@article{AtakLanglhoferSchaeferetal.2015, author = {Atak, Sinem and Langlhofer, Georg and Schaefer, Natascha and Kessler, Denise and Meiselbach, Heike and Delto, Carolyn and Schindelin, Hermann and Villmann, Carmen}, title = {Disturbances of ligand potency and enhanced degradation of the human glycine receptor at affected positions G160 and T162 originally identified in patients suffering from hyperekplexia}, series = {Frontiers in Molecular Neuroscience}, volume = {8}, journal = {Frontiers in Molecular Neuroscience}, number = {79}, doi = {10.3389/fnmol.2015.00079}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144818}, year = {2015}, abstract = {Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GIyR) undergoes direct interaction with the incoming ligand via a cation-π interaction. Recently, we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GIyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GIyR α1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 α1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER toward the ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is delivered to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, 1162 affects primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof.}, language = {en} } @article{BotheHaenzelmannBoehleretal.2022, author = {Bothe, Sebastian and H{\"a}nzelmann, Petra and B{\"o}hler, Stephan and Kehrein, Josef and Zehe, Markus and Wiedemann, Christoph and Hellmich, Ute A. and Brenk, Ruth and Schindelin, Hermann and Sotriffer, Christoph}, title = {Fragment screening using biolayer interferometry reveals ligands targeting the SHP-motif binding site of the AAA+ ATPase p97}, series = {Communications Chemistry}, volume = {5}, journal = {Communications Chemistry}, number = {1}, doi = {10.1038/s42004-022-00782-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-300821}, year = {2022}, abstract = {Biosensor techniques have become increasingly important for fragment-based drug discovery during the last years. The AAA+ ATPase p97 is an essential protein with key roles in protein homeostasis and a possible target for cancer chemotherapy. Currently available p97 inhibitors address its ATPase activity and globally impair p97-mediated processes. In contrast, inhibition of cofactor binding to the N-domain by a protein-protein-interaction inhibitor would enable the selective targeting of specific p97 functions. Here, we describe a biolayer interferometry-based fragment screen targeting the N-domain of p97 and demonstrate that a region known as SHP-motif binding site can be targeted with small molecules. Guided by molecular dynamics simulations, the binding sites of selected screening hits were postulated and experimentally validated using protein- and ligand-based NMR techniques, as well as X-ray crystallography, ultimately resulting in the first structure of a small molecule in complex with the N-domain of p97. The identified fragments provide insights into how this region could be targeted and present first chemical starting points for the development of a protein-protein interaction inhibitor preventing the binding of selected cofactors to p97.}, language = {en} } @unpublished{BrennerZinkWitzingeretal.2024, author = {Brenner, Marian and Zink, Christoph and Witzinger, Linda and Keller, Angelika and Hadamek, Kerstin and Bothe, Sebastian and Neuenschwander, Martin and Villmann, Carmen and von Kries, Jens Peter and Schindelin, Hermann and Jeanclos, Elisabeth and Gohla, Antje}, title = {7,8-Dihydroxyflavone is a direct inhibitor of pyridoxal phosphatase}, series = {eLife}, journal = {eLife}, doi = {10.7554/eLife.93094.2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350446}, year = {2024}, abstract = {Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5'-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small molecule screening, protein crystallography and biolayer interferometry, we discover and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.}, language = {en} } @article{DieboldSchoenemannEilersetal.2023, author = {Diebold, Mathias and Sch{\"o}nemann, Lars and Eilers, Martin and Sotriffer, Christoph and Schindelin, Hermann}, title = {Crystal structure of a covalently linked Aurora-A-MYCN complex}, series = {Acta Crystallographica}, volume = {D79}, journal = {Acta Crystallographica}, doi = {10.1107/s2059798322011433}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-318855}, pages = {1 -- 9}, year = {2023}, abstract = {Formation of the Aurora-A-MYCN complex increases levels of the oncogenic transcription factor MYCN in neuroblastoma cells by abrogating its degradation through the ubiquitin proteasome system. While some small-molecule inhibitors of Aurora-A were shown to destabilize MYCN, clinical trials have not been satisfactory to date. MYCN itself is considered to be `undruggable' due to its large intrinsically disordered regions. Targeting the Aurora-A-MYCN complex rather than Aurora-A or MYCN alone will open new possibilities for drug development and screening campaigns. To overcome the challenges that a ternary system composed of Aurora-A, MYCN and a small molecule entails, a covalently cross-linked construct of the Aurora-A-MYCN complex was designed, expressed and characterized, thus enabling screening and design campaigns to identify selective binders.}, language = {en} } @article{DietschreitWagnerLeetal.2020, author = {Dietschreit, Johannes C. B. and Wagner, Annika and Le, T. Anh and Klein, Philipp and Schindelin, Hermann and Opatz, Till and Engels, Bernd and Hellmich, Ute A. and Ochsenfeld, Christian}, title = {Predicting \(^{19}\)F NMR Chemical Shifts: A Combined Computational and Experimental Study of a Trypanosomal Oxidoreductase-Inhibitor Complex}, series = {Angewandte Chemie International Edition}, volume = {59}, journal = {Angewandte Chemie International Edition}, number = {31}, doi = {10.1002/anie.202000539}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-214879}, pages = {12669 -- 12673}, year = {2020}, abstract = {The absence of fluorine from most biomolecules renders it an excellent probe for NMR spectroscopy to monitor inhibitor-protein interactions. However, predicting the binding mode of a fluorinated ligand from a chemical shift (or vice versa) has been challenging due to the high electron density of the fluorine atom. Nonetheless, reliable \(^{19}\)F chemical-shift predictions to deduce ligand-binding modes hold great potential for in silico drug design. Herein, we present a systematic QM/MM study to predict the \(^{19}\)F NMR chemical shifts of a covalently bound fluorinated inhibitor to the essential oxidoreductase tryparedoxin (Tpx) from African trypanosomes, the causative agent of African sleeping sickness. We include many protein-inhibitor conformations as well as monomeric and dimeric inhibitor-protein complexes, thus rendering it the largest computational study on chemical shifts of \(^{19}\)F nuclei in a biological context to date. Our predicted shifts agree well with those obtained experimentally and pave the way for future work in this area.}, language = {en} } @article{EisenbergAlbertTeuffeletal.2022, author = {Eisenberg, Philip and Albert, Leon and Teuffel, Jonathan and Zitzow, Eric and Michaelis, Claudia and Jarick, Jane and Sehlke, Clemens and Große, Lisa and Bader, Nicole and Nunes-Alves, Ariane and Kreikemeyer, Bernd and Schindelin, Hermann and Wade, Rebecca C. and Fiedler, Tomas}, title = {The Non-phosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase GapN Is a Potential New Drug Target in Streptococcus pyogenes}, series = {Frontiers in Microbiology}, volume = {13}, journal = {Frontiers in Microbiology}, issn = {1664-302X}, doi = {10.3389/fmicb.2022.802427}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-262869}, year = {2022}, abstract = {The strict human pathogen Streptococcus pyogenes causes infections of varying severity, ranging from self-limiting suppurative infections to life-threatening diseases like necrotizing fasciitis or streptococcal toxic shock syndrome. Here, we show that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase GapN is an essential enzyme for S. pyogenes. GapN converts glyceraldehyde 3-phosphate into 3-phosphoglycerate coupled to the reduction of NADP to NADPH. The knock-down of gapN by antisense peptide nucleic acids (asPNA) significantly reduces viable bacterial counts of S. pyogenes laboratory and macrolide-resistant clinical strains in vitro. As S. pyogenes lacks the oxidative part of the pentose phosphate pathway, GapN appears to be the major NADPH source for the bacterium. Accordingly, other streptococci that carry a complete pentose phosphate pathway are not prone to asPNA-based gapN knock-down. Determination of the crystal structure of the S. pyogenes GapN apo-enzyme revealed an unusual cis-peptide in proximity to the catalytic binding site. Furthermore, using a structural modeling approach, we correctly predicted competitive inhibition of S. pyogenes GapN by erythrose 4-phosphate, indicating that our structural model can be used for in silico screening of specific GapN inhibitors. In conclusion, the data provided here reveal that GapN is a potential target for antimicrobial substances that selectively kill S. pyogenes and other streptococci that lack the oxidative part of the pentose phosphate pathway.}, language = {en} } @article{ImamChoudhuryHeinzeetal.2022, author = {Imam, Nasir and Choudhury, Susobhan and Heinze, Katrin G. and Schindelin, Hermann}, title = {Differential modulation of collybistin conformational dynamics by the closely related GTPases Cdc42 and TC10}, series = {Frontiers in Synaptic Neuroscience}, volume = {14}, journal = {Frontiers in Synaptic Neuroscience}, issn = {1663-3563}, doi = {10.3389/fnsyn.2022.959875}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-282816}, year = {2022}, abstract = {Interneuronal synaptic transmission relies on the proper spatial organization of presynaptic neurotransmitter release and its reception on the postsynaptic side by cognate neurotransmitter receptors. Neurotransmitter receptors are incorporated into and arranged within the plasma membrane with the assistance of scaffolding and adaptor proteins. At inhibitory GABAergic postsynapses, collybistin, a neuronal adaptor protein, recruits the scaffolding protein gephyrin and interacts with various neuronal factors including cell adhesion proteins of the neuroligin family, the GABAA receptor α2-subunit and the closely related small GTPases Cdc42 and TC10 (RhoQ). Most collybistin splice variants harbor an N-terminal SH3 domain and exist in an autoinhibited/closed state. Cdc42 and TC10, despite sharing 67.4\% amino acid sequence identity, interact differently with collybistin. Here, we delineate the molecular basis of the collybistin conformational activation induced by TC10 with the aid of recently developed collybistin FRET sensors. Time-resolved fluorescence-based FRET measurements reveal that TC10 binds to closed/inactive collybistin leading to relief of its autoinhibition, contrary to Cdc42, which only interacts with collybistin when forced into an open state by the introduction of mutations destabilizing the closed state of collybistin. Taken together, our data describe a TC10-driven signaling mechanism in which collybistin switches from its autoinhibited closed state to an open/active state.}, language = {en} } @article{JeanclosKnoblochHoffmannetal.2020, author = {Jeanclos, Elisabeth and Knobloch, Gunnar and Hoffmann, Axel and Fedorchenko, Oleg and Odersky, Andrea and Lamprecht, Anna-Karina and Schindelin, Hermann and Gohla, Antje}, title = {Ca\(^{2+}\) functions as a molecular switch that controls the mutually exclusive complex formation of pyridoxal phosphatase with CIB1 or calmodulin}, series = {FEBS Letters}, volume = {594}, journal = {FEBS Letters}, number = {13}, doi = {10.1002/1873-3468.13795}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-217963}, pages = {2099 -- 2115}, year = {2020}, abstract = {Pyridoxal 5′-phosphate (PLP) is an essential cofactor for neurotransmitter metabolism. Pyridoxal phosphatase (PDXP) deficiency in mice increases PLP and γ-aminobutyric acid levels in the brain, yet how PDXP is regulated is unclear. Here, we identify the Ca\(^{2+}\)- and integrin-binding protein 1 (CIB1) as a PDXP interactor by yeast two-hybrid screening and find a calmodulin (CaM)-binding motif that overlaps with the PDXP-CIB1 interaction site. Pulldown and crosslinking assays with purified proteins demonstrate that PDXP directly binds to CIB1 or CaM. CIB1 or CaM does not alter PDXP phosphatase activity. However, elevated Ca\(^{2+}\) concentrations promote CaM binding and, thereby, diminish CIB1 binding to PDXP, as both interactors bind in a mutually exclusive way. Hence, the PDXP-CIB1 complex may functionally differ from the PDXP-Ca\(^{2+}\)-CaM complex.}, language = {en} } @article{JeanclosSchloetzerHadameketal.2022, author = {Jeanclos, Elisabeth and Schl{\"o}tzer, Jan and Hadamek, Kerstin and Yuan-Chen, Natalia and Alwahsh, Mohammad and Hollmann, Robert and Fratz, Stefanie and Yesilyurt-Gerhards, Dilan and Frankenbach, Tina and Engelmann, Daria and Keller, Angelika and Kaestner, Alexandra and Schmitz, Werner and Neuenschwander, Martin and Hergenr{\"o}der, Roland and Sotriffer, Christoph and von Kries, Jens Peter and Schindelin, Hermann and Gohla, Antje}, title = {Glycolytic flux control by drugging phosphoglycolate phosphatase}, series = {Nature Communications}, volume = {13}, journal = {Nature Communications}, number = {1}, doi = {10.1038/s41467-022-34228-2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-300928}, year = {2022}, abstract = {Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates.}, language = {en} } @article{KasaragodSchindelin2019, author = {Kasaragod, Vikram Babu and Schindelin, Hermann}, title = {Structure of Heteropentameric GABAA Receptors and Receptor-Anchoring Properties of Gephyrin}, series = {Frontiers in Molecular Neuroscience}, volume = {12}, journal = {Frontiers in Molecular Neuroscience}, issn = {1662-5099}, doi = {10.3389/fnmol.2019.00191}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-189308}, pages = {191}, year = {2019}, abstract = {γ-Aminobutyric acid type A receptors (GABAARs) mediate the majority of fast synaptic inhibition in the central nervous system (CNS). GABAARs belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGIC) and are assembled from 19 different subunits. As dysfunctional GABAergic neurotransmission manifests itself in neurodevelopmental disorders including epilepsy and anxiety, GABAARs are key drug targets. The majority of synaptic GABAARs are anchored at the inhibitory postsynaptic membrane by the principal scaffolding protein gephyrin, which acts as the central organizer in maintaining the architecture of the inhibitory postsynaptic density (iPSD). This interaction is mediated by the long intracellular loop located in between transmembrane helices 3 and 4 (M3-M4 loop) of the receptors and a universal receptor-binding pocket residing in the C-terminal domain of gephyrin. In 2014, the crystal structure of the β3-homopentameric GABAAR provided crucial information regarding the architecture of the receptor; however, an understanding of the structure and assembly of heteropentameric receptors at the atomic level was lacking. This review article will highlight recent advances in understanding the structure of heteropentameric synaptic GABAARs and how these structures have provided fundamental insights into the assembly of these multi-subunit receptors as well as their modulation by diverse ligands including the physiological agonist GABA. We will further discuss the role of gephyrin in the anchoring of synaptic GABAARs and glycine receptors (GlyRs), which are crucial for maintaining the architecture of the iPSD. Finally, we will also summarize how anti-malarial artemisinin drugs modulate gephyrin-mediated inhibitory neurotransmission.}, language = {en} }