CIISB Research Highlights Archive

A) Confocal microscopy images of cells transfected with aptamer–ligand complex. The green color indicates the localization of (FAM)-aptamer/(FAM)-aptamer–ligand complex. The blue color corre- sponds to a cell nucleus stained by Hoechst 33342. B) Double-staining (PI/FAM) FCM analysis of transfected HeLa cells with the aptamer– ligand complex. Percentages of a viable non-transfected cells, viable aptamer–ligand complex containing cells, non-transfected dead/com- promised cells, and transfected dead/compromised cells with apta- mer–ligand complex are indicated in left-bottom, right-bottom (red), left-top, and right-top quadrants, respectively. C) Imino region of 1D ¹H NMR spectra of aptamer–ligand complex in vitro (TOP) and corresponding spectrum of HeLa cells transfected with aptamer– ligand complex (MIDDLE). Imino region of 1D ¹H NMR spectrum of extracellular fluid (supernatant) taken from the in-cell NMR samples after completion of the spectra acquisition (BOTTOM). D) 1D ¹³C- edited NMR spectra of the aptamer–ligand complex in vitro in EB- buffer (TOP) and corresponding spectra of HeLa cells transfected with aptamer–ligand complex.

Lukáš Trantírek and Harald Schwalbe Research Groups

Significance

L. Trantírek and H. Schwalbe research groups report here the in-cell NMR-spectroscopic observation of the binding of the cognate ligand 2’-deoxyguanosine to the aptamer domain of the bacterial 2’-deoxyguanosine-sensing riboswitch in eukaryotic cells, namely Xenopus laevis oocytes and in human HeLa cells. The riboswitch is sufficiently stable in both cell types to allow for detection of binding of the ligand to the riboswitch. Most importantly, they show that the binding mode established by in vitro characterization of this prokaryotic riboswitch is maintained in eukaryotic cellular environment. Data also bring important methodological insights: Thus far, in-cell NMR studies on RNA in mammalian cells have been limited to investigations of short (< 15 nt) RNA fragments that were extensively modified by protecting groups to limit their degradation in the intra-cellular space. Here, they show that the in-cell NMR setup can be adjusted for characterization of much larger (~ 70 nt) functional and chemically non-modified RNA.

Broft, P., S.; Dzatko, S.; Krafcikova, M.; Wacker, A.; Hänsel-Hertsch, R.; Dötsch, V.; Trantirek, L. & Schwalbe, H.: In-Cell NMR Spectroscopy of Functional Riboswitch Aptamers in Eukaryotic Cells, Angew. Chem. Int. Edit. 2020, 59, 2-11 doi.org/10.1002/anie.202007184

(−)-Bactobolin A and selected related natural products.

(A) Overall structure of ovine complex I. Core subunits necessary for the reaction of complex I are labeled with corresponding colors, and mammalian supernumerary subunits are shown in gray. NADH and quinone binding sites are indicated. The membrane arm contains four separate proton-pumping channels: three in the antiporter-like subunits ND2, ND4, and ND5 and one in the E-channel, composed of subunits ND1, ND6, and ND4L. Q, quinone. 

Leonid A. Sazanov Research Group

Significance

Complex I is the first and, with 45 subunits and a total mass of ~1 MDa, the most elaborate of the mitochondrial electron transfer chain enzymes. Complex I converts energy stored in chemical bonds into a proton gradient across the membrane that drives the synthesis of adenosine triphosphate (ATP), the universal energy currency of the cell. In each catalytic cycle, the transfer of two electrons from nicotinamide adenine dinucleotide (NADH) to a hydrophobic electron carrier quinone, which happens in the peripheral arm of the enzyme, is coupled to the translocation of four protons across the inner mitochondrial membrane in the membrane arm. The exact mechanism of this energy conversion currently presents an enigma because of complex I’s size and the spatial separation between the two reactions.

To understand the coupling mechanism of complex I, we solved its cryo–electron microscopy (cryo-EM) structures in five different conditions, including the substrate- and inhibitor-bound states and during active turnover, unlocking the various conformations that the enzyme goes through during the catalytic cycle. We also improved the resolution to up to 2.3 to 2.5 Å, allowing us to directly observe water molecules critical for proton pumping.

Kampjut, D. & Sazanov, L. A. The coupling mechanism of mammalian respiratory complex I, Science, 2020, 370, eabc4209, https://doi.org/10.1126/science.abc4209

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