Modulation of the Allosteric Communication between the Polo-Box Domain and the Catalytic Domain in Plk1 by Small Compounds

The Polo-like kinases (Plks) are an evolutionary conserved family of Ser/Thr protein kinases that possess, in addition to the classical kinase domain at the N-terminus, a C-terminal polo-box domain (PBD) that binds to phosphorylated proteins and modulates the kinase activity and its localization. Plk1, which regulates the formation of the mitotic spindle, has emerged as a validated drug target for the treatment of cancer, because it is required for numerous types of cancer cells but not for the cell division in noncancer cells. Here, we employed chemical biology methods to investigate the allosteric communication between the PBD and the catalytic domain of Plk1. We identified small compounds that bind to the catalytic domain and inhibit or enhance the interaction of Plk1 with the phosphorylated peptide PoloBoxtide in vitro. In cells, two new allosteric Plk1 inhibitors affected the proliferation of cancer cells in culture and the cell cycle but had distinct phenotypic effects on spindle formation. Both compounds inhibited Plk1 signaling, indicating that they specifically act on Plk1 in cultured cells.Fil: Raab, Monika. Goethe Universitat Frankfurt; AlemaniaFil: Sanhaji, Mourad. Goethe Universitat Frankfurt; AlemaniaFil: Pietsch, Larissa. German Cancer Research Center; Alemania. Goethe Universitat Frankfurt; AlemaniaFil: Béquignon, Isabelle. Goethe Universitat Frankfurt; AlemaniaFil: Herbrand, Amanda K.. Goethe Universitat Frankfurt; AlemaniaFil: Süß, Evelyn. Goethe Universitat Frankfurt; AlemaniaFil: Gande, Santosh L.. German Cancer Research Center; Alemania. Goethe Universitat Frankfurt; AlemaniaFil: Caspar, Birgit. Goethe Universitat Frankfurt; AlemaniaFil: Kudlinzki, Denis. Goethe Universitat Frankfurt; Alemania. German Cancer Research Center; AlemaniaFil: Saxena, Krishna. Goethe Universitat Frankfurt; AlemaniaFil: Sreeramulu, Sridhar. Goethe Universitat Frankfurt; AlemaniaFil: Schwalbe, Harald. Goethe Universitat Frankfurt; Alemania. German Cancer Research Center; AlemaniaFil: Strebhardt, Klaus. Goethe Universitat Frankfurt; Alemania. German Cancer Research Center; AlemaniaFil: Biondi, Ricardo Miguel. German Cancer Research Center; Alemania. Goethe Universitat Frankfurt; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentin

Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development

12 pags., 4 figs., 3 tabs.SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG through CRC902: “Molecular Principles of RNA-based regulation.” and through infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Union’s Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement number 101046203. “INSPIRED” (MIS 5002550) project, implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950—“SEE-DRUG” project (purchase of UPAT’s 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020—Grant Agreement Number 101017536. Open Access funding enabled and organized by Projekt DEALPeer reviewe

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form

Mechanismus der retention von pFGE und FGE im Endoplasmatischen Reticulum

Das Formylglycin bildende Enzym FGE, codiert durch das Gen SUMF1 (Sulfatase modifying factor1), verursacht in Sulfatasen eine neuartige co-translationale Modifikation eines spezifischen Cysteinrestes zu Formylglycin. Mutationen im FGE-codierenden Gen sind die Ursache der Multiplen Sulfatase- Defizienz, bei welcher die enzymatischen Aktivitäten sämtlicher Sulfatasen drastisch reduziert sind. Im humanen Genom wurde ein weiteres Gen identifiziert, welches eine starke Sequenzhomlogie zu FGE aufweist. Es wird als SUMF2 und das Protein als paraloges FGE (pFGE) bezeichnet, wobei die genaue Funktion dieses Proteins noch immer unbekannt ist. Die topologische Verteilung beider Proteine lässt auf eine ER(Endoplasmatischen Reticulum)-Lokalisation schließen, wobei ihnen in den meisten Spezies jedoch ein klassisches ER-Retentionssignal fehlt. Die vorliegende Studie konzentrierte sich auf die Identifikation des ER-Retentionsmechanismus von pFGE und FGE. Es konnte gezeigt werden, dass der Retentionsmechanimus von pFGE ein sättigbarer Prozess ist, bei welchem durch die Gegenwart eines C-terminalen Tags die Retention des Proteins behindert werden konnte. Weitergehende Untersuchungen führten zur Identifikation des Tetrapeptides PGEL als mutmaßliche C-terminale Determinate für die Retention von pFGE. Die Trunkierung der Sequenz PGEL führte zu einer drastisch verminderten Retention von pFGE. Das Einfügen der PGEL-Sequenz an den C-terminalen Teil des Enzyms Lysozym führte zu einer Retention des normalerweise sekretierten Porteins. Diese Beobachtungen zeigen, dass es sich bei PGEL um eine autonome Retentionssignal-Sequenz handelt.Die Retention von FGE im ER ist ebenfalls ein sättigbarer Mechanismus, allerdings besitzt FGE keine C-terminale Determinante, welche die Lokalisation im ER bestimmen könnte. Die starke Sequenzhomologie, die Lokalisation und die strukturellen Ähnlichkeiten zu pFGE führten zu weitergehenden Studien über eine mögliche Interaktion beider Proteine, durch die dann die ER-Retention von FGE vermittelt werden könnte. Obwohl Yeast-two-hybride-Tests eine Interaktion zwischen pFGE und FGE zeigten, konnte kein Einfluss auf die Retention von FGE festgestellt werden. Weiterführende biochemische Untersuchungen zur Indentifikation möglicher Interaktionsparter, die eine Retention von FGE im ER vermitteln könnten, lieferten keine adäquaten Kandidatenproteine. Schließlich wurde der aminoterminale Teil von FGE genauer untersucht. Hierbei konnte eine drastische Verminderung der Retention und der biologischen Aktivität (ohne dabei die katalytische in vitro-Aktivität signifikant zu beeinflussen) festgestellt werden, wenn die FGE-Aminosäurereste 34-69 trunkiert wurden. Diese Beobachtungen lassen darauf schließen dass der aminoterminale Teil von FGE eine Rolle bei der Vermittlung der ER-Retention von FGE spielt.Zusammenfassend kann man sagen, dass die Retention von pFGE durch sein C-terminales Tetrapeptid PGEL vermittelt wird, welches notwendig und ausreichend ist für die Retention im ER. Die Retention des FGE Retention dagegen wird durch den aminoterminalen Teil des FGE-Proteins vermittelt.The formylglycine generating enzyme FGE, encoded by SUMF1 gene (Sulfatase modifying factor 1) performs a novel co-translational modification of a specific cysteine residue to formylglycine in sulfatases. Mutations in the gene encoding FGE is the causes for multiple sulfatase deficiency disease in which the activity of all sulfatases is severely lowered. In the human genome, another gene that shares high sequence homology to FGE has been identified. It is designated as SUMF2 and the protein as paralog of FGE, pFGE, whose function is still ill- defined. Topological distribution of these proteins revealed them as ER(Endoplasmic reticulum)-resident proteins but in most species they lack the conventional ER retention signals. This study was focused on identifying the retention mechanism of pFGE and FGE. We have found that retention of pFGE occurs by a saturable mechanism and the presence of a C-terminal tag hinders the retention of pFGE. This observation led us to identify PGEL tetrapeptide as the putative C-terminal determinant of pFGE and truncation of PGEL lowers the retention of pFGE significantly. Further we found that PGEL when tagged to C-terminus of lysozyme, a secretory protein, could be retained, suggesting that PGEL is an autonomous retention signal sequence.FGE retention in ER is carried out by a saturable mechanism but it has no C-terminal determinant that could govern its localization to ER. Its high sequence homology, localization, and structural similarities to pFGE led us to study whether their interaction could be the means for the retention of FGE in the ER. Although, by yeast two-hybrid assay we found that pFGE interacts with FGE, it showed no influence on FGE retention. Biochemical approaches for identifying interacting proteins of FGE that may suggest for its retention were unsuccessful in obtaining appropriate candidate proteins. Finally we checked the amino terminus of FGE and we found that amino-terminal truncation of 34-69 residues in FGE lowers its retention and in vivo biological activity without effecting its in vitro catalytic activity significantly, suggesting that the amino-terminus of FGE might have some role in mediating its retention.In summary pFGE retention is mediated by its C-terminal tetrapeptide PGEL, which is necessary and sufficient to mediate retention in ER. While, FGE retention could be mediated by its amino-terminus residues

Design and isolation of temperature-sensitive mutants of Gal4 in yeast and Drosophila

Little is known about mechanisms responsible for the temperature-sensitive (ts) phenotype, or of the transferability of ts mutants of a specific gene between organisms. Using a structure-based approach, nine ts mutants of Gal4 were generated in yeast by mutating four DNA binding residues. Two of these nine yeast ts mutants were cloned into P element vectors under control of the Elav and GMR promoters and transgenic Drosophila lines were generated. These were crossed to UAS reporter lines and progeny were characterized for reporter gene expression as a function of temperature. Both of these yeast ts mutants show a ts phenotype in Drosophila and result in rapid induction of reporter gene expression upon shifting to the permissive temperature. Exposed, functional residues involved in protein-ligand or protein-protein interactions appear to be attractive candidate sites for generating ts mutants that are transferable between organisms

Light dynamics of the retinal-disease-relevant G90D bovine rhodopsin mutant

The RHO gene encodes the G-protein-coupled receptor (GPCR) rhodopsin. Numerous mutations associated with impaired visual cycle have been reported; the G90D mutation leads to a constitutively active mutant form of rhodopsin that causes CSNB disease. We report on the structural investigation of the retinal configuration and conformation in the binding pocket in the dark and light-activated state by solution and MAS-NMR spectroscopy. We found two long-lived dark states for the G90D mutant with the 11-cis retinal bound as Schiff base in both populations. The second minor population in the dark state is attributed to a slight shift in conformation of the covalently bound 11-cis retinal caused by the mutation-induced distortion on the salt bridge formation in the binding pocket. Time-resolved UV/Vis spectroscopy was used to monitor the functional dynamics of the G90D mutant rhodopsin for all relevant time scales of the photocycle. The G90D mutant retains its conformational heterogeneity during the photocycle

Design and Isolation of Temperature-sensitive Mutants of Gal4 in Yeast and Drosophila

Little is known about mechanisms responsible for the temperature-sensitive (ts) phenotype, or of the transferability of ts mutants of a specific gene between organisms. Using a structure-based approach, nine ts mutants of Gal4 were generated in yeast by mutating four DNA binding residues. Two of these nine yeast ts mutants were cloned into P element vectors under control of the Elav and GMR promoters and transgenic Drosophila lines were generated. These were crossed to UAS reporter lines and progeny were characterized for reporter gene expression as a function of temperature. Both of these yeast ts mutants show a ts phenotype in Drosophila and result in rapid induction of reporter gene expression upon shifting to the permissive temperature. Exposed, functional residues involved in protein–ligand or protein–protein interactions appear to be attractive candidate sites for generating ts mutants that are transferable between organisms

Characterization of the simultaneous decay kinetics of metarhodopsin states II and III in rhodopsin by solution-state NMR spectroscopy

The mammalian visual dim-light photoreceptor rhodopsin is considered a prototype G protein-coupled receptor. Here, we characterize the kinetics of its light-activation process. Milligram quantities of α,ε-15N-labeled tryptophan rhodopsin were produced in stably transfected HEK293 cells. Assignment of the chemical shifts of the indole signals was achieved by generating the single-point-tryptophan to phenylalanine mutants, and the kinetics of each of the five tryptophan residues were recorded. We find kinetic partitioning in rhodopsin decay, including three half-lives, that reveal two parallel processes subsequent to rhodopsin activation that are related to the photocycle. The meta II and meta III states emerge in parallel with a relative ratio of about 3:1. Transient formation of the meta III state was confirmed by flash photolysis experiments. From analysis of the site-resolved kinetic data we propose the involvement of the E2-loop in the formation of the meta III state