CIISB Research Highlights Archive

The catalytic center of [FeFe] hydrogenases, in which the molecular hydrogen oxidation or synthesis takes place, is called the H-cluster. It is a highly complex metallo-organic structure that is composed of a canonical [4Fe4S] cubane-cluster that, through a cysteine residue, binds a unique [FeFe] subcluster, which is coordinated with three CO, two CN⁻, and one unusual azadithiolate bridging ligand.

Jan Tachezy Research Group

Significance

Trichomonads, represented by the highly prevalent sexually transmitted human parasite Trichomonas vaginalis, are anaerobic eukaryotes with hydrogenosomes in the place of the standard mitochondria. Hydrogenosomes form indispensable FeS-clusters, synthesize ATP, and release molecular hydrogen as a waste product. Hydrogen formation is catalyzed by [FeFe] hydrogenase, the hallmark enzyme of all hydrogenosomes found in various eukaryotic anaerobes. Eukaryotic hydrogenases were originally thought to be exclusively localized within organelles, but today few eukaryotic anaerobes are known that possess hydrogenase in their cytosol. We identified a thus-far unknown hydrogenase in T. vaginalis cytosol that cannot use ferredoxin as a redox partner but can use cytochrome b5 as an electron acceptor. Trichomonads overexpressing the cytosolic hydrogenase, while maintaining the carbon flux through hydrogenosomes, show decreased excretion of hydrogen and increased excretion of methylated alcohols, suggesting that the cytosolic hydrogenase uses the hydrogen gas as a source of reducing power for the reactions occurring in the cytoplasm and thus accounts for the overall redox balance. This is the first evidence of hydrogen uptake in a eukaryote, although further work is needed to confirm it. Assembly of the catalytic center of [FeFe] hydrogenases (H-cluster) requires the activity of three dedicated maturases, and these proteins in T. vaginalis are exclusively localized in hydrogenosomes, where they participate in the maturation of organellar hydrogenases. Despite the different subcellular localization of cytosolic hydrogenase and maturases, the H-cluster is present in the cytosolic enzyme, suggesting the existence of an alternative mechanism of H-cluster assembly.

Smutna, T.; Dohnalkova, A.; Sutak, R.; Narayanasamy, R. K.; Tachezy, J. & Hrdy, I.: A cytosolic ferredoxin-independent hydrogenase possibly mediates hydrogen uptake in Trichomonas vaginalis, Current Biol. 2022, 32, R49-R51, https://doi.org/10.1016/j.cub.2021.10.050

Cryo-EM structures of CspA27 inside the exit tunnel

A. Structure and nascent chain contacts in the exit tunnel for CspA27-1.
B. Structure and nascent chain contacts in the exit tunnel for CspA27-2.
C. Structure and nascent chain contacts in the exit tunnel for CspA27-3.

Marina V. Rodnina Research Group

Significance

Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo-EM structure determination to show that folding of a β-barrel protein begins with formation of a dynamic α-helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N-terminal part of the nascent chain refolds to a β-hairpin structure that remains dynamic until its release from the ribosome. Contacts with the ribosome and structure of the peptidyl transferase center depend on nascent chain conformation. These results indicate that proteins may start out as α-helices inside the tunnel and switch into their native folds only as they emerge from the ribosome. Moreover, the correlation of nascent chain conformations with reorientation of key residues of the ribosomal peptidyl-transferase center suggest that protein folding could modulate ribosome activity.

Agirrezabala, X.; Samatova, E.; Macher, M.; Liutkute, M.; Maiti, M.; Gil-Carton, D.; Novacek, J.; Valle. M. & Rodnina, M. V.: EMBO J. (2022) e109175, https://doi.org/10.15252/embj.2021109175

Snapshots from simulations representing the release pathways. The rapid pathway category (R1–R3) was divided into (R1) burst genome release, where capsids disintegrate into fragments; (R2) rupture genome release, where capsids split open (most often into two halves); and (R3) bloom genome release, where capsids open wide in one hemisphere without breaking the other. In the rupture and bloom pathways, a majority of the capsid reassembles after the genome release. Occasionally, pentamers of capsid proteins may be detached. The subcategory leaky release (L) started with a slow release and ended with a rapid release. The slow pathway (S1–S3) category was divided into (S1) release through a pore on a twofold axis, (S2) release through a pore on a threefold axis, and (S3) release through multiple pores. Note that slow release was only observed for the noncompact genome. All release examples are shown with the noncompact genome. Color coding: Beads forming the body of the capsid are orange on the outside and purple on the inside of the capsid. The genome is represented by blue beads. Red and green beads represent attractive beads between pentamers.

Robert Vácha Research Group

Significance

Virus-like nanoparticles are protein shells similar to wild-type viruses, and both aim to deliver their content into a cell. Unfortunately, the release mechanism of their cargo/ genome remains elusive. Pores on the symmetry axes were proposed to enable the slow release of the viral genome. In contrast, cryo-EM images showed that capsids of nonenveloped RNA viruses can crack open and rapidly release the genome. We combined in vitro cryo-EM observations of the genome release of three viruses with coarse-grained simulations of generic virus-like nanoparticles to investigate the cargo/genome release pathways. Simulations provided details on both slow and rapid release pathways, including the success rates of individual releases. Moreover, the simulated structures from the rapid release pathway were in agreement with the experiment. Slow release occurred when interactions between capsid subunits were long-ranged, and the cargo/genome was noncompact. In contrast, rapid release was preferred when the interaction range was short and/or the cargo/genome was compact. These findings indicate a design strategy of virus-like nanoparticles for drug delivery.

Sukeník, L.; Mukhamedova, L.; Procházková, M.; Škubník, K.; Plevka, P. & Vácha, R.: Cargo Release from Nonenveloped Viruses and Virus-like Nanoparticles: Capsid Rupture or Pore Formation, ABC Nano 2021, 15, 12, 19233–19243, https://doi.org/10.1021/acsnano.1c04814

Loss of PrimPol S538 phosphorylation affects genomic stability after UV-C damage and is dependent on RPA interaction

(A) Damage sensitivity was measured by colony survival after increasing doses of UV-C (left) or cisplatin (right). (B) Quantification of cell cycle recovery after damage was measured by flow cytometry, using EdU and PI labeling 24 hours after treatment with 5 J/m² UV-C, images shown in fig. S4B. (C) Cells with one or more micronuclei were counted 48 hours after 5 J/m² UV-C treatment. (D) CldU/IdU ratios show replication changes after a pulse of 20 J/m² UV-C was given between labels. (E) Undamaged replication fork speeds were measured in the different cell lines at least 16 hours after PrimPol expression by labeling cells consecutively with CldU and IdU. (F) UV sensitivity was analyzed by colony survival in ΔPP-1 cells expressing RAB mutated forms of PrimPol also carrying the 538 mutations. (G) The effect of loss of PrimPol’s RPA interaction in micronuclei formation was compared in cells expressing PrimPol mutants additionally carrying the RAB mutations, 48 hours after exposure to 5 J/m² UV-C.

Aidan J. Doherty Research Group

Significance

Replication stress and DNA damage stall replication forks and impede genome synthesis. During S phase, damage tolerance pathways allow lesion bypass to ensure efficient genome duplication. One such pathway is repriming, mediated by Primase-Polymerase (PrimPol) in human cells. However, the mechanisms by which PrimPol is regulated are poorly understood. Here, A. Doherty et al. demonstrate that PrimPol is phosphorylated by Polo-like kinase 1 (PLK1) at a conserved residue between PrimPol’s RPA binding motifs. This phosphorylation is differentially modified throughout the cell cycle, which prevents aberrant recruitment of PrimPol to chromatin. Phosphorylation can also be delayed and reversed in response to replication stress. The absence of PLK1-dependent regulation of PrimPol induces phenotypes including chromosome breaks, micronuclei, and decreased survival after treatment with camptothecin, olaparib, and UV-C. Together, these findings establish that deregulated repriming leads to genomic instability, highlighting the importance of regulating this damage tolerance pathway following fork stalling and throughout the cell cycle.

Bailey, L. J.; Teague, R.; Kolesar, P.; Bainbridge, L. J.; Lindsay, H. D. & Doherty, A. J.: PLK1 regulates the PrimPol damage tolerance pathway during the cell cycle, Sci. Adv. 2021, 7, eabh1004, https://doi.org/10.1126/sciadv.abh1004

Visualization of the regions with the highest degree of modification and the difference between apo- and holomyoglobin for all reagents (A). Structure of apo-/holomyoglobin with highlighted modified residues after the reaction with reagent 3 (B). The red regions/residues are less modified in holomyoglobin, and the blue regions/residues are more modified in holomyoglobin.

Petr Novák Research Group

Significance

Covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X-ray, NMR, or cryogenic electron microscopy (Cryo-EM), used for protein structure determination. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resolution to be monitored, there is still high demand for alternative, less complicated, andinexpensive approaches. Here, we introduce a new covalent labeling method based on fast fluoroalkylation of proteins (FFAP). FFAP uses fluoroalkyl radicals formed by reductive decomposition of Togni reagents with ascorbic acid to label proteins on a time scale of seconds. The feasibility of FFAP to effectively label proteins was demonstrated by monitoring the differential amino acids modification of native horse heart apomyoglobin/holomyoglobin and the human haptoglobin−hemoglobin complex. The obtained data confirmed the Togni reagent-mediated FFAP is an advantageous alternative method for covalent labeling in applications such as protein footprinting and epitope mapping of proteins (and their complexes) in general. Data are accessible via the ProteomeXchange server with the data set identifier PXD027310.

Fojtík, L.; Fiala, J.; Pompach, P.; Chmelík, J.; Matoušek, V.; Beier, P.; Kukačka^*, Z. & Novák^*, P.: Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules, J. Amer. Chem. 2021, https://doi.org/10.1021/jacs.1c07771

Experimental setup of the MAS solid-state NMR experiment of insoluble proteins.

(A) The sample is placed inside a rotor that is oriented at 54.7° with respect to the static magnetic field and rotated within a solenoid coil, which allows the application of rf pulses to manipulate nuclear magnetization. Upon sample rotation, molecules experience periodical modulations of the rf field due to spatial inhomogeneity. Magnetic field lines are drawn schematically. (B) Protein molecules are contained in microcrystals that are randomly oriented in a powder. (C) Atomic level protein structure with arrows illustrating NCA and NCO magnetization transfer pathways between the amide nitrogen and Cα/C′ carbons (NCA/NCO transfers) of the protein backbone. The relative orientation of a bond vector with respect to the external static magnetic field determines the size of the dipolar interaction between the two atoms. The scale on the right indicates typical order of magnitude for object dimensions.

Zdeněk Tošner and Bernd Reif Research Groups

Significance

Dipolar recoupling is a central concept in the nuclear magnetic resonance spectroscopy of powdered solids and is used to establish correlations between different nuclei by magnetization transfer. The efficiency of conventional cross-polarization methods is low because of the inherent radio frequency (rf) field inhomogeneity present in the magic angle spinning (MAS) experiments and the large chemical shift anisotropies at high magnetic fields. Very high transfer efficiencies can be obtained using optimal control–derived experiments. These sequences had to be optimized individually for a particular MAS frequency. We show that by adjusting the length and the rf field amplitude of the shaped pulse synchronously with sample rotation, optimal control sequences can be successfully applied over a range of MAS frequencies without the need of reoptimization. This feature greatly enhances their applicability on spectrometers operating at differing external fields where the MAS frequency needs to be adjusted to avoid detrimental resonance effects.

Tosner, Z.; Brandl, M. J.; Blahut, J.; Glaser, S. .J. & Reif, B.: Maximizing efficiency of dipolar recoupling in solid-state NMR using optimal control sequences, Sci. Adv. 2021, 7(42), abj5913, https://doi.org/10.1126/sciadv.abj5913