Type 1 secretion necessitates a tight interplay between all domains of the ABC transporter.

Type I secretion systems (T1SS) facilitate the secretion of substrates in one step across both membranes of Gram-negative bacteria. A prime example is the hemolysin T1SS which secretes the toxin HlyA. Secretion is energized by the ABC transporter HlyB, which forms a complex together with the membrane fusion protein HlyD and the outer membrane protein TolC. HlyB features three domains: an N-terminal C39 peptidase-like domain (CLD), a transmembrane domain (TMD) and a C-terminal nucleotide binding domain (NBD). Here, we created chimeric transporters by swapping one or more domains of HlyB with the respective domain(s) of RtxB, a HlyB homolog from Kingella kingae. We tested all chimeric transporters for their ability to secrete pro-HlyA when co-expressed with HlyD. The CLD proved to be most critical, as a substitution abolished secretion. Swapping only the TMD or NBD reduced the secretion efficiency, while a simultaneous exchange abolished secretion. These results indicate that the CLD is the most critical secretion determinant, while TMD and NBD might possess additional recognition or interaction sites. This mode of recognition represents a hierarchical and extreme unusual case of substrate recognition for ABC transporters and optimal secretion requires a tight interplay between all domains.

The metagenome-derived esterase PET40 is highly promiscuous and hydrolyses polyethylene terephthalate (PET).

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, ß-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.

Calcium binding of AtCBL1: Structural and functional insights.

CBL1 is an EF hand Ca2+ binding protein from A. thaliana that is involved in the detection of cellular Ca2+ signals and the downstream signal transmission by interaction with the protein kinase CIPK23. So far, the structure and calcium ion binding affinities of CBL1 remain elusive. In this study it was observed that CBL1 tends to form higher oligomeric states due to an intrinsic hydrophobicity and the presence of the detergent BriJ35 was required for the purification of monomeric and functional protein. Functional insights into the in vitro Ca2+ binding capabilities of CBL1 were obtained by isothermal titration calorimetry (ITC) of the wildtype protein as well as single site EF hand mutants. Based on our results, a binding model of CBL1 for Ca2+in vivo is proposed. Additionally, upon both, ITC measurements and the analysis of an AlphaFold2 model of CBL1, we could gain first insights into the formation of the dimer interface. We could identify an area around EF hand 4 to be relevant for the structural and functional integrity of monomeric CBL1 and likely EF hand 1 to be involved in the dimer interface.

Integrative modeling of guanylate binding protein dimers.

Guanylate-binding proteins (GBPs) are essential interferon-γ-activated large GTPases that play a crucial role in host defense against intracellular bacteria and parasites. While their protective functions rely on protein polymerization, our understanding of the structural intricacies of these multimerized states remains limited. To bridge this knowledge gap, we present dimer models for human GBP1 (hGBP1) and murine GBP2 and 7 (mGBP2 and mGBP7) using an integrative approach, incorporating the crystal structure of hGBP1's GTPase domain dimer, crosslinking mass spectrometry, small-angle X-ray scattering, protein-protein docking, and molecular dynamics simulations. Our investigation begins by comparing the protein dynamics of hGBP1, mGBP2, and mGBP7. We observe that the M/E domain in all three proteins exhibits significant mobility and hinge motion, with mGBP7 displaying a slightly less pronounced motion but greater flexibility in its GTPase domain. These dynamic distinctions can be attributed to variations in the sequences of mGBP7 and hGBP1/mGBP2, resulting in different dimerization modes. Unlike hGBP1 and its close ortholog mGBP2, which exclusively dimerize through their GTPase domains, we find that mGBP7 exhibits three equally probable alternative dimer structures. The GTPase domain of mGBP7 is only partially involved in its dimerization, primarily due to an accumulation of negative charge, allowing mGBP7 to dimerize independently of GTP. Instead, mGBP7 exhibits a strong tendency to dimerize in an antiparallel arrangement across its stalks. The results of this work go beyond the sequence-structure-function relationship, as the sequence differences in mGBP7 and mGBP2/hGBP1 do not lead to different structures, but to different protein dynamics and dimerization. The distinct GBP dimer structures are expected to encode specific functions crucial for disrupting pathogen membranes.

Respiratory syncytial virus-approved mAb Palivizumab as ligand for anti-idiotype nanobody-based synthetic cytokine receptors.

Synthetic cytokine receptors can modulate cellular functions based on an artificial ligand to avoid off-target and/or unspecific effects. However, ligands that can modulate receptor activity so far have not been used clinically because of unknown toxicity and immunity against the ligands. Here, we developed a fully synthetic cytokine/cytokine receptor pair based on the antigen-binding domain of the respiratory syncytial virus-approved mAb Palivizumab as a synthetic cytokine and a set of anti-idiotype nanobodies (AIPVHH) as synthetic receptors. Importantly, Palivizumab is neither cross-reactive with human proteins nor immunogenic. For the synthetic receptors, AIPVHH were fused to the activating interleukin-6 cytokine receptor gp130 and the apoptosis-inducing receptor Fas. We found that the synthetic cytokine receptor AIPVHHgp130 was efficiently activated by dimeric Palivizumab single-chain variable fragments. In summary, we created an in vitro nonimmunogenic full-synthetic cytokine/cytokine receptor pair as a proof of concept for future in vivo therapeutic strategies utilizing nonphysiological targets during immunotherapy.

An archaeal lid-containing feruloyl esterase degrades polyethylene terephthalate.

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.

A step forward to the optimized HlyA type 1 secretion system through directed evolution.

Secretion of proteins into the extracellular space has great advantages for the production of recombinant proteins. Type 1 secretion systems (T1SS) are attractive candidates to be optimized for biotechnological applications, as they have a relatively simple architecture compared to other classes of secretion systems. A paradigm of T1SS is the hemolysin A type 1 secretion system (HlyA T1SS) from Escherichia coli harboring only three membrane proteins, which makes the plasmid-based expression of the system easy. Although for decades the HlyA T1SS has been successfully applied for secretion of a long list of heterologous proteins from different origins as well as peptides, but its utility at commercial scales is still limited mainly due to low secretion titers of the system. To address this drawback, we engineered the inner membrane complex of the system, consisting of HlyB and HlyD proteins, following KnowVolution strategy. The applied KnowVolution campaign in this study provided a novel HlyB variant containing four substitutions (T36L/F216W/S290C/V421I) with up to 2.5-fold improved secretion for two hydrolases, a lipase and a cutinase. KEY POINTS: • An improvement in protein secretion via the use of T1SS • Reaching almost 400 mg/L of soluble lipase into the supernatant • A step forward to making E. coli cells more competitive for applying as a secretion host.

Functional reconstitution and structural characterization of the plant hormone receptor ETR1 in lipid nanodiscs.

The plant hormone receptor ETR1 regulates many highly relevant agronomic processes. Today, significant functional and structural questions remain unanswered regarding its multi-pass transmembrane sensor domain able to bind and respond to the gaseous plant hormone ethylene at femtomolar concentrations. A significant reason for this is the lack of structural data on full-length ETR1 in a lipid environment. Herein, we present the functional reconstitution of recombinant full-length ETR1 purified and solubilized from a bacterial host into lipid nanodiscs, allowing the study of the purified plant receptor for the first time in a detergent-free membrane-like environment.

DNA-binding and protein structure of nuclear factors likely acting in genetic information processing in the Paulinella chromatophore.

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.

Investigations on the substrate binding sites of hemolysin B, an ABC transporter, of a type 1 secretion system.

The ABC transporter hemolysin B (HlyB) is the key protein of the HlyA secretion system, a paradigm of type 1 secretion systems (T1SS). T1SS catalyze the one-step substrate transport across both membranes of Gram-negative bacteria. The HlyA T1SS is composed of the ABC transporter (HlyB), the membrane fusion protein (HlyD), and the outer membrane protein TolC. HlyA is a member of the RTX (repeats in toxins) family harboring GG repeats that bind Ca2+ in the C-terminus upstream of the secretion signal. Beside the GG repeats, the presence of an amphipathic helix (AH) in the C-terminus of HlyA is essential for secretion. Here, we propose that a consensus length between the GG repeats and the AH affects the secretion efficiency of the heterologous RTX secreted by the HlyA T1SS. Our in silico studies along with mutagenesis and biochemical analysis demonstrate that there are two binding pockets in the nucleotide binding domain of HlyB for HlyA. The distances between the domains of HlyB implied to interact with HlyA indicated that simultaneous binding of the substrate to both cytosolic domains of HlyB, the NBD and CLD, is possible and required for efficient substrate secretion.

The periplasmic chaperone Skp prevents misfolding of the secretory lipase A from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a wide-spread opportunistic human pathogen and a high-risk factor for immunodeficient people and patients with cystic fibrosis. The extracellular lipase A belongs to the virulence factors of P. aeruginosa. Prior to the secretion, the lipase undergoes folding and activation by the periplasmic foldase LipH. At this stage, the enzyme is highly prone to aggregation in mild and high salt concentrations typical for the sputum of cystic fibrosis patients. Here, we demonstrate that the periplasmic chaperone Skp of P. aeruginosa efficiently prevents misfolding of the lipase A in vitro. In vivo experiments in P. aeruginosa show that the lipase secretion is nearly abolished in absence of the endogenous Skp. Small-angle X-ray scattering elucidates the trimeric architecture of P. aeruginosa Skp and identifies two primary conformations of the chaperone, a compact and a widely open. We describe two binding modes of Skp to the lipase, with affinities of 20 nM and 2 µM, which correspond to 1:1 and 1:2 stoichiometry of the lipase:Skp complex. Two Skp trimers are required to stabilize the lipase via the apolar interactions, which are not affected by elevated salt concentrations. We propose that Skp is a crucial chaperone along the lipase maturation and secretion pathway that ensures stabilization and carry-over of the client to LipH.

Heterologously secreted MbxA from Moraxella bovis induces a membrane blebbing response of the human host cell.

Many proteins of the Repeats in Toxins (RTX) protein family are toxins of Gram-negative pathogens including hemolysin A (HlyA) of uropathogenic E. coli. RTX proteins are secreted via Type I secretion systems (T1SS) and adopt their native conformation in the Ca2+-rich extracellular environment. Here we employed the E. coli HlyA T1SS as a heterologous surrogate system for the RTX toxin MbxA from the bovine pathogen Moraxella bovis. In E. coli the HlyA system successfully activates the heterologous MbxA substrate by acylation and secretes the precursor proMbxA and active MbxA allowing purification of both species in quantities sufficient for a variety of investigations. The activating E. coli acyltransferase HlyC recognizes the acylation sites in MbxA, but unexpectedly in a different acylation pattern as for its endogenous substrate HlyA. HlyC-activated MbxA shows host species-independent activity including a so-far unknown toxicity against human lymphocytes and epithelial cells. Using live-cell imaging, we show an immediate MbxA-mediated permeabilization and a rapidly developing blebbing of the plasma membrane in epithelial cells, which is associated with immediate cell death.

Guilty by association: Importers, exporters and MscS-type mechanosensitive channels encoded in biosynthetic gene clusters for the stress-protectant ectoine.

Ectoine and its derivative hydroxyectoine are widely synthesized or imported by bacteria to fend off the detrimental effects of high osmolarity on cellular hydration and growth. Genes that are connected to a particular physiological process are often found in the same genomic context. We exploited this feature in a comprehensive bioinformatical analysis of 1103 ectoine biosynthetic gene clusters from Bacteria and Archaea through which we identified 415 ect operons that colocalize with genes encoding potential osmolyte transporters. These belong to various importer families. Focusing on the complex ect gene clusters of the alpha-proteobacteria Hyphomonas neptunium and Novoshingobium sp. LH128, we analysed several transporters with respect to their substrate specificities through physiological, molecular and modelling approaches. Accordingly, we identified an MFS-type uptake system specific for ectoines (EctU) and a novel SSS-type ectoine/hydroxyectoine importer (EctI) with a broader substrate profile for osmostress protectants. Furthermore, some ect gene clusters encode a MscS/YbdG-type mechanosensitive channel protein, whose functionality was assessed through down-shock assays. Moreover, our analysis identified the gene for the first putative ectoine/hydroxyectoine-specific efflux system (EctX), a member of the MFS superfamily. Our findings make substantial contributions to the understanding of the ecophysiology of ectoines, key players in microbial osmostress adjustment systems.

Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects.

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.75 ± 1.22 µM. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

A New Twist in ABC Transporter Mediated Multidrug Resistance - Pdr5 is a Drug/proton Co-transporter.

The two major efflux pump systems that are involved in multidrug resistance (MDR) are (i) ATP binding cassette (ABC) transporters and (ii) secondary transporters. While the former use binding and hydrolysis of ATP to facilitate export of cytotoxic compounds, the latter utilize electrochemical gradients to expel their substrates. Pdr5 from Saccharomyces cerevisiae is a prominent member of eukaryotic ATP binding cassette (ABC) transporters that are involved in multidrug resistance (MDR) and used as a frequently studied model system. Although investigated for decades, the underlying molecular mechanisms of drug transport and substrate specificity remain elusive. Here, we provide electrophysiological data on the reconstituted Pdr5 demonstrating that this MDR efflux pump does not only actively translocate its substrates across the lipid bilayer, but at the same time generates a proton motif force in the presence of Mg2+-ATP and substrates by acting as a proton/drug co-transporter. Importantly, a strictly substrate dependent co-transport of protons was also observed in in vitro transport studies using Pdr5-enriched plasma membranes. We conclude from these results that the mechanism of MDR conferred by Pdr5 and likely other transporters is more complex than the sole extrusion of cytotoxic compounds and involves secondary coupled processes suitable to increase the effectiveness.

A Plurizyme with Transaminase and Hydrolase Activity Catalyzes Cascade Reactions.

Engineering dual-function single polypeptide catalysts with two abiotic or biotic catalytic entities (or combinations of both) supporting cascade reactions is becoming an important area of enzyme engineering and catalysis. Herein we present the development of a PluriZyme, TR2 E2 , with efficient native transaminase (kcat : 69.49±1.77 min-1 ) and artificial esterase (kcat : 3908-0.41 min-1 ) activities integrated into a single scaffold, and evaluate its utility in a cascade reaction. TR2 E2 (pHopt : 8.0-9.5; Topt : 60-65 °C) efficiently converts methyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate into 3-(R)-amino-4-(2,4,5-trifluorophenyl)butanoic acid, a crucial intermediate for the synthesis of antidiabetic drugs. The reaction proceeds through the conversion of the ß-keto ester into the ß-keto acid at the hydrolytic site and subsequently into the ß-amino acid (e.e. >99 %) at the transaminase site. The catalytic power of the TR2 E2 PluriZyme was proven with a set of ß-keto esters, demonstrating the potential of such designs to address bioinspired cascade reactions.

Development of a First-in-Class Small-Molecule Inhibitor of the C-Terminal Hsp90 Dimerization.

Heat shock proteins 90 (Hsp90) are promising therapeutic targets due to their involvement in stabilizing several aberrantly expressed oncoproteins. In cancerous cells, Hsp90 expression is elevated, thereby exerting antiapoptotic effects, which is essential for the malignant transformation and tumor progression. Most of the Hsp90 inhibitors (Hsp90i) under investigation target the ATP binding site in the N-terminal domain of Hsp90. However, adverse effects, including induction of the prosurvival resistance mechanism (heat shock response or HSR) and associated dose-limiting toxicity, have so far precluded their clinical approval. In contrast, modulators that interfere with the C-terminal domain (CTD) of Hsp90 do not inflict HSR. Since the CTD dimerization of Hsp90 is essential for its chaperone activity, interfering with the dimerization process by small-molecule protein-protein interaction inhibitors is a promising strategy for anticancer drug research. We have developed a first-in-class small-molecule inhibitor (5b) targeting the Hsp90 CTD dimerization interface, based on a tripyrimidonamide scaffold through structure-based molecular design, chemical synthesis, binding mode model prediction, assessment of the biochemical affinity, and efficacy against therapy-resistant leukemia cells. 5b reduces xenotransplantation of leukemia cells in zebrafish models and induces apoptosis in BCR-ABL1+ (T315I) tyrosine kinase inhibitor-resistant leukemia cells, without inducing HSR.

New insights into the resistance mechanism for the BceAB-type transporter SaNsrFP.

Treatment of bacterial infections is one of the major challenges of our time due to the evolved resistance mechanisms of pathogens against antibiotics. To circumvent this problem, it is necessary to understand the mode of action of the drug and the mechanism of resistance of the pathogen. One of the most potent antibiotic targets is peptidoglycan (PGN) biosynthesis, as this is an exclusively occurring and critical feature of bacteria. Lipid II is an essential PGN precursor synthesized in the cytosol and flipped into the outer leaflet of the membrane prior to its incorporation into nascent PGN. Antimicrobial peptides (AMPs), such as nisin and colistin, targeting PGN synthesis are considered promising weapons against multidrug-resistant bacteria. However, human pathogenic bacteria that were also resistant to these compounds evolved by the expression of an ATP-binding cassette transporter of the bacitracin efflux (BceAB) type localized in the membrane. In the human pathogen Streptococcus agalactiae, the BceAB transporter SaNsrFP is known to confer resistance to the antimicrobial peptide nisin. The exact mechanism of action for SaNsrFP is poorly understood. For a detailed characterization of the resistance mechanism, we heterologously expressed SaNsrFP in Lactococcus lactis. We demonstrated that SaNsrFP conferred resistance not only to nisin but also to a structurally diverse group of antimicrobial PGN-targeting compounds such as ramoplanin, lysobactin, or bacitracin/(Zn)-bacitracin. Growth experiments revealed that SaNsrFP-producing cells exhibited normal behavior when treated with nisin and/or bacitracin, in contrast to the nonproducing cells, for which growth was significantly reduced. We further detected the accumulation of PGN precursors in the cytoplasm after treating the cells with bacitracin. This did not appear when SaNsrFP was produced. Whole-cell proteomic protein experiments verified that the presence of SaNsrFP in L. lactis resulted in higher production of several proteins associated with cell wall modification. These included, for example, the N-acetylmuramic acid-6-phosphate etherase MurQ and UDP-glucose 4-epimerase. Analysis of components of the cell wall of SaNsrFP-producing cells implied that the transporter is involved in cell wall modification. Since we used an ATP-deficient mutant of the transporter as a comparison, we can show that SaNsrFP and its inactive mutant do not show the same phenotype, albeit expressed at similar levels, which demonstrates the ATP dependency of the mediated resistance processes. Taken together, our data agree to a target protection mechanism and imply a direct involvement of SaNsrFP in resistance by shielding the membrane-localized target of these antimicrobial peptides, resulting in modification of the cell wall.

Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region.

Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.

The structure of MadC from Clostridium maddingley reveals new insights into class I lanthipeptide cyclases.

The rapid emergence of microbial multi-resistance against antibiotics has led to intense search for alternatives. One of these alternatives are ribosomally synthesized and post-translationally modified peptides (RiPPs), especially lantibiotics. They are active in a low nanomolar range and their high stability is due to the presence of characteristic (methyl-) lanthionine rings, which makes them promising candidates as bacteriocides. However, innate resistance against lantibiotics exists in nature, emphasizing the need for artificial or tailor-made lantibiotics. Obviously, such an approach requires an in-depth mechanistic understanding of the modification enzymes, which catalyze the formation of (methyl-)lanthionine rings. Here, we determined the structure of a class I cyclase (MadC), involved in the modification of maddinglicin (MadA) via X-ray crystallography at a resolution of 1.7 Å, revealing new insights about the structural composition of the catalytical site. These structural features and substrate binding were analyzed by mutational analyses of the leader peptide as well as of the cyclase, shedding light into the mode of action of MadC.