Publications by authors named "Michael Groll"

166 Publications

Structural and Mechanistic Insights into C-S Bond Formation in Gliotoxin.

Angew Chem Int Ed Engl 2021 Jun 14;60(25):14188-14194. Epub 2021 May 14.

Technical University of Munich, Center for Protein Assemblies, Ernst-Otto-Fischer-Strasse 8, 85747, Garching, Germany.

Glutathione-S-transferases (GSTs) usually detoxify xenobiotics. The human pathogenic fungus Aspergillus fumigatus however uses the exceptional GST GliG to incorporate two sulfur atoms into its virulence factor gliotoxin. Because these sulfurs are essential for biological activity, glutathionylation is a key step of gliotoxin biosynthesis. Yet, the mechanism of carbon-sulfur linkage formation from a bis-hydroxylated precursor is unresolved. Here, we report structures of GliG with glutathione (GSH) and its reaction product cyclo[-l-Phe-l-Ser]-bis-glutathione, which has been purified from a genetically modified A. fumigatus strain. The structures argue for stepwise processing of first the Phe and second the Ser moiety. Enzyme-mediated dehydration of the substrate activates GSH and a helix dipole stabilizes the resulting anion via a water molecule for the nucleophilic attack. Activity assays with mutants validate the interactions of GliG with the ligands and enrich our knowledge about enzymatic C-S bond formation in gliotoxin and epipolythiodioxopiperazine (ETP) natural compounds in general.
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http://dx.doi.org/10.1002/anie.202104372DOI Listing
June 2021

Design of buried charged networks in artificial proteins.

Nat Commun 2021 03 25;12(1):1895. Epub 2021 Mar 25.

Center for Integrated Protein Science Munich (CIPSM) at the Department Chemie, Technische Universität München, Lichtenbergstraße 4, 85748, Garching, Germany.

Soluble proteins are universally packed with a hydrophobic core and a polar surface that drive the protein folding process. Yet charged networks within the central protein core are often indispensable for the biological function. Here, we show that natural buried ion-pairs are stabilised by amphiphilic residues that electrostatically shield the charged motif from its surroundings to gain structural stability. To explore this effect, we build artificial proteins with buried ion-pairs by combining directed computational design and biophysical experiments. Our findings illustrate how perturbation in charged networks can introduce structural rearrangements to compensate for desolvation effects. We validate the physical principles by resolving high-resolution atomic structures of the artificial proteins that are resistant towards unfolding at extreme temperatures and harsh chemical conditions. Our findings provide a molecular understanding of functional charged networks and how point mutations may alter the protein's conformational landscape.
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http://dx.doi.org/10.1038/s41467-021-21909-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994573PMC
March 2021

Activation, Structure, Biosynthesis and Bioactivity of Glidobactin-like Proteasome Inhibitors from Photorhabdus laumondii.

Chembiochem 2021 May 3;22(9):1582-1588. Epub 2021 Mar 3.

Molecular Biotechnology, Department of Biosciences, Goethe University Frankfurt, 60438, Frankfurt am Main, Germany.

The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881-1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.
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http://dx.doi.org/10.1002/cbic.202100014DOI Listing
May 2021

A monodomain class II terpene cyclase assembles complex isoprenoid scaffolds.

Nat Chem 2020 10 10;12(10):968-972. Epub 2020 Aug 10.

Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zürich, Zurich, Switzerland.

Class II terpene cyclases, such as oxidosqualene and squalene-hopene cyclases, catalyse some of the most complex polycyclization reactions. They minimally exhibit a β,γ-didomain architecture that has been evolutionarily repurposed in a wide range of terpene-processing enzymes and likely resulted from a fusion of unidentified monodomain proteins. Although single domain class I terpene cyclases have already been identified, the corresponding class II counterparts have not been previously reported. Here we present high-resolution X-ray structures of a monodomain class II cyclase, merosterolic acid synthase (MstE). With a minimalistic β-domain architecture, this cyanobacterial enzyme is able to construct four rings in cytotoxic meroterpenoids with a sterol-like topology. The structures with bound substrate, product, and inhibitor provide detailed snapshots of a cyclization mechanism largely governed by residues located in a noncanonical enzyme region. Our results complement the few known class II cyclase crystal structures, while also indicating that archaic monodomain cyclases might have already catalyzed complex reaction cascades.
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http://dx.doi.org/10.1038/s41557-020-0515-3DOI Listing
October 2020

Structural snapshots of the minimal PKS system responsible for octaketide biosynthesis.

Nat Chem 2020 08 6;12(8):755-763. Epub 2020 Jul 6.

Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Technische Universität München, Garching, Germany.

Type II polyketide synthases (PKSs) are multi-enzyme complexes that produce secondary metabolites of medical relevance. Chemical backbones of such polyketides are produced by minimal PKS systems that consist of a malonyl transacylase, an acyl carrier protein and an α/β heterodimeric ketosynthase. Here, we present X-ray structures of all ternary complexes that constitute the minimal PKS system for anthraquinone biosynthesis in Photorhabdus luminescens. In addition, we characterize this invariable core using molecular simulations, mutagenesis experiments and functional assays. We show that malonylation of the acyl carrier protein is accompanied by major structural rearrangements in the transacylase. Principles of an ongoing chain elongation are derived from the ternary complex with a hexaketide covalently linking the heterodimeric ketosynthase with the acyl carrier protein. Our results for the minimal PKS system provide mechanistic understanding of PKSs and a fundamental basis for engineering PKS pathways for future applications.
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http://dx.doi.org/10.1038/s41557-020-0491-7DOI Listing
August 2020

Structural basis of HapE-linked antifungal triazole resistance in .

Life Sci Alliance 2020 07 28;3(7). Epub 2020 May 28.

Center for Integrated Protein Science Munich at the Department Chemistry, Technical University of Munich, Garching, Germany

Azoles are first-line therapeutics for human and plant fungal infections, but their broad use has promoted the development of resistances. Recently, a pan-azole-resistant clinical isolate was identified to carry the mutation P88L in subunit HapE of the CCAAT-binding complex (CBC), a conserved eukaryotic transcription factor. Here, we define the mechanistic basis for resistance in this isolate by showing that the HapE mutation interferes with the CBC's ability to bend and sense CCAAT motifs. This failure leads to transcriptional derepression of the gene, which encodes the target of azoles, the 14-α sterol demethylase Cyp51A, and ultimately causes drug resistance. In addition, we demonstrate that the CBC-associated transcriptional regulator HapX assists repression in low-iron environments and that this iron-dependent effect is lost in the HapE mutant. Altogether, these results indicate that the mutation HapE confers increased resistance to azoles compared with wt , particularly in low-iron clinical niches such as the lung.
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http://dx.doi.org/10.26508/lsa.202000729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266990PMC
July 2020

effector AnkX displaces the switch II region for Rab1b phosphocholination.

Sci Adv 2020 May 15;6(20):eaaz8041. Epub 2020 May 15.

Department of Biochemistry and Signal Transduction, University Medical Centre Hamburg-Eppendorf (UKE), Martinistr. 52, 20246 Hamburg, Germany.

The causative agent of Legionnaires disease, , translocates the phosphocholine transferase AnkX during infection and thereby posttranslationally modifies the small guanosine triphosphatase (GTPase) Rab1 with a phosphocholine moiety at S76 using cytidine diphosphate (CDP)-choline as a cosubstrate. The molecular basis for Rab1 binding and enzymatic modification have remained elusive because of lack of structural information of the low-affinity complex with AnkX. We combined thiol-reactive CDP-choline derivatives with recombinantly introduced cysteines in the AnkX active site to covalently capture the heterocomplex. The resulting crystal structure revealed that AnkX induces displacement of important regulatory elements of Rab1 by placing a β sheet into a conserved hydrophobic pocket, thereby permitting phosphocholine transfer to the active and inactive states of the GTPase. Together, the combination of chemical biology and structural analysis reveals the enzymatic mechanism of AnkX and the family of filamentation induced by cyclic adenosine monophosphate (FIC) proteins.
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http://dx.doi.org/10.1126/sciadv.aaz8041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228754PMC
May 2020

Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase.

Front Microbiol 2020 28;11:742. Epub 2020 Apr 28.

KAUST Catalysis Center, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.

Environments previously thought to be uninhabitable offer a tremendous wealth of unexplored microorganisms and enzymes. In this paper, we present the discovery and characterization of a novel γ-carbonic anhydrase (γ-CA) from the polyextreme Red Sea brine pool Discovery Deep (2141 m depth, 44.8°C, 26.2% salt) by single-cell genome sequencing. The extensive analysis of the selected gene helps demonstrate the potential of this culture-independent method. The enzyme was expressed in the bioengineered haloarchaeon sp. NRC-1 and characterized by X-ray crystallography and mutagenesis. The 2.6 Å crystal structure of the protein shows a trimeric arrangement. Within the γ-CA, several possible structural determinants responsible for the enzyme's salt stability could be highlighted. Moreover, the amino acid composition on the protein surface and the intra- and intermolecular interactions within the protein differ significantly from those of its close homologs. To gain further insights into the catalytic residues of the γ-CA enzyme, we created a library of variants around the active site residues and successfully improved the enzyme activity by 17-fold. As several γ-CAs have been reported without measurable activity, this provides further clues as to critical residues. Our study reveals insights into the halophilic γ-CA activity and its unique adaptations. The study of the polyextremophilic carbonic anhydrase provides a basis for outlining insights into strategies for salt adaptation, yielding enzymes with industrially valuable properties, and the underlying mechanisms of protein evolution.
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http://dx.doi.org/10.3389/fmicb.2020.00742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7199487PMC
April 2020

PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2-mediated phosphorylation at Threonine72.

Biochem J 2020 05;477(9):1651-1668

MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, U.K.

Loss of function mutations in the PTEN-induced kinase 1 (PINK1) kinase are causal for autosomal recessive Parkinson's disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies, we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor and GTPase activating protein. In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2 in vitro. Strikingly, we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 of recombinant nucleotide-bound Rab8A in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in transfected HEK293 cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and nuclear magnetic resonance structure of Ser111-phosphorylated Rab1B. The structures reveal that the phosphorylated SF3 motif does not induce any major changes, but may interfere with effector-Switch II interactions through intramolecular H-bond formation and/or charge effects with Arg79. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A indicating a potential cross-talk between PINK1-regulated mitochondrial homeostasis and LRRK2 signalling that requires further investigation in vivo.
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http://dx.doi.org/10.1042/BCJ20190664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7219890PMC
May 2020

Functional Characterisation of ClpP Mutations Conferring Resistance to Acyldepsipeptide Antibiotics in Firmicutes.

Chembiochem 2020 07 9;21(14):1997-2012. Epub 2020 Apr 9.

Interfaculty Institute of Microbiology and Infection Medicine, Dept. of Microbial Bioactive Compounds, University of Tübingen, Auf der Morgenstelle 28, 72076, Tuebingen, Germany.

Acyldepsipeptide (ADEP) is an exploratory antibiotic with a novel mechanism of action. ClpP, the proteolytic core of the caseinolytic protease, is deregulated towards unrestrained proteolysis. Here, we report on the mechanism of ADEP resistance in Firmicutes. This bacterial phylum contains important pathogens that are relevant for potential ADEP therapy. For Staphylococcus aureus, Bacillus subtilis, enterococci and streptococci, spontaneous ADEP-resistant mutants were selected in vitro at a rate of 10 . All isolates carried mutations in clpP. All mutated S. aureus ClpP proteins characterised in this study were functionally impaired; this increased our understanding of the mode of operation of ClpP. For molecular insights, crystal structures of S. aureus ClpP bound to ADEP4 were determined. Well-resolved N-terminal domains in the apo structure allow the pore-gating mechanism to be followed. The compilation of mutations presented here indicates residues relevant for ClpP function and suggests that ADEP resistance will occur at a lower rate during the infection process.
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http://dx.doi.org/10.1002/cbic.201900787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496096PMC
July 2020

Fatal amyloid formation in a patient's antibody light chain is caused by a single point mutation.

Elife 2020 03 10;9. Epub 2020 Mar 10.

Center for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, Garching, Germany.

In systemic light chain amyloidosis, an overexpressed antibody light chain (LC) forms fibrils which deposit in organs and cause their failure. While it is well-established that mutations in the LC's V domain are important prerequisites, the mechanisms which render a patient LC amyloidogenic are ill-defined. In this study, we performed an in-depth analysis of the factors and mutations responsible for the pathogenic transformation of a patient-derived λ LC, by recombinantly expressing variants in . We show that proteolytic cleavage of the patient LC resulting in an isolated V domain is essential for fibril formation. Out of 11 mutations in the patient V, only one, a leucine to valine mutation, is responsible for fibril formation. It disrupts a hydrophobic network rendering the C-terminal segment of V more dynamic and decreasing domain stability. Thus, the combination of proteolytic cleavage and the destabilizing mutation trigger conformational changes that turn the LC pathogenic.
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http://dx.doi.org/10.7554/eLife.52300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064341PMC
March 2020

Structures in Tetrahydrofolate Methylation in Desulfitobacterial Glycine Betaine Metabolism at Atomic Resolution.

Chembiochem 2020 03 18;21(6):776-779. Epub 2019 Nov 18.

Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, 85748, Garching, Germany.

Enzymes orchestrating methylation between tetrahydrofolate (THF) and cobalamin (Cbl) are abundant among all domains of life. During energy production in Desulfitobacterium hafniense, MtgA catalyzes the methyl transfer from methylcobalamin (Cbl-CH ) to THF in the catabolism of glycine betaine (GB). Despite its lack of sequence identity with known structures, we could show that MtgA forms a homodimeric complex of two TIM barrels. Atomic crystallographic insights into the interplay of MtgA with THF as well as analysis of a trapped reaction intermediate (THF-CH ) reveal conformational rearrangements during the transfer reaction. Whereas residues for THF methylation are conserved, the binding mode for the THF glutamyl-p-aminobenzoate moiety (THF tail) is unique. Apart from snapshots of individual reaction steps of MtgA, structure-based mutagenesis combined with enzymatic activity assays allowed a mechanistic description of the methyl transfer between Cbl-CH and THF. Altogether, the THF-tail-binding motion observed in MtgA is unique compared to other THF methyltransferases and therefore contributes to the general understanding of THF-mediated methyl transfer.
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http://dx.doi.org/10.1002/cbic.201900515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154762PMC
March 2020

Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen.

Sci Rep 2019 08 19;9(1):11971. Epub 2019 Aug 19.

King Abdullah University of Science and Technology (KAUST), KAUST Catalysis Center (KCC), Physical Sciences and Engineering Division (PSE), Thuwal, 23955-6900, Saudi Arabia.

The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA are extensively used to add non-canonical amino acids (ncAAs) to the genetic code of bacterial and eukaryotic cells. However, new ncAAs often require a cumbersome de novo engineering process to generate an appropriate PylRS/tRNA pair. We here report a strategy to predict a PylRS variant with novel properties. The designed polyspecific PylRS variant HpRS catalyzes the aminoacylation of 31 structurally diverse ncAAs bearing clickable, fluorinated, fluorescent, and for the first time biotinylated entities. Moreover, we demonstrated a site-specific and copper-free conjugation strategy of a nanobody by the incorporation of biotin. The design of polyspecific PylRS variants offers an attractive alternative to existing screening approaches and provides insights into the complex PylRS-substrate interactions.
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http://dx.doi.org/10.1038/s41598-019-48357-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700097PMC
August 2019

Molecular mechanism of polyketide shortening in anthraquinone biosynthesis of .

Chem Sci 2019 Jul 14;10(25):6341-6349. Epub 2019 May 14.

Molekulare Biotechnologie , Fachbereich Biowissenschaften , Buchmann Institute for Molecular Life Sciences (BMLS) , Goethe Universität Frankfurt , Max-von-Laue-Str. 15, Max-von-Laue-Str. 9 , 60438 Frankfurt am Main , Germany . Email:

Anthraquinones, a widely distributed class of aromatic natural products, are produced by a type II polyketide synthase system in the Gram-negative bacterium . Heterologous expression of the anthraquinone biosynthetic gene cluster in identified AntI as an unusual lyase, catalysing terminal polyketide shortening prior to formation of the third aromatic ring. Functional and analysis of AntI using X-ray crystallography, structure-based mutagenesis, and molecular simulations revealed that AntI converts a defined octaketide to the tricyclic anthraquinone ring retro-Claisen and Dieckmann reactions. Thus, AntI catalyses a so far unobserved multistep reaction in this PKS system.
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http://dx.doi.org/10.1039/c9sc00749kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601290PMC
July 2019

An Uncommon Type II PKS Catalyzes Biosynthesis of Aryl Polyene Pigments.

J Am Chem Soc 2019 10 3;141(42):16615-16623. Epub 2019 Apr 3.

Molekulare Biotechnologie, Fachbereich Biowissenschaften, Goethe-Universität Frankfurt am Main and Buchmann Institute for Molecular Life Sciences (BMLS) , Goethe-Universität Frankfurt , Max-von-Laue-Straße 9 and 15 , 60438 Frankfurt am Main , Germany.

Aryl polyene (APE) pigments are a widely distributed class of bacterial polyketides. So far, little is known about the biosynthesis of these compounds, which are produced by a novel type II polyketide synthase (PKS). We have identified all enzymes involved in APE biosynthesis and determined their peculiar functions. The biosynthesis was reconstituted , and ACP-bound intermediates were assigned for each reaction step by HPLC-MS. Native mass spectrometry experiments identified four stable complexes: the acyl-carrier proteins ApeE and ApeF bound to the thioesterase ApeK, the dehydratases ApeI and ApeP, and the ketosynthase ApeO in complex with its chain-length factor ApeC. X-ray structures of the heterodimeric ApeO:ApeC and ApeI:ApeP complexes depict striking protein-protein interactions. Altogether, our study elucidated mechanistic aspects of APE biosynthesis that unifies elements of type II fatty acid and PKS systems, but in addition includes novel enzyme complexes.
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http://dx.doi.org/10.1021/jacs.8b10776DOI Listing
October 2019

Genetically Encoded Biotin Analogues: Incorporation and Application in Bacterial and Mammalian Cells.

Chembiochem 2019 07 26;20(14):1795-1798. Epub 2019 Jun 26.

Division of Physical Sciences and Engineering, KAUST Catalysis Center (KCC), King Abdullah University of Science and Technology, Thuwal, 23955, Saudi Arabia.

The biotin-streptavidin interaction is among the strongest known in nature. Herein, the site-directed incorporation of biotin and 2-iminobiotin composed of noncanonical amino acids (ncAAs) into proteins is reported. 2-Iminobiotin lysine was employed for protein purification based on the pH-dependent dissociation constant to streptavidin. By using the high-affinity binding of biotin lysine, the bacterial protein RecA could be specifically isolated and its interaction partners analyzed. Furthermore, the biotinylation approach was successfully transferred to mammalian cells. Stringent control over the biotinylation site and the tunable affinity between ncAAs and streptavidin of the different biotin analogues make this approach an attractive tool for protein interaction studies, protein immobilization, and the generation of well-defined protein-drug conjugates.
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http://dx.doi.org/10.1002/cbic.201900015DOI Listing
July 2019

The Co-chaperone Cns1 and the Recruiter Protein Hgh1 Link Hsp90 to Translation Elongation via Chaperoning Elongation Factor 2.

Mol Cell 2019 04 12;74(1):73-87.e8. Epub 2019 Mar 12.

Center for Integrated Protein Science at the Department Chemie, Technische Universität München, Lichtenbergstrasse 4, 85748 Garching, Germany. Electronic address:

The Hsp90 chaperone machinery in eukaryotes comprises a number of distinct accessory factors. Cns1 is one of the few essential co-chaperones in yeast, but its structure and function remained unknown. Here, we report the X-ray structure of the Cns1 fold and NMR studies on the partly disordered, essential segment of the protein. We demonstrate that Cns1 is important for maintaining translation elongation, specifically chaperoning the elongation factor eEF2. In this context, Cns1 interacts with the novel co-factor Hgh1 and forms a quaternary complex together with eEF2 and Hsp90. The in vivo folding and solubility of eEF2 depend on the presence of these proteins. Chaperoning of eEF2 by Cns1 is essential for yeast viability and requires a defined subset of the Hsp90 machinery as well as the identified eEF2 recruiting factor Hgh1.
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http://dx.doi.org/10.1016/j.molcel.2019.02.011DOI Listing
April 2019

Structure-Based Design of Inhibitors Selective for Human Proteasome β2c or β2i Subunits.

J Med Chem 2019 02 5;62(3):1626-1642. Epub 2019 Feb 5.

Gorlaeus Laboratories , Leiden Institute of Chemistry and Netherlands Proteomics Centre , Einsteinweg 55 , 2333 CC Leiden , Netherlands.

Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the β1c, β1i, β5c, and β5i subunits exploit the differences in the substrate-binding channels identified by X-ray crystallography, compounds selectively targeting β2c or β2i could not yet be rationally designed because of the high structural similarity of these two subunits. Here, we report the development, chemical synthesis, and biological screening of a compound library that led to the identification of the β2c- and β2i-selective compounds LU-002c (4; IC β2c: 8 nM, IC β2i/β2c: 40-fold) and LU-002i (5; IC β2i: 220 nM, IC β2c/β2i: 45-fold), respectively. Co-crystal structures with β2 humanized yeast proteasomes visualize protein-ligand interactions crucial for subunit specificity. Altogether, organic syntheses, activity-based protein profiling, yeast mutagenesis, and structural biology allowed us to decipher significant differences of β2 substrate-binding channels and to complete the set of subunit-selective proteasome inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378654PMC
February 2019

Discovery of the First-in-Class Dual Histone Deacetylase-Proteasome Inhibitor.

J Med Chem 2018 11 8;61(22):10299-10309. Epub 2018 Nov 8.

Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Medical Faculty , Leipzig University , Brüderstraße 34 , 04103 Leipzig , Germany.

Dual- or multitarget drugs have emerged as a promising alternative to combination therapies. Proteasome inhibitors (PIs) possess synergistic activity with histone deacetylase (HDAC) inhibitors due to the simultaneous blockage of the ubiquitin degradation and aggresome pathways. Here, we present the design, synthesis, binding modes, and anticancer properties of RTS-V5 as the first-in-class dual HDAC-proteasome ligand. The inhibition of both targets was confirmed by biochemical and cellular assays as well as X-ray crystal structures of the 20S proteasome and HDAC6 complexed with RTS-V5. Cytotoxicity assays with leukemia and multiple myeloma cell lines as well as therapy refractory primary patient-derived leukemia cells demonstrated that RTS-V5 possesses potent and selective anticancer activity. Our results will thus guide the structure-based optimization of dual HDAC-proteasome inhibitors for the treatment of hematological malignancies.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249066PMC
November 2018

Design, synthesis, and evaluation of cystargolide-based β-lactones as potent proteasome inhibitors.

Eur J Med Chem 2018 Sep 20;157:962-977. Epub 2018 Aug 20.

Department of Chemistry, New Mexico Institute of Mining and Technology, Socorro, NM 87801, USA. Electronic address:

The peptidic β-lactone proteasome inhibitors (PIs) cystargolides A and B were used to conduct structure-activity relationship (SAR) studies in order to assess their anticancer potential. A total of 24 different analogs were designed, synthesized and evaluated for proteasome inhibition, for cytotoxicity towards several cancer cell lines, and for their ability to enter intact cells. X-ray crystallographic analysis and subunit selectivity was used to determine the specific subunit binding associated with the structural modification of the β-lactone (P), peptidic core, (P and P), and end-cap (P) of our scaffold. The cystargolide derivative 5k, structurally unique at both P and P, exhibited the most promising inhibitory activity for the β5 subunit of human proteasomes (IC = 3.1 nM) and significant cytotoxicity towards MCF-7 (IC = 416 nM), MDA-MB-231 (IC = 74 nM) and RPMI 8226 (IC = 41 nM) cancer cell lines. Cellular infiltration assays revealed that minor structural modifications have significant effects on the ability of our PIs to inhibit intracellular proteasomes, and we identified 5k as a promising candidate for continued therapeutic studies. Our novel drug lead 5k is a more potent proteasome inhibitor than carfilzomib with mid-to-low nanomolar IC measurements and it is cytotoxic against multiple cancer cell lines at levels approaching those of carfilzomib.
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http://dx.doi.org/10.1016/j.ejmech.2018.08.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168434PMC
September 2018

Structural and Mechanistic Features of ClyA-Like α-Pore-Forming Toxins.

Toxins (Basel) 2018 08 23;10(9). Epub 2018 Aug 23.

Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany.

Recent technological advances have seen increasing numbers of complex structures from diverse pore-forming toxins (PFT). The ClyA family of α-PFTs comprises a broad variety of assemblies including single-, two- and three-component toxin systems. With crystal structures available for soluble subunits of all major groups in this extended protein family, efforts now focus on obtaining molecular insights into physiological pore formation. This review provides an up-to-date discussion on common and divergent structural and functional traits that distinguish the various ClyA family PFTs. Open questions of this research topic are outlined and discussed.
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http://dx.doi.org/10.3390/toxins10090343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162564PMC
August 2018

Iron Scavenging in Aspergillus Species: Structural and Biochemical Insights into Fungal Siderophore Esterases.

Angew Chem Int Ed Engl 2018 10 27;57(44):14624-14629. Epub 2018 Aug 27.

Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Technische Universität München, Lichtenbergstraße 4, 85748, Garching, Germany.

Fungi utilize high-affinity chelators termed siderophores with chemically diverse structures to scavenge the essential nutrient iron from their surroundings. Since they are among the strongest known Fe binding agents, intracellular release of the heavy metal atom is facilitated by the activity of specific hydrolases. In this work, we report the characterization and X-ray crystal structures of four siderophore esterases: AfEstB and AfSidJ from Aspergillus fumigatus, as well as AnEstB and AnEstA from Aspergillus nidulans. Even though they all display the conserved α/β-hydrolase fold, we found significant structural and enzymatic discrepancies in their adaption to both related and chemically diverse substrates. A structure of AfEstB in complex with its substrate triacetylfusarinine C gives insight into the active enzyme and shows tetrahedral coordination between the catalytic serine and the scissile ester bond.
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http://dx.doi.org/10.1002/anie.201807093DOI Listing
October 2018

(-)-Homosalinosporamide A and Its Mode of Proteasome Inhibition: An X-ray Crystallographic Study.

Mar Drugs 2018 Jul 19;16(7). Epub 2018 Jul 19.

Department of Chemistry, Texas A&M University, College Station, TX 77843, USA.

Upon acylation of the proteasome by the β-lactone inhibitor salinosporamide A (SalA), tetrahydrofuran formation occurs by intramolecular alkylation of the incipient alkoxide onto the choroethyl sidechain and irreversibly blocks the active site. Our previously described synthetic approach to SalA, utilizing a bioinspired, late-stage, aldol-β-lactonization strategy to construct the bicyclic β-lactone core, enabled synthesis of (⁻)-homosalinosporamide A (homoSalA). This homolog was targeted to determine whether an intramolecular tetrahydropyran is formed in a similar manner to SalA. Herein, we report the X-ray structure of the yeast 20S proteasome:homoSalA-complex which reveals that tetrahydropyran ring formation does not occur despite comparable potency at the chymotrypsin-like active site in a luminogenic enzyme assay. Thus, the natural product derivative homoSalA blocks the proteasome by a covalent reversible mode of action, opening the door for further fine-tuning of proteasome inhibition.
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http://dx.doi.org/10.3390/md16070240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071143PMC
July 2018

Impacts of dam draining on the mobility of heavy metals and arsenic in water and basin bottom sediments of three studied dams in Germany.

Sci Total Environ 2018 Nov 6;640-641:1072-1081. Epub 2018 Jun 6.

Philipps-University of Marburg, Faculty of Pharmacy, Mass Spectrometry, Marbacher Weg 6-10, 35037 Marburg, Germany. Electronic address:

The draining of a dam is a relatively rare event, however, it can have severe consequences for a watercourse connected to that reservoir. In order to understand the effects of the draining on the mobility of pollutants stored in the bottom sediments, the concentrations of heavy metals and arsenic were measured in pore water, river water, and sediments sampled from three emptied reservoirs in Germany. Two of these sites were analyzed immediately after the draining, while the third reservoir was studied one and a half years after the complete discharge of the stored water. Heavy metal and arsenic concentrations within the sediments varied among the studied dams as a result of different geological characteristics and the degree of anthropogenic impacts. Based on the analysis of pore water samples, the concentrations of heavy metals and arsenic were not significantly altered shortly after the draining. However, increased concentrations of As, Fe, Cd, Ni, and Zn were measured in pore water samples after a longer duration of sediment exposure, which altered the redox conditions and sediment properties. The changes in Cu, Cr, Mn, and Pb concentrations in pore water samples were less pronounced. As a result of the pore water drainage increased dissolved heavy metal concentrations were found in the discharge water immediately after draining. At the third site, which had been emptied for one and a half years, only for Mn, a concentration increase was detected in the reservoir discharge water, which emphasizes the strong temporal dynamic of the mobilization of the analyzed elements.
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http://dx.doi.org/10.1016/j.scitotenv.2018.05.295DOI Listing
November 2018

Structure and mechanism of the two-component α-helical pore-forming toxin YaxAB.

Nat Commun 2018 05 4;9(1):1806. Epub 2018 May 4.

Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Chair of Biochemistry, Technische Universität München, Lichtenbergstrasse 4, 85747, Garching, Germany.

Pore-forming toxins (PFT) are virulence factors that transform from soluble to membrane-bound states. The Yersinia YaxAB system represents a family of binary α-PFTs with orthologues in human, insect, and plant pathogens, with unknown structures. YaxAB was shown to be cytotoxic and likely involved in pathogenesis, though the molecular basis for its two-component lytic mechanism remains elusive. Here, we present crystal structures of YaxA and YaxB, together with a cryo-electron microscopy map of the YaxAB complex. Our structures reveal a pore predominantly composed of decamers of YaxA-YaxB heterodimers. Both subunits bear membrane-active moieties, but only YaxA is capable of binding to membranes by itself. YaxB can subsequently be recruited to membrane-associated YaxA and induced to present its lytic transmembrane helices. Pore formation can progress by further oligomerization of YaxA-YaxB dimers. Our results allow for a comparison between pore assemblies belonging to the wider ClyA-like family of α-PFTs, highlighting diverse pore architectures.
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http://dx.doi.org/10.1038/s41467-018-04139-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935710PMC
May 2018

Defective immuno- and thymoproteasome assembly causes severe immunodeficiency.

Sci Rep 2018 04 13;8(1):5975. Epub 2018 Apr 13.

Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Trogerstr. 30, 81675, Munich, Germany.

By N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated the mutant mouse line TUB6 that is characterised by severe combined immunodeficiency (SCID) and systemic sterile autoinflammation in homozygotes, and a selective T cell defect in heterozygotes. The causative missense point mutation results in the single amino acid exchange G170W in multicatalytic endopeptidase complex subunit-1 (MECL-1), the β2i-subunit of the immuno- and thymoproteasome. Yeast mutagenesis and crystallographic data suggest that the severe TUB6-phenotype compared to the MECL-1 knockout mouse is caused by structural changes in the C-terminal appendage of β2i that prevent the biogenesis of immuno- and thymoproteasomes. Proteasomes are essential for cell survival, and defective proteasome assembly causes selective death of cells expressing the mutant MECL-1, leading to the severe immunological phenotype. In contrast to the immunosubunits β1i (LMP2) and β5i (LMP7), mutations in the gene encoding MECL-1 have not yet been assigned to human disorders. The TUB6 mutant mouse line exemplifies the involvement of MECL-1 in immunopathogenesis and provides the first mouse model for primary immuno- and thymoproteasome-associated immunodeficiency that may also be relevant in humans.
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http://dx.doi.org/10.1038/s41598-018-24199-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899138PMC
April 2018

Catalytic mechanism and molecular engineering of quinolone biosynthesis in dioxygenase AsqJ.

Nat Commun 2018 03 21;9(1):1168. Epub 2018 Mar 21.

Center for Integrated Protein Science Munich (CIPSM), Department Chemie, Technische Universität München, Lichtenbergstraße 4, 85748, Garching, Germany.

The recently discovered Fe/α-ketoglutarate-dependent dioxygenase AsqJ from Aspergillus nidulans stereoselectively catalyzes a multistep synthesis of quinolone alkaloids, natural products with significant biomedical applications. To probe molecular mechanisms of this elusive catalytic process, we combine here multi-scale quantum and classical molecular simulations with X-ray crystallography, and in vitro biochemical activity studies. We discover that methylation of the substrate is essential for the activity of AsqJ, establishing molecular strain that fine-tunes π-stacking interactions within the active site. To rationally engineer AsqJ for modified substrates, we amplify dispersive interactions within the active site. We demonstrate that the engineered enzyme has a drastically enhanced catalytic activity for non-methylated surrogates, confirming our computational data and resolved high-resolution X-ray structures at 1.55 Å resolution. Our combined findings provide crucial mechanistic understanding of the function of AsqJ and showcase how combination of computational and experimental data enables to rationally engineer enzymes.
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http://dx.doi.org/10.1038/s41467-018-03442-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862883PMC
March 2018

A Predictive Approach for the Optical Control of Carbonic Anhydrase II Activity.

ACS Chem Biol 2018 03 9;13(3):793-800. Epub 2018 Feb 9.

Department of Chemistry , Ludwig-Maximilian-University Munich and Munich Center for Integrated Protein Science (CIPSM) , Butenandtstrasse 5-13 , 83177 Munich , Germany.

Optogenetics and photopharmacology are powerful approaches to investigating biochemical systems. While the former is based on genetically encoded photoreceptors that utilize abundant chromophores, the latter relies on synthetic photoswitches that are either freely diffusible or covalently attached to specific bioconjugation sites, which are often native or engineered cysteines. The identification of suitable cysteine sites and appropriate linkers for attachment is generally a lengthy and cumbersome process. Herein, we describe an in silico screening approach that is designed to propose a small number of optimal combinations. By applying this computational approach to human carbonic anhydrase and a set of three photochromic tethered ligands, the number of potential site-ligand combinations was narrowed from over 750 down to 6, which we then evaluated experimentally. Two of these six combinations resulted in light-responsive human Carbonic Anhydrases (LihCAs), which were characterized with enzymatic activity assays, mass spectrometry, and X-ray crystallography. Our study also provides insights into the reactivity of cysteines toward maleimides and the hydrolytic stability of the adducts obtained.
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http://dx.doi.org/10.1021/acschembio.7b00862DOI Listing
March 2018

On the Trails of the Proteasome Fold: Structural and Functional Analysis of the Ancestral β-Subunit Protein Anbu.

J Mol Biol 2018 03;430(5):628-640

Center for Integrated Protein Science Munich at the Department Chemie, Lehrstuhl für Biochemie, Technische Universität München, Garching, Germany. Electronic address:

The 20S proteasome is a key player in eukaryotic and archaeal protein degradation, but its progenitor in eubacteria is unknown. Recently, the ancestral β-subunit protein (Anbu) was predicted to be the evolutionary precursor of the proteasome. We crystallized Anbu from Hyphomicrobium sp. strain MC1 in four different space groups and solved the structures by SAD-phasing and Patterson search calculation techniques. Our data reveal that Anbu adopts the classical fold of Ntn-hydrolases, but its oligomeric state differs from that of barrel-shaped proteases. In contrast to their typical architecture, the Anbu protomer is a tightly interacting dimer that can assemble into a helical superstructure. Although Anbu features a catalytic triad of Thr1O, Asp17O and Lys32N, it is unable to hydrolyze standard protease substrates. The lack of activity might be caused by the incapacity of Thr1NH to function as a Brønsted acid during substrate cleavage due to its missing activation via hydrogen bonding. Altogether, we demonstrate that the topology of the proteasomal fold is conserved in Anbu, but whether it acts as a protease still needs to be clarified.
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http://dx.doi.org/10.1016/j.jmb.2018.01.004DOI Listing
March 2018

The protease GtgE from Salmonella exclusively targets inactive Rab GTPases.

Nat Commun 2018 01 3;9(1):44. Epub 2018 Jan 3.

Center for Integrated Protein Science Munich (CIPSM), Department Chemistry, Technical University of Munich, Lichtenbergstrasse 4, 85747, Garching, Germany.

Salmonella infections require the delivery of bacterial effectors into the host cell that alter the regulation of host defense mechanisms. The secreted cysteine protease GtgE from S. Typhimurium manipulates vesicular trafficking by modifying the Rab32 subfamily via cleaving the regulatory switch I region. Here we present a comprehensive biochemical, structural, and computational characterization of GtgE in complex with Rab32. Interestingly, GtgE solely processes the inactive GDP-bound GTPase. The crystal structure of the Rab32:GDP substrate in complex with the inactive mutant GtgE reveals the molecular basis of substrate recognition. In combination with atomistic molecular dynamics simulations, the structural determinants for protein and activity-state specificity are identified. Mutations in a central interaction hub lead to loss of the strict GDP specificity. Our findings shed light on the sequence of host cell manipulation events during Salmonella infection and provide an explanation for the dependence on the co-secreted GTPase activating protein SopD2.
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http://dx.doi.org/10.1038/s41467-017-02110-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752668PMC
January 2018