Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric basic structure of the nuclear egress complex (core NEC). These core NECs serve as a hexameric lattice-structured platform for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina- and membrane-rearranging functions (multicomponent NEC). Here, we report the X-ray structures of β- and γ-herpesvirus core NECs obtained through an innovative recombinant expression strategy based on NEC-hook::NEC-groove protein fusion constructs. This approach yielded the first structure of γ-herpesviral core NEC, namely the 1.56 Å structure of Epstein-Barr virus (EBV) BFRF1–BFLF2, as well as an increased resolution 1.48 Å structure of human cytomegalovirus (HCMV) pUL50-pUL53. Detailed analysis of these structures revealed that the prominent hook segment is absolutely required for core NEC formation and contributes approximately 80% of the interaction surface of the globular domains of NEC proteins. Moreover, using HCMV::EBV hook domain swap constructs, computational prediction of the roles of individual hook residues for binding, and quantitative binding assays with synthetic peptides presenting the HCMV- and EBV-specific NEC hook sequences, we characterized the unique hook-into-groove NEC interaction at various levels. Although the overall physicochemical characteristics of the protein interfaces differ considerably in these β- and γ-herpesvirus NECs, the binding free energy contributions of residues displayed from identical positions are similar. In summary, the results of our study reveal critical details of the molecular mechanism of herpesviral NEC interactions and highlight their potential as an antiviral drug target.

MicroScale Thermophoresis (MST) is a frequently used method for the quantitative characterization of intermolecular interactions with several advantages over other technologies. One of these is its capability to determine equilibrium constants in solution including complex biological matrices such as cell lysates. MST requires one binding partner to be fluorescent, which is typically achieved by labeling target proteins with a suitable fluorophore. Here, we present a near-native, site-specific in situ labeling strategy for MST experiments that enables reliable measurements in cell lysates and that has distinct advantages over routine covalent labeling techniques. To this end, we exploited the high-affinity interaction of tris-NTA with oligohistidine-tags, which are popular for purification, immobilization or detection of recombinant proteins. We used various DYE-tris-NTA conjugates to successfully label His-tagged proteins that were either purified or a component of cell lysate. The RED-tris-NTA was identified as the optimal dye conjugate with a high affinity towards oligohistidine-tags, a high fluorescence signal and an optimal signal-to-noise ratio in MST binding experiments. Owing to its emission in the red region of the spectrum, it also enables reliable measurements in complex biological matrices such as cell lysates allowing a more physiologically realistic assessment and eliminating the need for protein purification.

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.

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The handling of plastic waste and the associated ubiquitous occurrence of microplastic poses one of the biggest challenges of our time. Recent investigations of plastic degrading enzymes have opened new prospects for biological microplastic decomposition as well as recycling applications. For polyethylene terephthalate, in particular, several natural and engineered enzymes are known to have such promising properties. From a previous study that identified new PETase candidates by homology search, we chose the candidate PET6 from the globally distributed, halophilic organism Vibrio gazogenes for further investigation. By mapping the occurrence of Vibrios containing PET6 homologs we demonstrated their ubiquitous prevalence in the pangenome of several Vibrio strains. The biochemical characterization of PET6 showed that PET6 has a comparatively lower activity than other enzymes but also revealed a superior turnover at very high salt concentrations. The crystal structure of PET6 provides structural insights into this adaptation to saline environments. By grafting only a few beneficial mutations from other PET degrading enzymes onto PET6, we increased the activity up to three‐fold, demonstrating the evolutionary potential of the enzyme. MD simulations of the variant helped rationalize the mutational effects of those mutants and elucidate the interaction of the enzyme with a PET substrate. With tremendous amounts of plastic waste in the Ocean and the prevalence of Vibrio gazogenes in marine biofilms and estuarine marshes, our findings suggest that Vibrio and the PET6 enzyme are worthy subjects to study the PET degradation in marine environments.