CIISB Research Highlights Archive

Automated structure determination using 4D-CHAINS/autoNOE-Rosetta. (a) Logo of 4D-CHAINS algorithm depicting its powerfulness. Chains squeeze the NMR spectrometer to unleash high-quality structures by using a minimal set of 4D spectra and fully automated data analysis. (b) 4D-CHAINS utilizes two complementary experimental datasets, a 4D-TOCSY and a 4D-NOESY, to yield correct assignments for at least 95% of residues and an error rate of less than 1.5% (middle bar; TOCSY-NOESY). (c-d) Performance of different 4D-CHAINS assignment scenarios for a 20 kDa protein structure, α-lytic protease, calculated using autoNOE-Rosetta. (c) Goodness of structure ensembles is measured using the Rosetta all-atom energy function, backbone heavy atom RMSD to X-ray structure and degree of structural convergence. (d) Lowest-energy structures in each ensemble colored as the points in (c) superimposed on the X-ray reference structure (gray).

Konstantinos Tripsianes Research Group

Significance

The automation of NMR structure determination remains a significant bottleneck towards increasing the throughput and accessibility of NMR as a structural biology tool to study proteins. The chief barrier currently is that obtaining NMR assignments at sufficient levels of completeness to accurately define the structures by conventional methods requires a significant amount of spectrometer time (several weeks), and effort by a trained expert (up to several months). Here, we describe 4D-CHAINS/autoNOE-Rosetta, a complete pipeline for NOE-driven structure determination of medium- to larger-sized proteins. The 4D-CHAINS algorithm analyzes two 4D spectra in an iterative ansatz where common NOEs between different spin systems supplement conventional through-bond connectivities to establish assignments of sidechain and backbone resonances at high levels of completeness and with a minimum error rate. The 4D-CHAINS assignments are then used to guide automated assignment of long-range NOEs and structure refinement in autoNOE-Rosetta. Our results on four targets ranging in size from 15.5 to 27.3 kDa illustrate that the NMR structures of proteins can be determined accurately and in an unsupervised manner in a matter of days.

4D-CHAINS software is free for non-commercial usage and can be downloaded from https://github.com/tevang/4D-CHAINS

Evangelidis, T. et al. Automated NMR resonance assignments and structure determination using a minimal set of 4D spectra. Nature Communications 9, 13, doi:10.1038/s41467-017-02592-z (2018).

Schematic of an intramolecular A) i-motif DNA structure and B) C.C+ base pair. C) Double-staining (PI/FAM) FCM analysis of transfected HeLa cells with the (FAM)-DAP construct (upper left corner). Percentages of viable DNA non-transfected cells, viable DNA-containing cells, non-transfected dead/compromised cells, and transfected dead/compromised cells with DNA are indicated in left-bottom, right-bottom, left-top, and right-top quadrants, respectively. Confocal microscope images of cells transfected with (FAM)-DAP (upper right corner). The green color indicates the localization of (FAM)-DAP. The blue color corresponds to a cell nucleus stained by Hoechst 33342. Imino region of 1D 1H NMR spectra of DAP in vitro in T-buffer (140 mm sodium phosphate, 5 mm KCl, 10 mm MgCl2, pH 7.0) (black) and in-cell (red). Imino region of 1D 1H NMR spectrum of extracellular fluid taken from the in-cell NMR samples after completion of the spectra acquisition (gray). The (in-cell) NMR spectra were acquired at 20oC.

Lukáš Trantírek Research Group

Significance

C-rich DNA has the capacity to form a tetra-stranded structure known as an i-motif. The i-motifs within genomic DNA have been proposed to contribute to the regulation of DNA transcription. However, direct experimental evidence for the existence of these structures in vivo has been missing. Whether i-motif structures form in complex environment of living cells is not currently known. Using state-of- the-art in-cell NMR spectroscopy, Lukáš Trantírek and his colleagues from CEITEC Masaryk University in Brno has evaluated the stabilities of i-motif structures in the complex cellular environment. They showed that i-motifs formed from naturally occurring C-rich sequences in the human genome are stable and persist in the nuclei of living human cells. The obtained data show that i-motif stabilities in vivo are generally distinct from those in vitro. Results are the first to interlink the stability of DNA i-motifs in vitro with their stability in vivo and provide essential information for the design and development of i-motif-based DNA biosensors for intracellular applications.

Dzatko, S.; Krafcikova, M.; Hänsel-Hertsch, R.; Fessl, T.; Fiala, R.; Loja, T.; Krafcik, D.; Mergny, J.-L.; Foldynova-Trantirkova, S. & Trantirek, L.: Evaluation of the Stability of DNA i-Motifs in the Nuclei of Living Mammalian Cells, Angew. Chem. Int. Edit. 2018, in press, DOI: 10.1002/anie.201712284

Cryo-EM Structures of Polyproline-Stalled Ribosomes in the Presence of EF-P (A-C) Schematic representation (A) and cryo-EM reconstructions (B and C) of PPP-stalled ribosome complexes with (B) or without (C) of EF-P (salmon) bound in the E site. (D and E) Cryo-EM density (mesh) of the CCA end of the P-site tRNA (green) from cryo-EM maps in (C) without EF-P (D) and in (B) with EF-P (E), respectively, with aligned fMet (cyan, PDB: 1VY4) (Polikanov et al., 2014).

Daniel N. Wilson Research Group

Significance

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.

Huter, P.; Arenz, S.; Bock, L. V.; Frister, J. O.; Heuer, A.; Peil, L.; Starosta, A. L.; Peske, F.; Nováček, J.; Berninghausen, O.; Grubmüller, H.; Tenson, T.; Beckmann, R.; Rodina, M. V.; Vaiana, A. C. & Wilson, D. N.: Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P, Moll Cell 68, No. 3., 515-527.e6 DOI: dx.doi.org/10.1016/j.molcel.2017.10.014

Structural changes in crystal structure of of AfGcHK sensor protein induced by sodium dithionite soaking (PDB ID 5OHF) and conformational changes revealed by HDX-MS. (A) Two protein chains of the heme domain of the sensor protein observed in crystal structure including alternative B (yellow) occurring with dithionite soaking. (B) Conformational changes revealed by HDX-MS after 60 min of deuteration of the full-length AfGcHK proteins visualized on the protein structure. Differences between the Fe(III)-OH- form (active) and inactive Fe(II) form are color coded: grey - no difference, red - higher and blue - lower levels of deuteration.

• Heme-Containing Sensor Proteins Group of Markéta Martínková
• Laboratory of Structure and Function of Biomolecules, Jan Dohnálek
• Laboratory of Structural Biology and Cell Signaling, Petr Man 

Significance

The heme-based oxygen sensor histidine kinase AfGcHK is part of a two-component signal transduction system in bacteria. O₂ binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His-183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH^- and -CN^- complexes of AfGcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. The crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN^- and inactive 5-coordinate Fe(II) forms were determined, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry (HDX–MS) the intramolecular signal transduction mechanisms was investigated in full length AfGcHK.  The results suggest that structural changes at the heme proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of AfGcHK. For the first time, the conformational changes associated with signal transduction were studied in a full-length globin-coupled oxygen sensor protein and linked to directly observed structural changes in the globin domain.

Stranava, M.; Man, P.; Skálová, T.; Kolenko, P.; Blaha, J.; Fojtikova, V.; Martínek, V.; Dohnálek, J.; Lengalova, A.; Rosůlek, M.; Shimizu, T. & Martínková, M.: Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction. J. Biol. Chem. first Published on November 1, 2017, doi: 10.1074/jbc.M117.817023jbc.M117.817023.

High-resolution structure of a stable G-hairpin calculated from NMR data (PDB ID: 5M1W). (A) Ten lowest-energy structures. Loop residues are colored orange and O4′ atoms are colored red. (B) Schematic representation of hairpin folding topology. Chain reversal arrangement of the backbone and the 3′-to-5′ stacking of the terminal residues are indicated by dark green and magenta arrows, respectively. Anti and syn guanines that form G:G base pairs are colored dark and light blue, respectively.

Lukáš Trantírek Research Group

Significance

The first atomic resolution structure of a stable G-hairpin formed by a natively occurring DNA sequence is reported. An 11-nt long G-rich DNA oligonucleotide, 5′-d(GTGTGGGTGTG)-3′, corresponding to the most abundant sequence motif in irregular telomeric DNA from Saccharomyces cerevisiae adopts a novel type of mixed parallel/antiparallel fold-back DNA structure, which is stabilized by dynamic G:G base pairs that transit between N1-carbonyl symmetric and N1-carbonyl, N7-amino base-pairing arrangements. The structure reveals previously unknown principles of the folding of G-rich oligonucleotides that could be applied to the prediction of natural and/or the design of artificial recognition DNA elements. The structure also demonstrates that the folding landscapes of short DNA single strands is much more complex than previously assumed. 

Gajarsky, M.; Zivkovic, M. L.; Stadlbauer, P.; Pagano, B.; Fiala, R.; Amato, J.; Tomaska, L.; Sponer, J.; Plavec, J. &  Trantirek, L.: Structure of a Stable G-Hairpin JACS 139, 3591-3594, doi:10.1021/jacs.6b10786 (2017)

A model of RNA polymerase II bound to the transcription termination factor Rtt103. The structural model was created using integrative structural biology.

Richard Štefl Research Group

Significance

RNA polymerase II (RNAPII) not only transcribes protein coding genes and many noncoding RNA, but also coordinates transcription and RNA processing. This coordination is mediated by a long C-terminal domain (CTD) of the largest RNAPII subunit, which serves as a binding platform for many RNA/protein-binding factors involved in transcription regulation. In this work, we used a hybrid approach to visualize the architecture of the full-length CTD in complex with the transcription termination factor Rtt103. Specifically, we first solved the structures of the isolated subcomplexes at high resolution and then arranged them into the overall envelopes determined at low resolution by small-angle X-ray scattering. The reconstructed overall architecture of the Rtt103–CTD complex reveals how Rtt103 decorates the CTD platform.

Jasnovidova, O.; Klumpler, T.; Kubicek, K.; Kalynych, S.; Plevka, P. & Stefl, R.: Structure and dynamics of the RNAPII CTDsome with Rtt103, PNAS 2017 114 (42) 11133-11138; doi:10.1073/pnas.1712450114