Publications by authors named "Arne Skerra"

160 Publications

The role of changing loop conformations in streptavidin versions engineered for high-affinity binding of the Strep-tag II peptide.

J Mol Biol 2021 Feb 24:166893. Epub 2021 Feb 24.

Lehrstuhl für Biologische Chemie, Technische Universität München, 85354 Freising, Germany. Electronic address:

The affinity system based on the artificial peptide ligand Strep-tag® II and engineered tetrameric streptavidin, known as Strep-Tactin®, offers attractive applications for the study of recombinant proteins, from detection and purification to functional immobilization. To further improve binding of the Strep-tag II to streptavidin we have subjected two protruding loops that shape its ligand pocket for the peptide - instead of D-biotin recognized by the natural protein - to iterative random mutagenesis. Sequence analyses of hits from functional screening assays revealed several unexpected structural motifs, such as a disulfide bridge at the base of one loop, replacement of the crucial residue Trp120 by Gly and a two-residue deletion in the second loop. The mutant m1-9 (dubbed Strep-Tactin XT) showed strongly enhanced affinity towards the Strep-tag II, which was further boosted in case of the bivalent Twin-Strep-tag®. Four representative streptavidin mutants were crystallized in complex with the Strep-tag II and their X-ray structures were solved at high resolutions. In addition, the crystal structure of the complex between Strep-Tactin XT and the Twin-Strep-tag was elucidated, indicating a bivalent mode of binding and explaining the experimentally observed avidity effect. Our study illustrates the structural plasticity of streptavidin as a scaffold for ligand binding and reveals interaction modes that would have been difficult to predict. As result, Strep-Tactin XT offers a convenient reagent for the kinetically stable immobilization of recombinant proteins fused with the Twin-Strep-tag. The possibility of reversibly dissociating such complexes simply with D-biotin enables functional studies in protein science as well as cell biology.
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http://dx.doi.org/10.1016/j.jmb.2021.166893DOI Listing
February 2021

The IMiD target CRBN determines HSP90 activity toward transmembrane proteins essential in multiple myeloma.

Mol Cell 2021 Feb 3. Epub 2021 Feb 3.

Department of Medicine III, Klinikum rechts der Isar, Technical University of Munich, 81675 Munich, Germany; TranslaTUM, Center for Translational Cancer Research, Technical University of Munich, 81675 Munich, Germany; German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany. Electronic address:

The complex architecture of transmembrane proteins requires quality control (QC) of folding, membrane positioning, and trafficking as prerequisites for cellular homeostasis and intercellular communication. However, it has remained unclear whether transmembrane protein-specific QC hubs exist. Here we identify cereblon (CRBN), the target of immunomodulatory drugs (IMiDs), as a co-chaperone that specifically determines chaperone activity of HSP90 toward transmembrane proteins by means of counteracting AHA1. This function is abrogated by IMiDs, which disrupt the interaction of CRBN with HSP90. Among the multiple transmembrane protein clients of CRBN-AHA1-HSP90 revealed by cell surface proteomics, we identify the amino acid transporter LAT1/CD98hc as a determinant of IMiD activity in multiple myeloma (MM) and present an Anticalin-based CD98hc radiopharmaceutical for MM radio-theranostics. These data establish the CRBN-AHA1-HSP90 axis in the biogenesis of transmembrane proteins, link IMiD activity to tumor metabolism, and nominate CD98hc and LAT1 as attractive diagnostic and therapeutic targets in MM.
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http://dx.doi.org/10.1016/j.molcel.2020.12.046DOI Listing
February 2021

Complement inhibition at the level of C3 or C5: mechanistic reasons for ongoing terminal pathway activity.

Blood 2021 Jan;137(4):443-455

Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, Ulm, Germany.

Blocking the terminal complement pathway with the C5 inhibitor eculizumab has revolutionized the clinical management of several complement-mediated diseases and has boosted the clinical development of new inhibitors. Data on the C3 inhibitor Compstatin and the C5 inhibitors eculizumab and Coversin reported here demonstrate that C3/C5 convertases function differently from prevailing concepts. Stoichiometric C3 inhibition failed to inhibit C5 activation and lytic activity during strong classical pathway activation, demonstrating a "C3 bypass" activation of C5. We show that, instead of C3b, surface-deposited C4b alone can also recruit and prime C5 for consecutive proteolytic activation. Surface-bound C3b and C4b possess similar affinities for C5. By demonstrating that the fluid phase convertase C3bBb is sufficient to cleave C5 as long as C5 is bound on C3b/C4b-decorated surfaces, we show that surface fixation is necessary only for the C3b/C4b opsonins that prime C5 but not for the catalytic convertase unit C3bBb. Of note, at very high C3b densities, we observed membrane attack complex formation in absence of C5-activating enzymes. This is explained by a conformational activation in which C5 adopts a C5b-like conformation when bound to densely C3b-opsonized surfaces. Stoichiometric C5 inhibitors failed to prevent conformational C5 activation, which explains the clinical phenomenon of residual C5 activity documented for different inhibitors of C5. The new insights into the mechanism of C3/C5 convertases provided here have important implications for the development and therapeutic use of complement inhibitors as well as the interpretation of former clinical and preclinical data.
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http://dx.doi.org/10.1182/blood.2020005959DOI Listing
January 2021

PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology.

Int J Mol Sci 2020 Dec 24;22(1). Epub 2020 Dec 24.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.

Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.
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http://dx.doi.org/10.3390/ijms22010124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795856PMC
December 2020

Translating the Concept of Bispecific Antibodies to Engineering Heterodimeric Phosphotriesterases with Broad Organophosphate Substrate Recognition.

Biochemistry 2020 Nov 4;59(45):4395-4406. Epub 2020 Nov 4.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.

We have adopted the concept of bispecific antibodies, which can simultaneously block or cross-link two different biomolecular targets, to create bispecific enzymes by exploiting the homodimeric quaternary structure of bacterial phosphotriesterases (PTEs). The PTEs from and , whose engineered variants can efficiently hydrolyze organophosphorus (OP) nerve agents and pesticides, respectively, have attracted considerable interest for the treatment of the corresponding intoxications. OP nerve agents and pesticides still pose a severe toxicological threat in military conflicts, including acts of terrorism, as well as in agriculture, leading to >100000 deaths per year. In principle, engineered conventional homodimeric PTEs may provoke hydrolytic inactivation of individual OPs , and their application as catalytic bioscavengers via administration into the bloodstream has been proposed. However, their narrow substrate specificity would necessitate therapeutic application of a set or mixture of different enzymes, which complicates biopharmaceutical development. We succeeded in combining subunits from both enzymes and to stabilize their heterodimerization by rationally designing electrostatic steering mutations, thus breaking the natural C2 symmetry. The resulting bispecific enzyme from two PTEs with different bacterial origin exhibits an ultrabroad OP substrate profile and allows the efficient detoxification of both nerve agents and pesticides. Our approach of combining two active sites with distinct substrate specificities within one artificial dimeric biocatalyst-retaining the size and general properties of the original enzyme without utilizing protein mixtures or much larger fusion proteins-not only should facilitate biological drug development but also may be applicable to oligomeric enzymes with other catalytic activities.
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http://dx.doi.org/10.1021/acs.biochem.0c00751DOI Listing
November 2020

Anticalin® proteins: from bench to bedside.

Expert Opin Biol Ther 2020 Nov 1:1-10. Epub 2020 Nov 1.

Chair of Biological Chemistry, School of Life Sciences, Technical University of Munich , Freising, Germany.

Introduction: Anticalin proteins are engineered versions of lipocalins that constitute a novel class of clinical-stage biopharmaceuticals. The lipocalins exhibit a central β-barrel with eight antiparallel β-strands and an α-helix attached to its side. Four structurally variable loops at the open end of the β-barrel form a pronounced binding pocket, which can be reshaped to generate specificities toward diverse disease-relevant molecular targets.

Areas Covered: This article reviews the current status of Anticalin engineering, from the basic principles to the development of Anticalins with high target affinity and specificity via combinatorial protein design and directed evolution, including examples of Anticalin-based drug candidates under preclinical and clinical development.

Expert Opinion: Combinatorial gene libraries together with powerful molecular selection techniques have enabled the expansion of the natural ligand specificities of lipocalins from small molecules to peptides and proteins. This biomolecular concept has been validated by structural analyses of a series of Anticalin•target complexes. Promising Anticalin lead candidates have reached different preclinical and clinical development stages in the areas of (immuno)oncology, metabolic, and respiratory diseases, as antidotes to treat intoxications and as novel antibiotics. Thus, Anticalins offer an alternative to antibodies with promising and potentially superior features as next-generation biologics.
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http://dx.doi.org/10.1080/14712598.2021.1839046DOI Listing
November 2020

Proline/alanine-rich sequence (PAS) polypeptides as an alternative to PEG precipitants for protein crystallization.

Acta Crystallogr F Struct Biol Commun 2020 Jul 1;76(Pt 7):320-325. Epub 2020 Jul 1.

Lehrstuhl für Biologische Chemie, Technische Universität München, 85354 Freising, Germany.

Proline/alanine-rich sequence (PAS) polypeptides represent a novel class of biosynthetic polymers comprising repetitive sequences of the small proteinogenic amino acids L-proline, L-alanine and/or L-serine. PAS polymers are strongly hydrophilic and highly soluble in water, where they exhibit a natively disordered conformation without any detectable secondary or tertiary structure, similar to polyethylene glycol (PEG), which constitutes the most widely applied precipitant for protein crystallization to date. To investigate the potential of PAS polymers for structural studies by X-ray crystallography, two proteins that were successfully crystallized using PEG in the past, hen egg-white lysozyme and the Fragaria × ananassa O-methyltransferase, were subjected to crystallization screens with a 200-residue PAS polypeptide. The PAS polymer was applied as a precipitant using a vapor-diffusion setup that allowed individual optimization of the precipitant concentration in the droplet in the reservoir. As a result, crystals of both proteins showing high diffraction quality were obtained using the PAS precipitant. The genetic definition and precise macromolecular composition of PAS polymers, both in sequence and in length, distinguish them from all natural and synthetic polymers that have been utilized for protein crystallization so far, including PEG, and facilitate their adaptation for future applications. Thus, PAS polymers offer potential as novel precipitants for biomolecular crystallography.
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http://dx.doi.org/10.1107/S2053230X20008328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336357PMC
July 2020

Design of a surrogate Anticalin protein directed against CD98hc for preclinical studies in mice.

Protein Sci 2020 08 16;29(8):1774-1783. Epub 2020 Jun 16.

Lehrstuhl für Biologische Chemie, Technische Universität München, Freising, Germany.

The human CD98 heavy chain (CD98hc) offers a promising biomedical target both for tumor therapy and for drug delivery to the brain. We have previously developed a cognate Anticalin protein with picomolar affinity and demonstrated its effectiveness in a xenograft animal model. Due to the lack of cross-reactivity with the murine ortholog, we now report the development and X-ray structural analysis of an Anticalin with high affinity toward CD98hc from mouse. This binding protein recognizes the same protruding epitope loop-despite distinct structure-in the membrane receptor ectodomain as the Anticalin selected against human CD98hc. Thus, this surrogate Anticalin should be useful for the preclinical assessment of CD98hc targeting in vivo and support the translational development for medical application in humans.
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http://dx.doi.org/10.1002/pro.3894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380666PMC
August 2020

First In-Human Medical Imaging with a PASylated Zr-Labeled Anti-HER2 Fab-Fragment in a Patient with Metastatic Breast Cancer.

Nucl Med Mol Imaging 2020 Apr 20;54(2):114-119. Epub 2020 Apr 20.

5Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.

Purpose: PASylation® offers the ability to systematically tune and optimize the pharmacokinetics of protein tracers for molecular imaging. Here we report the first clinical translation of a PASylated Fab fragment (Zr∙Df-HER2-Fab-PAS) for the molecular imaging of tumor-related HER2 expression.

Methods: A patient with HER2-positive metastatic breast cancer received 37 MBq of Zr∙Df-HER2-Fab-PAS at a total mass dose of 70 μg. PET/CT was carried out 6, 24, and 45 h after injection, followed by image analysis of biodistribution, normal organ uptake, and lesion targeting.

Results: Images show a biodistribution typical for protein tracers, characterized by a prominent blood pool 6 h p.i., which decreased over time. Lesions were detectable as early as 24 h p.i. Zr∙Df-HER2-Fab-PAS was tolerated well.

Conclusion: This study demonstrates that a PASylated Fab tracer shows appropriate blood clearance to allow sensitive visualization of small tumor lesions in a clinical setting.
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http://dx.doi.org/10.1007/s13139-020-00638-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198682PMC
April 2020

Development of a Chimeric Antigen-Binding Fragment Directed Against Human Galectin-3 and Validation as an Immuno-Positron Emission Tomography Tracer for the Sensitive Imaging of Thyroid Cancer.

Thyroid 2020 09 30;30(9):1314-1326. Epub 2020 Apr 30.

Klinikum rechts der Isar, Department of Nuclear Medicine, Technical University Munich, Munich, Germany.

The lack of facile methods for the specific characterization of malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Due to its restricted expression in such nodules, the cell-associated lectin galectin-3 (Gal3) has emerged as a marker for TC with growing interest for imaging as well as targeted radionuclide therapy. To accelerate translation into clinical application, we have developed a cognate chimeric human antigen-binding fragment (Fab) derived from the rat anti-Gal3 monoclonal antibody M3/38. The variable immunoglobulin (Ig) light and heavy chain sequences were cloned from the hybridoma cell line, and the corresponding Fab carrying human IgG1/κ constant genes was functionally produced in the periplasm of and purified to homogeneity. To moderately prolong its plasma half-life and, thus, increase tumor uptake, the recombinant Fab was fused with a long disordered amino acid chain comprising in total 200 Pro, Ala, and Ser residues (PASylation). This novel tracer was subjected to characterization and validation by using two thyroid cancer orthotopic murine models. To this end, the αGal3-Fab-PAS was conjugated with deferoxamine (Dfo), labeled with Zr under mild conditions and tested for binding on TC cell lines. Athymic nude mice were inoculated either with FRO82-1 or with CAL62 tumor cells into the left thyroid lobe. After intravenous injection with ∼3.0 MBq of Zr-Dfo-PAS-Fab, these mice were subjected to positron emission tomography (PET)/computed tomography imaging followed by quantification of tumor accumulation and immunohistochemical analysis. The αGal3-Fab-PAS revealed high affinity toward the recombinant Gal3 antigen, with a dissociation constant ≤1 nM as measured via enzyme-linked immunosorbent assay, surface plasmon resonance spectroscopy, and radioactive cell binding assay. The Gal3-targeting by the Zr(IV)-labeled protein tracer, as investigated by immuno-PET, demonstrated highly selective and fast accumulation in orthotopically implanted tumors, with strong contrast images achieved 24 hours postinjection, and no uptake in the tumor-free thyroid lobe, as also confirmed by biodistribution studies. The chimeric αGal3 Zr-Dfo-PAS-Fab tracer exhibits selective accumulation in the tumor-bearing thyroid lobe of xenograft mice. Thus, this novel radioactive probe offers potential to change TC management, in addition to current diagnostic procedures, and to reduce unnecessary thyroidectomies.
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http://dx.doi.org/10.1089/thy.2019.0670DOI Listing
September 2020

Engineering a Highly Active Sucrose Isomerase for Enhanced Product Specificity by Using a "Battleship" Strategy.

Chembiochem 2020 08 16;21(15):2161-2169. Epub 2020 Apr 16.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354, Freising, Germany.

The sucrose isomerase SmuA from Serratia plymuthica efficiently catalyses the isomerisation of sucrose into isomaltulose, an artificial sweetener used in the food industry. However, the formation of a hygroscopic by-product, trehalulose, necessitates additional separation to obtain a crystalline product. Therefore, we have improved the product specificity of SmuA by first introducing a few exploratory amino acid exchanges around the active site and investigating their influence. Then, we devised a second set of mutations, either at promising positions from the preceding cycle, but with a different side chain, or at alternative positions in the vicinity. After seven iterative cycles involving just 55 point mutations, we obtained the triple mutant Y219L/D398G/V465E which showed 2.3 times less trehalulose production but still had high catalytic efficiency (k /K =11.8 mM  s ). Not only does this mutant SmuA appear attractive as an industrial biocatalyst, but our semirational protein-engineering strategy, which resembles the battleship board game, should be of interest for other challenging enzyme optimization endeavours.
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http://dx.doi.org/10.1002/cbic.202000007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496859PMC
August 2020

Development of a high affinity Anticalin directed against human CD98hc for theranostic applications.

Theranostics 2020 12;10(5):2172-2187. Epub 2020 Jan 12.

Lehrstuhl für Biologische Chemie, Technische Universität München, 85354 Freising, Germany.

Enhanced amino acid supply and dysregulated integrin signaling constitute two hallmarks of cancer and are pivotal for metastatic transformation of cells. In line with its function at the crossroads of both processes, overexpression of CD98hc is clinically observed in various cancer malignancies, thus rendering it a promising tumor target. : We describe the development of Anticalin proteins based on the lipocalin 2 (Lcn2) scaffold against the human CD98hc ectodomain (hCD98hcED) using directed evolution and protein design. X-ray structural analysis was performed to identify the epitope recognized by the lead Anticalin candidate. The Anticalin - with a tuned plasma half-life using PASylation technology - was labeled with Zr and investigated by positron emission tomography (PET) of CD98-positive tumor xenograft mice. : The Anticalin P3D11 binds CD98hc with picomolar affinity and recognizes a protruding loop structure surrounded by several glycosylation sites within the solvent exposed membrane-distal part of the hCD98hcED. studies revealed specific binding activity of the Anticalin towards various CD98hc-expressing human tumor cell lines, suggesting broader applicability in cancer research. PET/CT imaging of mice bearing human prostate carcinoma xenografts using the optimized and Zr-labeled Anticalin demonstrated strong and specific tracer accumulation (8.6 ± 1.1 %ID/g) as well as a favorable tumor-to-blood ratio of 11.8. : Our findings provide a first proof of concept to exploit CD98hc for non-invasive biomedical imaging. The novel Anticalin-based αhCD98hc radiopharmaceutical constitutes a promising tool for preclinical and, potentially, clinical applications in oncology.
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http://dx.doi.org/10.7150/thno.38968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019167PMC
January 2020

FluoroCalins: engineered lipocalins with novel binding functions fused to a fluorescent protein for applications in biomolecular imaging and detection.

Protein Eng Des Sel 2019 12;32(6):289-296

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.

FluoroCalins represent novel bifunctional protein reagents derived from engineered lipocalins fused to a fluorescent reporter protein, here the enhanced green fluorescent protein (eGFP). We demonstrate the construction, facile bacterial production and broad applicability of FluoroCalins using two Anticalin® molecules directed against the tumor vasculature-associated extra domain B of fibronectin (ED-B) and the vascular endothelial growth factor receptor 3, a marker of tumor and lymphangiogenesis. FluoroCalins were prepared with two different spacers: (i) a short Ser3Ala linker and (ii) a long hydrophilic and conformationally unstructured PASylation® polypeptide comprising 200 Pro, Ala and Ser residues. These FluoroCalins were applied for direct target quantification in enzyme-linked immunosorbent assay as well as target detection by flow cytometry and fluorescence microscopy of live and fixed cells, respectively, demonstrating high specificity and signal-to-noise ratio. Hence, FluoroCalins offer a promising alternative to antibody-based reagents for state of the art fluorescent in vitro detection and biomolecular imaging.
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http://dx.doi.org/10.1093/protein/gzz047DOI Listing
December 2019

Engineered Protein Scaffolds as Next-Generation Therapeutics.

Annu Rev Pharmacol Toxicol 2020 01;60:391-415

Lehrstuhl für Biologische Chemie, Technische Universität München, 85354 Freising, Germany; email:

The concept of engineering robust protein scaffolds for novel binding functions emerged 20 years ago, one decade after the advent of recombinant antibody technology. Early examples were the Affibody, Monobody (Adnectin), and Anticalin proteins, which were derived from fragments of streptococcal protein A, from the tenth type III domain of human fibronectin, and from natural lipocalin proteins, respectively. Since then, this concept has expanded considerably, including many other protein templates. In fact, engineered protein scaffolds with useful binding specificities, mostly directed against targets of biomedical relevance, constitute an area of active research today, which has yielded versatile reagents as laboratory tools. However, despite strong interest from basic science, only a handful of those protein scaffolds have undergone biopharmaceutical development up to the clinical stage. This includes the abovementioned pioneering examples as well as designed ankyrin repeat proteins (DARPins). Here we review the current state and clinical validation of these next-generation therapeutics.
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http://dx.doi.org/10.1146/annurev-pharmtox-010818-021118DOI Listing
January 2020

A catalytic bioscavenger with improved stability and reduced susceptibility to oxidation for treatment of acute poisoning with neurotoxic organophosphorus compounds.

Toxicol Lett 2020 Mar 28;321:138-145. Epub 2019 Dec 28.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany. Electronic address:

Organophosphorus (OP) nerve agents pose a severe toxicological threat, both after dissemination in military conflicts and by terrorists. Hydrolytic enzymes, which may be administered into the blood stream of victims by injection and can decompose the circulating nerve agent into non-toxic metabolites in vivo, could offer a treatment. Indeed, for the phosphotriesterase found in the bacterium Brevundimonas diminuta (BdPTE), engineered versions with improved catalytic efficiencies have been described; yet, their biochemical stabilities are insufficient for therapeutic use. Here, we describe the application of rational protein design to develop novel mutants of BdPTE that are less susceptible to oxidative damage. In particular, the replacement of two unpaired cysteine residues by more inert amino acids led to higher stability while maintaining high catalytic activity towards a broad spectrum of substrates, including OP pesticides and V-type nerve agents. The mutant BdPTE enzymes were produced in Escherichia coli, purified to homogeneity, and their biochemical and enzymological properties were assessed. Several candidates both revealed enhanced thermal stability and were less susceptible to oxidative stress, as demonstrated by mass spectrometry. These mutants of BdPTE may show promise for the treatment of acute intoxications by nerve agents as well as OP pesticides.
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http://dx.doi.org/10.1016/j.toxlet.2019.12.030DOI Listing
March 2020

PASylation of IL-1 receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis.

J Biol Chem 2020 01 9;295(3):868-882. Epub 2019 Dec 9.

Department of Medicine, University of Colorado, Aurora, Colorado 80045.

Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS-IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS-IL-1Ra molecules, PAS600-IL-1Ra and PAS800-IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600-IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600-IL-1Ra and PAS800-IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800-IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800-IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600-IL-1Ra, and PAS800-IL-Ra reduced IL-1α-induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed -induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS-IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS-IL-1Ra as an active IL-1 antagonist with marked potency and a significantly extended half-life compared with anakinra.
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http://dx.doi.org/10.1074/jbc.RA119.010340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6970921PMC
January 2020

Scavenging Bacterial Siderophores with Engineered Lipocalin Proteins as an Alternative Antimicrobial Strategy.

Chembiochem 2020 03 13;21(5):601-606. Epub 2019 Dec 13.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354, Freising, Germany.

Iron acquisition mediated by siderophores, high-affinity chelators for which bacteria have evolved specific synthesis and uptake mechanisms, plays a crucial role in microbiology and in host-pathogen interactions. In the ongoing fight against bacterial infections, this area has attracted biomedical interest. Beyond several approaches to interfere with siderophore-mediated iron uptake from medicinal and immunochemistry, the development of high-affinity protein scavengers that tightly complex the siderophores produced by pathogenic bacteria has appeared as a novel strategy. Such binding proteins have been engineered based on siderocalin-also known as lipocalin 2-an endogenous human scavenger of enterobactin and bacillibactin that controls the systemic spreading of commensal bacteria such as Escherichia coli. By using combinatorial protein design, siderocalin was reshaped to bind several siderophores from Pseudomonas aeruginosa and, in particular, petrobactin from Bacillus anthracis, none of which is recognized by the natural protein. Such engineered versions of siderocalin effectively suppress the growth of corresponding pathogenic bacteria by depriving them of their iron supply and offer the potential to complement antibiotic therapy in situations of acute or persistent infection.
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http://dx.doi.org/10.1002/cbic.201900564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079049PMC
March 2020

A Tetrahedral Boronic Acid Diester Formed by an Unnatural Amino Acid in the Ligand Pocket of an Engineered Lipocalin.

Chembiochem 2020 02 7;21(4):469-472. Epub 2019 Nov 7.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354, Freising, Germany.

Boronic acids have long been known to form cyclic diesters with cis-diol compounds, including many carbohydrates. This phenomenon was previously exploited to create an artificial lectin by incorporating p-borono-l-phenylalanine (Bpa) into the ligand pocket of an engineered lipocalin, resulting in a so-called Borocalin. Here we describe the X-ray analysis of its covalent complex with 4-nitrocatechol as a high-affinity model ligand. As expected, the crystal structure reveals the formation of a cyclic diester between the biosynthetic boronate side chain and the two ortho-hydroxy substituents of the benzene ring. Interestingly, the boron also has a hydroxide ion associated, despite an only moderately basic pH 8.5 in the crystallization buffer. The complex is stabilized by a polar contact to the side chain of Asn134 within the ligand pocket, thus validating the functional design of the Borocalin as an artificial sugar-binding protein. Our structural analysis demonstrates how a boronate can form a thermodynamically stable diester with a vicinal diol in a tetrahedral configuration in aqueous solution near physiological pH. Moreover, our data provide a basis for the further engineering of the Borocalin with the goal of specific recognition of biologically relevant glycans.
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http://dx.doi.org/10.1002/cbic.201900405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065090PMC
February 2020

Different Levels of Incomplete Terminal Pathway Inhibition by Eculizumab and the Clinical Response of PNH Patients.

Front Immunol 2019 18;10:1639. Epub 2019 Jul 18.

Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, Ulm, Germany.

Eculizumab blocks the lytic complement pathway by inhibiting C5 and has become the standard of care for certain complement-mediated diseases. Previously, we have shown that strong complement activation overrides the C5 inhibition by Eculizumab, which accounts for residual terminal pathway activity. Here we show that the levels of residual hemolysis in assays differ markedly (up to 3.4-fold) across sera collected from different paroxysmal nocturnal hemoglobinuria (PNH) patients on Eculizumab treatment. This large variability of residual activity was also found in sera of healthy donors, thus cross-validating the findings in patients. While PNH patients with residual lytic activities of 11-30% exhibited hemolysis levels around the upper limit of normal (i.e., plasma LDH of ~250 u/L), as expected for PNH patients on Eculizumab therapy, we found sustained and markedly increased LDH levels of around 400 u/L for the patient with the highest residual activity of 37%. Furthermore, the clinical history of nine out of 14 PNH patients showed intravascular breakthrough hemolysis at the time of documented infections despite ample amounts of administered Eculizumab and/or experimentally determined excess over C5. The occurrence of extraordinary high levels of residual terminal pathway activity in PNH patients receiving Eculizumab is rare, but can impair the suppression of hemolysis. The commonly observed low levels of residual terminal pathway activity seen for most PNH patients can exacerbate during severe infections and, thus, can cause pharmacodynamic breakthrough hemolysis in PNH patients treated with Eculizumab.
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http://dx.doi.org/10.3389/fimmu.2019.01639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657537PMC
October 2020

Engineering of binding functions into proteins.

Curr Opin Biotechnol 2019 12 14;60:230-241. Epub 2019 Jun 14.

Lehrstuhl für Biologische Chemie, Technische Universität München, 85354 Freising, Germany. Electronic address:

Initially emerging as a highly innovative concept in the late 1990s, the concept of creating novel binding reagents based on stable protein scaffolds from outside the immunoglobulin (Ig) superfamily has become a well-developed area of research and discovery today. Numerous scaffolds based on extracellular, membrane-bound or intracellular proteins (or their domains) have been recruited, yielding versatile research reagents and even biological drug candidates to serve as a viable alternative to antibodies. This minireview discusses both established and novel concepts in this field and summarizes the current state of clinical development of the more advanced protein scaffolds, in particular Affibody, Adnectin, Anticalin and DARPin drug candidates.
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http://dx.doi.org/10.1016/j.copbio.2019.05.007DOI Listing
December 2019

Half-life extension and non-human primate pharmacokinetic safety studies of i-body AD-114 targeting human CXCR4.

MAbs 2019 10 23;11(7):1331-1340. Epub 2019 Aug 23.

The Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University , Bundoora , Melbourne , Australia.

Single domain antibodies that combine antigen specificity with high tissue penetration are an attractive alternative to conventional antibodies. However, rapid clearance from the bloodstream owing to their small size can be a limitation of therapeutic single domain antibodies. Here, we describe and evaluate the conjugation of a single domain i-body, AD-114, which targets CXCR4, to a panel of half-life extension technologies including a human serum albumin-binding peptide, linear and branched PEG, and PASylation (PA600). The conjugates were assessed in murine, rat and cynomolgus monkey pharmacokinetic studies and showed that the branched PEG was most effective at extending circulating half-life in mice; however, manufacturing limitations of PEGylated test material precluded scale-up and assessment in larger animals. PA600, by comparison, was amenable to scale-up and afforded considerable half-life improvements in mice, rats and cynomolgus monkeys. In mice, the circulating half-life of AD-114 was extended from 0.18 h to 7.77 h following conjugation to PA600, and in cynomolgus monkeys, the circulating half-life of AD-114-PA600 was 24.27 h. AD-114-PA600 was well tolerated in cynomolgus monkeys at dose rates up to 100 mg/kg with no mortalities or drug-related clinical signs.
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http://dx.doi.org/10.1080/19420862.2019.1626652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748587PMC
October 2019

Structural differences between the ectodomains of murine and human CD98hc.

Proteins 2019 08 23;87(8):693-698. Epub 2019 Apr 23.

Lehrstuhl für Biologische Chemie, Technische Universität München, Freising, Germany.

The CD98 heavy chain (CD98hc) constitutes both a promising cell surface target for the treatment of cancers and a transcytosis receptor potentially useful for the brain delivery of therapeutics. However, pharmacokinetic studies and safety assessment of cognate antibodies or nonimmunoglobulin binding proteins in rodents is hampered by cross-species variability of both amino acid sequence and glycosylation pattern. Here, we report the crystal structure of the murine CD98hc extracellular domain and a comprehensive comparison with its human ortholog, revealing only one conserved surface patch that is neither shielded by glycosylation nor by the cell membrane with an accessible surface area typical for an antibody epitope. Our results imply the necessity of a surrogate approach for CD98hc-specific binding proteins with predictive power for clinical investigations.
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http://dx.doi.org/10.1002/prot.25686DOI Listing
August 2019

Consistent success in life-supporting porcine cardiac xenotransplantation.

Nature 2018 12 5;564(7736):430-433. Epub 2018 Dec 5.

Department of Anaesthesiology, University Hospital, LMU Munich, Munich, Germany.

Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need. Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative. Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945 days after heterotopic abdominal transplantation in baboons. This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25 years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57 days and this was achieved, to our knowledge, only once. Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195 days is a milestone on the way to clinical cardiac xenotransplantation.
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http://dx.doi.org/10.1038/s41586-018-0765-zDOI Listing
December 2018

Regeneration After Radiation- and Immune-Mediated Tissue Injury Is Not Enhanced by Type III Interferon Signaling.

Int J Radiat Oncol Biol Phys 2019 03 29;103(4):970-976. Epub 2018 Nov 29.

Klinik und Poliklinik für Innere Medizin 3, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.

Purpose: Type I interferon (IFN-I) and interleukin (IL)-22 modulate regeneration of the thymus and intestinal epithelial cells (IECs) after cytotoxic stress such as irradiation. Radiation-induced damage to thymic tissues and IECs is a crucial aspect during the pathogenesis of inadequate immune reconstitution and acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with myeloablative total body irradiation (TBI), respectively. IL-22 and IFN-I reduce the severity of acute GVHD after allo-HSCT with myeloablative TBI. However, the role of biologically related type III interferon (IFN-III), also known as interferon lambda (IFN-λ) or IL-28, in this context is unclear. We therefore studied the role of the IFN-III pathway in thymic regeneration and GVHD after TBI and allo-HSCT.

Methods And Materials: Cohoused wild-type (WT) and IFN-III receptor-deficient (IL-28 receptor alpha subunit-deficient/IL-28Ra) mice were analyzed in models of TBI-induced thymus damage and a model of GVHD after allo-HSCT with myeloablative TBI. PASylated IFN-III (PASylated IL-28A, XL-protein GmbH) was generated to prolong the plasma half-life of IFN-III. Pharmacologic activity and the effects of PASylated IL-28A on radiation-induced thymus damage and the course of GVHD after allo-HSCT with myeloablative TBI were tested.

Results: The course and severity of GVHD after myeloablative TBI and allo-HSCT in IL-28Ra mice was comparable to those in WT mice. Activation of the IFN-III pathway by PASylated IL-28A did not significantly modulate GVHD after allo-HSCT with TBI. Furthermore, IL28Ra mice and WT mice showed similar thymus regeneration after radiation, which could also not be significantly modulated by IFN-III receptor engagement using PASylated IL-28A.

Conclusions: We analyzed the role of IFN-III signaling during radiation-mediated acute tissue injury. Despite molecular and biologic homologies with IFN-I and IL-22, IFN-III signaling did not improve thymus regeneration after radiation or the course of GVHD after myeloablative TBI and allo-HSCT.
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http://dx.doi.org/10.1016/j.ijrobp.2018.11.038DOI Listing
March 2019

An engineered lipocalin that tightly complexes the plant poison colchicine for use as antidote and in bioanalytical applications.

Biol Chem 2019 02;400(3):351-366

Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85354 Freising (Weihenstephan), Germany.

Colchicine is a toxic alkaloid prevalent in autumn crocus (Colchicum autumnale) that binds to tubulin and inhibits polymerization of microtubules. Using combinatorial and rational protein design, we have developed an artificial binding protein based on the human lipocalin 2 that binds colchicine with a dissociation constant of 120 pm, i.e. 10000-fold stronger than tubulin. Crystallographic analysis of the engineered lipocalin, dubbed Colchicalin, revealed major structural changes in the flexible loop region that forms the ligand pocket at the open end of the eight-stranded β-barrel, resulting in a lid-like structure over the deeply buried colchicine. A cis-peptide bond between residues Phe71 and Pro72 in loop #2 constitutes a peculiar feature and allows intimate contact with the tricyclic ligand. Using directed evolution, we achieved an extraordinary dissociation half-life of more than 9 h for the Colchicalin-colchicine complex. Together with the chemical robustness of colchicine and availability of activated derivatives, this also opens applications as a general-purpose affinity reagent, including facile quantification of colchicine in biological samples. Given that engineered lipocalins, also known as Anticalin® proteins, represent a class of clinically validated biopharmaceuticals, Colchicalin may offer a therapeutic antidote to scavenge colchicine and reverse its poisoning effect in situations of acute intoxication.
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http://dx.doi.org/10.1515/hsz-2018-0342DOI Listing
February 2019

PASylated interferon α efficiently suppresses hepatitis B virus and induces anti-HBs seroconversion in HBV-transgenic mice.

Antiviral Res 2019 01 12;161:134-143. Epub 2018 Nov 12.

Institute of Virology, Technische Universität München/Helmholtz Zentrum München, 81675, Munich, Germany; German Center for Infection Research (DZIF), Munich Partner Site, 81675, Munich, Germany. Electronic address:

Interferon α (IFNα) so far is the only therapeutic option for chronic hepatitis B virus (HBV) infection that can lead to virus clearance. Unfortunately, its application is limited by side effects and response rates are low. The aim of this study was to generate a novel long-acting IFNα with the help of PASylation technology that adds a polypeptide comprising Proline, Alanine and Serine (PAS) to increase plasma half-life. Following evaluation of four selected recombinant murine IFNα (mIFNα) subtypes in cell culture, the most active subtype, mIFNα11, was fused with a 600 amino acid PAS chain. The activity of PAS-mIFNα was assessed by interferon bioassay and further evaluated for induction of interferon-stimulated genes (ISG) and antiviral efficacy in cell culture as well as in HBV-transgenic mice. PAS-mIFNα induced expression of ISG comparable to unmodified mIFNα and, likewise, evoked dose-dependent reduction of HBV replication in vitro. In vivo, PAS-mIFNα led to pronounced suppression of HBV replication without detectable liver damage whereas conventional mIFNα treatment only had a modest antiviral effect. Importantly, all PAS-mIFNα treated mice showed an anti-HBs antibody response, lost HBsAg and achieved seroconversion after three weeks. PASylated IFNα showed a profoundly increased antiviral effect in vivo compared to the non-modified version without toxicity, providing proof-of-concept that an improved IFNα can achieve higher rates of HBV antiviral and immune control.
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http://dx.doi.org/10.1016/j.antiviral.2018.11.003DOI Listing
January 2019

Type I IFN signaling blockade by a PASylated antagonist during chronic SIV infection suppresses specific inflammatory pathways but does not alter T cell activation or virus replication.

PLoS Pathog 2018 08 24;14(8):e1007246. Epub 2018 Aug 24.

Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, United States of America.

Chronic activation of the immune system in HIV infection is one of the strongest predictors of morbidity and mortality. As such, approaches that reduce immune activation have received considerable interest. Previously, we demonstrated that administration of a type I interferon receptor antagonist (IFN-1ant) during acute SIV infection of rhesus macaques results in increased virus replication and accelerated disease progression. Here, we administered a long half-life PASylated IFN-1ant to ART-treated and ART-naïve macaques during chronic SIV infection and measured expression of interferon stimulated genes (ISG) by RNA sequencing, plasma viremia, plasma cytokines, T cell activation and exhaustion as well as cell-associated virus in CD4 T cell subsets sorted from peripheral blood and lymph nodes. Our study shows that IFN-1ant administration in both ART-suppressed and ART-untreated chronically SIV-infected animals successfully results in reduction of IFN-I-mediated inflammation as defined by reduced expression of ISGs but had no effect on plasma levels of IL-1β, IL-1ra, IL-6 and IL-8. Unlike in acute SIV infection, we observed no significant increase in plasma viremia up to 25 weeks after IFN-1ant administration or up to 15 weeks after ART interruption. Likewise, cell-associated virus measured by SIV gag DNA copies was similar between IFN-1ant and placebo groups. In addition, evaluation of T cell activation and exhaustion by surface expression of CD38, HLA-DR, Ki67, LAG-3, PD-1 and TIGIT, as well as transcriptome analysis showed no effect of IFN-I blockade. Thus, our data show that blocking IFN-I signaling during chronic SIV infection suppresses IFN-I-related inflammatory pathways without increasing virus replication, and thus may constitute a safe therapeutic intervention in chronic HIV infection.
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http://dx.doi.org/10.1371/journal.ppat.1007246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126880PMC
August 2018

A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery.

Sci Rep 2018 08 22;8(1):12538. Epub 2018 Aug 22.

Telethon Kids Institute, University of Western Australia, Subiaco, Western Australia, Australia.

Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell's endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple "Phylomer" CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne's muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics.
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http://dx.doi.org/10.1038/s41598-018-30790-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105642PMC
August 2018

Reprogramming Human Siderocalin To Neutralize Petrobactin, the Essential Iron Scavenger of Anthrax Bacillus.

Angew Chem Int Ed Engl 2018 10 2;57(44):14619-14623. Epub 2018 Oct 2.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354, Freising, Germany.

Bacillus anthracis owes its pronounced virulence-apart from specific toxins-to a twofold import mechanism for Fe ions. This pathogenic bacterium secretes the siderophores bacillibactin (BB) and petrobactin (PB), of which only BB is neutralized by human siderocalin, an abundant lipocalin in plasma. We describe its reshaping via combinatorial protein design to bind PB⋅Fe instead of BB⋅Fe , and with even higher affinity (K ≈20 pm). X-ray crystallographic analysis of the resulting "petrocalin" in complex with PB⋅Ga reveals a positively charged ligand pocket while the extended butterfly-like conformation of the bound PB provides a rationale for the missing recognition by the natural siderocalin. In microbiological studies, a combination of petrocalin and siderocalin effectively suppressed the growth of a BB /PB strain of Bacillus cereus under iron-limiting culture conditions. Thus, our reprogrammed lipocalin may offer novel treatment options for devastating infections caused by B. anthracis.
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http://dx.doi.org/10.1002/anie.201807442DOI Listing
October 2018