pocket ligand guided the design of the bivalent CK2 inhibitor KN2. It binds to CK2 with low nanomolar affinity, is cell-permeable, and suppresses the intracellular phosphorylation of typical CK2 substrates. Kinase profiling revealed a high selectivity of KN2 for CK2 and emphasizes the selectivity-promoting potential of the αD pocket.

The human protein kinase CK2 is an emerging target not only in current cancer research but also in the pathophysiology of viral diseases, such as CoV-2 infection. Two isoforms of the catalytic subunit of human CK2, namely CK2α and CK2α’ were identified, exhibiting high similarity but minor functional and structural differences. Further, they differ in their expression profiles, with CK2α being ubiquitously expressed in every tissue while CK2α’ being mainly present in brain and testis. In the cell, CK2α and CK2α’ exist either as free subunits or in a tetrameric complex bound to a dimer of non-catalytic CK2β subunits. Inhibitors of CK2 which selectively target specific subunits of the kinase are advantageous for the examination of the different functions of the paralogous isoforms. Here we report on THN27B, an indeno[1,2-b]indole derivate that exhibits higher CK2α’ inhibitory activity (IC50 = 0.25 µM) in comparison to CK2α (IC50 = 0.61 µM). Co-crystal structures of CK2α and CK2α’ with THN27B reveled a different conformational viability in the interdomain hinge region explaining this behavior. Remarkably, this selective inhibitory behavior was eliminated by the addition of the regulatory subunit (CK2α2β2 IC50 = 0.12 µM; CK2α’2β2 IC50 = 0.12 µM). These results indicate that the preference of THN27B for CK2α’ can be further utilized to study the distinct functions of free catalytic subunit paralogs.

Selective inhibitors of protein kinase CK2 with significant cytotoxicity on tumor cells based on a 2-aminothiazole scaffold were described recently. Here, these studies are supplemented with representative CK2α/CK2α' complex struc-tures. They reveal that the 2-aminothiazole-based inhibitors occupy the ATP cavity, whereas preliminary data had indicated an allosteric binding site. The crystal structure findings are corroborated by subsequent enzyme kinetic studies; their atomic-resolution quality provides the basis for future optimization of these promising CK2 inhibitors.

CK2α and CK2α′ are the two isoforms of the catalytic subunit of human protein kinase CK2, an important target for cancer therapy. They have similar, albeit not identical functional and structural properties, and were occasionally reported to be inhibited with distinct efficacies by certain ATP-competitive ligands. Here, we present THN27, an indeno[1,2-b]indole derivative, as a further inhibitor with basal isoform selectivity. The selectivity disappears when measured using CK2α/CK2α′ complexes with CK2β, the regulatory CK2 subunit. Co-crystal structures of THN27 with CK2α and CK2α′ reveal that subtle differences in the conformational variability of the interdomain hinge region are correlated with the observed effect. In the case of CK2α′, a crystallographically problematic protein so far, this comparative structural analysis required the development of an experimental strategy that finally enables atomic resolution structure determinations with ab initio phasing of potentially any ATP-competitive CK2 inhibitor and possibly many non-ATP-competitive ligands as well bound to CK2α′.