CIISB Research Highlights Archive

Shape-Controlled Self-Assembly Capabilities of Light-Powered Hematite/Pt Janus Microrobots under UV-Light Irradiation

Martin Pumera Research Group

Significance

Nature presents the collective behavior of living organisms aiming to accomplish complex tasks, inspiring the development of cooperative micro/nanorobots. Herein, the spontaneous assembly of hematite-based microrobots with different shapes is presented. Autonomous motile light-driven hematite/Pt microrobots with cubic and walnut-like shapes are prepared by hydrothermal synthesis, followed by the deposition of a Pt layer to design Janus structures. Both microrobots show a fuel-free motion ability under light irradiation. Because of the asymmetric orientation of the magnetic dipole moment in the crystal, cubic hematite/Pt microrobots can self-assemble into ordered microchains, contrary to the random aggregation observed for walnut-like microrobots. The microchains exhibit different synchronized motions under light irradiation depending on the mutual orientation of the individual microrobots during the assembly, which allows them to accomplish multiple tasks, including capturing, picking up, and transporting microscale objects, such as yeast cells and suspended matter in water extracted from personal care products, as well as degrading polymeric materials. Such light-powered self-assembled microchains demonstrate an innovative cooperative behavior for small-scale multitasking artificial robotic systems, holding great potential toward cargo capture, transport, and delivery, and wastewater remediation.

Xia Peng, Urso, M., Ussia, M., and Pumera, M.:

Shape-Controlled Self-Assembly of Light-Powered Microrobots into Ordered Microchains for Cells Transport and Water Remediation, ACS Nano. 2022, 16, 5, 7615-7625, https://doi.org/10.1021/acsnano.1c11136

Extended mechanism of staphylokinase (SAK). (a) Simplified graphical schematic of the mechanism and (b) detailed kinetic pathway with all analyzed steps and parameters provided for clarity. In comparison to the general mechanism (Figure 1), the extended mechanism newly includes binding of staphylokinase (SAK) to plasminogen (Plg) to form an inactive complex, preventing plasminogen to plasmin (Plg to Plm) conversion and having a significant effect on the overall effectivity. The mechanism further includes a newly identified two-step induced-fit binding mechanism for both Plm and Plg binding by SAK. SAK·Plg* to SAK·Plm* conversion by Plm, reported previously (15) and marked with a gray dotted arrow, was not included in the kinetic model because its rate was very slow and not significantly detectable during the experimental time window. Forward and reverse rate constants of the ith step are denoted with the symbols k+i and k–i, respectively. The steady-state kinetic parameters Km, kcat, and Kp correspond to the Michaelis constant, turnover number, and product inhibition constant, respectively.

Zbyněk Prokop Research Group

Significance

The plasminogen activator staphylokinase is a fibrin-specific thrombolytic biomolecule and an attractive target for the development of effective myocardial infarction and stroke therapy. To engineer the protein rationally, a detailed understanding of the biochemical mechanism and limiting steps is essential. Conventional fitting to equations derived on the basis of simplifying approximations may be inaccurate for complex mechanisms such as that of staphylokinase. We employed a modern numerical approach of global kinetic data analysis whereby steady-state kinetics and binding affinity data sets were analyzed in parallel. Our approach provided an extended, revised understanding of the staphylokinase mechanism without simplifying approximations and determined the value of turnover number k_cat of 117 s^–1 that was 10000-fold higher than that reported in the literature. The model further showed that the rate-limiting step of the catalytic cycle is binding of staphylokinase to plasmin molecules, which occurs via an induced-fit mechanism. The overall staphylokinase effectivity is further influenced by the formation of an inactive staphylokinase·plasminogen complex. Here, Z. Prokop et.al. describe a quick and simplified guide for obtaining reliable estimates of key parameters whose determination is critical to fully understand the staphylokinase catalytic functionality and define rational strategies for its engineering. Their study provides an interesting example of how a global numerical analysis of kinetic data can be used to better understand the mechanism and limiting factors of complex biochemical processes. The high catalytic activity of staphylokinase (more than 1000-fold higher than that of the clinically used drug alteplase) determined herein makes this thrombolytic agent a very attractive target for further engineering.

Toul, M., Nikitin, D., Marek, M., Damborsky, J., and Prokop, Z:

Extended Mechanism of the Plasminogen Activator Staphylokinase Revealed by Global Kinetic Analysis: 1000-fold Higher Catalytic Activity than That of Clinically Used Alteplase, ACS Catal. 2022, 12, 7, 3807-3814, https://doi.org/10.1021/acscatal.1c05042

Overview of hcis-PT structure and reaction scheme

(A) Cartoon representation of a single DHDDS-NgBR heterodimer in complex with FPP [Protein Data Bank (PDB) 6Z1N]. DHDDS and NgBR are colored blue and yellow, respectively. Surface representations of the FPP and IPP [placed by superposition with PDB 6W2L] molecules are colored pink and green, respectively. The residue W3, at the DHDDS N terminus, is shown as spheres. (B) Condensation reaction scheme. At the first cycle, the allylic diphosphate primer, FPP (C₁₅, pink), undergoes a condensation with IPP (C₅, green) to produce geranylgeranyl-diphosphate (GGPP) (C₂₀). The cycle repeats with further condensations (14–17) of the allylic diphosphate at S₁, ultimately leading to a final product length of C_85–100.

Yoni Haitin Research Group

Significance

Isoprenoids are synthesized by the prenyltransferase superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, because of an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticulum membrane and playing a crucial role in protein glycosylation. Crystallographic investigation of hcis-PT along the reaction cycle revealed an outlet for the elongating product. Hydrogen-deuterium exchange mass spectrometry analysis showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Last, using a fluorescence substrate analog, we show that product elongation and membrane association are closely correlated. Together, our results support direct membrane insertion of the elongating isoprenoid during catalysis, uncoupling active site volume from product length.

Giladi, M., Bar-El, ML., Lisnyansky, M., Vankova, P., Ferofontov, A., Melvin, E., Alkaderi, S., Kavan, D., Redko, B., Haimov, E., Wiener, R., Man, P., and Haitin, Y.: Structural basis for long-chain isoprenoid synthesis by cis-prenyltransferases, Sci. Adv. 2022, 7, eabn1171, https://doi.10.1126/sciadv.abn1171

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