CIISB Research Highlights Archive

The concept of extended MCRs with indolealdehydes.

Rodolfo Lavilla Research Group

Significance

The participation of reactants undergoing a polarity inversion along a multicomponent reaction, allows the continuation of the transformation with productive domino processes. Thus, indole aldehydes in Groebke-Blackburn-Bienaymé reactions lead to an initial adduct which spontaneously triggers a series of events leading to the discovery of novel reaction pathways together with a direct access to a variety of linked, fused and bridged polyheterocyclic scaffolds. Indole 3- and 4-carbaldehydes with suitable isocyanides and aminoazines afford fused adducts through oxidative Pictet-Spengler processes, whereas indole 2-carbaldehyde yields linked indolocarbazoles under mild conditions, and a bridged macrocycle at high temperature. These novel structures are potent activators of the human aryl hydrocarbon receptor signaling pathway.

Ghashghaei, O.; Pedrola, M.; Seghetti, F.; Martin, V. V.; Zavarce, R.; Babiak, M.; Novacek, J.; Hartung, F.; Rolfes, K. M.; Haarmann-Stemmann, T. & Lavilla, R.: Extended Multicomponent Reactions with Indole Aldehydes: Access to Unprecedented Polyheterocyclic Scaffolds, Ligands of the Aryl Hydrocarbon Receptor. Angew. Chem. Int. Ed. online https://doi.org/10.1002/anie.202011253

Representative poses of Suprastat bound to the zHDAC6-CD2 catalytic pocket after molecular dynamics (MD) simulations. (A) Flexible hydroxylbutyl chain engages in H-bonding interaction with N530. (B) Transition state between each stable conformer.

Cyril Bařinka Research Group

Significance

Selective inhibition of histone deacetylase 6 (HDAC6) is being recognized as a therapeutic approach for cancers. In this study, we designed a new HDAC6 inhibitor, named Suprastat, using in silico simulations. X-ray crystallography and molecular dynamics simulations provide strong evidence to support the notion that the aminomethyl and hydroxyl groups in the capping group of Suprastat establish significant hydrogen bond interactions, either direct or water-mediated, with residues D460, N530, and S531, which play a vital role in regulating the deacetylase function of the enzyme and which are absent in other isoforms. In vitrocharacterization of Suprastat demonstrates subnanomolar HDAC6 inhibitory potency and a hundred- to a thousand-fold HDAC6 selectivity over the other HDAC isoforms. In vivo studies reveal that a combination of Suprastat and anti-PD1 immunotherapy enhances antitumor immune response, mediated by a decrease of protumoral M2 macrophages and increased infiltration of antitumor CD8+ effector and memory T-cells.

Noonepalle, S.; Shen, S.; Ptáček, J.; Tavares, M. T.; Zhang, G.; Stránský, J.; Pavlíček, J.; Ferreira, G. M.; Hadley, M.; Pelaez, G.; Bařinka*, C.; Kozikowski*, A. P. & Villagra*, A.: Rational Design of Suprastat: A Novel Selective Histone Deacetylase 6 Inhibitor with the Ability to Potentiate Immunotherapy in Melanoma Models, J. Med. Chem.  2020, 63, 10246-10262, https://doi.org/10.1021/acs.jmedchem.0c00567

Sinefungin recognition by the nsp16 MTase. A) SARS CoV-2 nsp10-nsp16 protein complex bound to sinefungin (white sticks), nsp16 in surface representation (cyan), nsp10 in cartoon representation (orange) and zinc ions as gray spheres. B) Detailed view of sinefungin recognition, important amino acid residues are shown in stick representation, water as red spheres and hydrogen bonds are shown as dashed lines.

Evžen Bouřa and Radim Nencka Research Groups

Significance

COVID-19 pandemic is caused by the SARS-CoV-2 virus that has several enzymes that could be targeted by antivirals including a 2'-O RNA methyltransferase (MTase) that is involved in the viral RNA cap formation; an essential process for RNA stability. This MTase is composed of two nonstructural proteins, the nsp16 catalytic subunit and the activating nsp10 protein. We have solved the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Based on the structural data we built a model of the MTase in complex with RNA that illustrates the catalytic reaction. A structural comparison to the Zika MTase revealed low conservation of the catalytic site between these two RNA viruses suggesting preparation of inhibitors targeting both these viruses will be very difficult. Together, our data will provide the information needed for structure-based drug design.

Krafčíková, P.; Šilhan, J.; Nencka, R. & Bouřa, E.: Structural analysis of the SARS-CoV-2 methyltransferase complex involved in coronaviral RNA cap creation, Nat. Commun. (2020) 11, 3717, https://doi.org/10.1038/s41467-020-17495-9

Schematic illustration of the TRAK1-mediated anchoring of KIF5B. a Top: in absence of TRAK1, KIF5B (green) can either continue its walk by rebinding the disengaged motor domain to the microtubule or dissociate from the microtubule when the engaged motor domain unbinds from the microtubule. Bottom: in presence of microtubule-bound TRAK1 (magenta), when both motor domains of KIF5B disengage from the microtubule, KIF5B remains tethered to the microtubule through a diffusive interaction of TRAK1 with the microtubule and thereby enables the rebinding of a motor domain of KIF5B to the microtubule. In this state, TRAK1 might facilitate navigation around obstacles by diffusion along the microtubule surface. b Overview of the functions of TRAK1. Top: TRAK1 activates auto-inhibited KIF5B, enabling its processive movement along microtubules. Middle: TRAK1 increases the processivity of KIF5B in crowded environments. Bottom: TRAK1 enables KIF5B-based transport of isolated mitochondria along microtubules in vitro.

Zdeněk Lánský Research Group

Significance

Intracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential cellular processes. In absence of other proteins on the microtubule surface, kinesin-1 performs micron-long runs. Under crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases robustness of kinesin- 1 stepping on crowded microtubule surfaces. Interaction with TRAK1 i) facilitates kinesin-1 navigation around obstacles, ii) increases the probability of kinesin-1 passing through cohesive islands of tau and iii) increases the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, pro- viding an additional anchor for the kinesin-1-TRAK1 complex. Furthermore, TRAK1 enables mitochondrial transport in vitro. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments.

Henrichs, V.; Grycova, L.; Bařinka, C.; Nahačka, Z.; Neužil, J.; Diez, S.; Rohlena, J.; Braun, M. & Lánský, Z.: Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments, Nat. Commun. (2020) 11, 3123, https://doi.org/10.1038/s41467-020-16972-5

Structure of the RcGTA particle and organization of segments of the R. capsulatus genome encoding protein components of RcGTA particles. a Cryo-EM reconstruction of a native particle of RcGTA from R. capsulatus strain DE442 calculated from 42,242 particle images. The left part of the panel shows the complete particle, whereas on the right the front half of the particle has been removed to show DNA and internal proteins. Individual proteins in the density map are colored according to the gene map in panel b. Yellow mesh highlights the structural organization of capsid proteins within the RcGTA head. The inset shows an example of a two-dimensional class average and an electron micrograph of an RcGTA particle. The scale bar within the inset represents 20 nm. b Gene map of three genome segments encoding fourteen structural proteins of RcGTA particles. c Cryo-EM reconstruction of an RcGTA particle from R. capsulatus strain DE442 with T = 3 quasi-icosahedral head. The reconstruction is based on 1076 particle images. The structure is at the scale of those shown in panel a. The inset shows an example of a two-dimensional class average and an electron micrograph of RcGTA particle with an icosahedral head. Scale bar represents 20 nm. d Organization of capsomers in the oblate capsid of RcGTA. Capsomers forming one fifth of the capsid are highlighted in different colors and marked with P for pentamer and H for hexamer.

Pavel Plevka Research Group

Significance

Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm.

Bárdy, P.; Füzik, T.; Hrebík, D.; Pantůček, R.; Beatty, T. .J. & Plevka, P: Structure and mechanism of DNA delivery of a gene transfer agent, Nat. Commun. (2020) 11, 3034. doi.org/10.1038/s41467-020-16669-9

Crystal structure of BP39L and CV39L. (A) Crystal structure of BP39L shown in secondary structure representation (β-strands in yellow and loops in green) (B) Surface representation (yellow) of BP39L color-coded according to putative binding sites (blue). (C) Crystal structure of CV39L shown in secondary structure representation (β-strands in violet, α-helices in cyan and loops in pink). (D) Surface representation (violet) of CV39L color-coded according to putative binding sites (blue), α-helices shown in cyan.

Michaela Wimmerova Research Group

Significance

Burkholderia pseudomallei and Chromobacterium violaceum are bacteria of tropical and subtropical soil and water that occasionally cause fatal infections in humans and animals. Microbial lectins mediate the adhesion of organisms to host cells, which is the first phase in the development of infection. Here we report the discovery of two novel lectins from the above-mentioned bacteria – BP39L and CV39L. The crystal structures revealed that the lectins possess a seven-bladed β-propeller fold. Functional studies conducted on a series of oligo- and polysaccharides confirmed the preference of BP39L for mannosylated saccharides and CV39L for rather more complex polysaccharides with a monosaccharide preference for β-l-fucose. The presented data indicate that the proteins belong to a currently unknown family of lectins.

Sykorova, P.; Novotna, J.; Demo, G.; Pompidor, G.; Dubska, E.; Komarek, J.; Fujdiarova, E.; Houser, J.; Haronikova, L.; Varrot, A.; Shilova, N.; Imberty, A.; Bovin, N.; Pokorna, M. & Wimmerova, M.: Characterization of novel lectins from Burkholderia pseudomallei and Chromobacterium violaceum with seven-bladed beta-propeller fold, Int. J. Biol. Macromol. 2019, 152, 1113-1124, doi.org/10.1016/j.ijbiomac.2019.10.200