a, Kinome tree representation of the selectivity of OTS964 (at 1 µM concentration) in the Eurofins panel of 412 human kinases. The size of the red circles shows the percentage of inhibition of kinase activity. The green circle corresponds to CDK11. The percentage of CDK11 inhibition was derived from IVKAs presented in b and Fig. 3f The blue circle corresponds to TOPK. The percentage of TOPK inhibition was estimated from the published IC₅₀ = 353 nM and Extended Data Fig. 1b. b, Immunoblots of IVKAs of Flag-tagged (F) CDK11 WT, G579S and kinase dead (KD) mutants (left), or Flag-tagged CDK9 WT and KD mutant (right) phosphorylation of glutathione-S-transferase (GST)-tagged RNAPII^CTD substrate with the indicated concentrations of OTS964. c, The percentage of normalized NanoBRET ratio for CDK11/cyclin L2 or CDK9/cyclin T1 after OTS964 or control dinaciclib treatment. n = 2 biologically independent replicates; a representative replicate is shown. d, Immunoblot analysis of proteins after treatment of cells with OTS964 for 4 h. Short and long indicate short and long exposures of the film.

Dalibor Blažek Research Group

Significance

RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA–protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex^. Splicing factor 3B subunit 1 (SF3B1) protein—a subunit of the U2 small nuclear ribonucleoprotein—is phosphorylated during spliceosome activation, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the B^act complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex B^act and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.

Hluchý, M., Gajdušková, P., Ruiz de los Mozos, I., Rájecký, M., Kluge, M., Berger, B-T., Slabá, Z., Potěšil, D., Weiß, E., Ule, J., Zdráhal, Z., Knapp, S., Paruch, K., Friedel, C.C., and Blažek, D.:

CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1, Nature. (2022) 609, 829-834. https://doi.org/10.1038/s41586-022-05204-z

a)Ribbon diagram of the NKR-P1 CTLD. Secondary structure elements are labeled in different colors: helix α1 is red, helix α2 is yellow, and β-strands and loops are cyan. b)Comparison between NKR-P1 dimers formed by the glycosylated (cyan), deglycosylated free (green), and LLT1-bound (blue) forms of NKR-P1. c) Comparison between helices α1- and α2-centered dimerization of murine dectin-1 (magenta) and human LLT1 (green), respectively; helices α1 and α2 are shown in red and yellow. Structural alignments of dectin-1 and NKR-P1 homodimers and LLT1 and NKR-P1 homodimers, prepared by aligning only one monomer from each dimer, are shown on the right-hand side. Although the CTLD fold is conserved in each pair of the aligned monomers, the helix α1- and helix α2-centered dimers show inverse arrangement.

Ondřej Vaněk Research Group

Significance

Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the receptor on the cell surface is necessary to understand its functions. Here we report the crystal structures of the NKR-P1 and NKR-P1:LLT1 complexes, which provides evidence that NKR-P1 forms homodimers in an unexpected arrangement to enable LLT1 binding in two modes, bridging two LLT1 molecules. These interaction clusters are suggestive of an inhibitory immune synapse. By observing the formation of these clusters in solution using SEC-SAXS analysis, by dSTORM super-resolution microscopy on the cell surface, and by following their role in receptor signaling with freshly isolated NK cells, we show that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. In summary, our findings collectively support a model of NKR-P1:LLT1 clustering, which allows the interacting proteins to overcome weak ligand-receptor affinity and to trigger signal transduction upon cellular contact in the immune synapse.

Blaha, J., Skalova, T. Kalouskova, B., Skorepa, O., Cmunt, D., Grobarova, V.,Pazicky, S., Polachova, E., Abreu, C., Stransky, J., Koval, T., Duskova, J.,Zhao, Y.,Harlos, K., Hasek, J. Dohnalek, J., and Vanek, O.:

Structure of the human NK cell NKR-P1:LLT1 receptor: ligand complex reveals clustering in the immune synapse, Nature Comm. (2022)13:5022, https://doi.org/10.1038/s41467-022-32577-6

Model for cooperation between the intrinsically disordered and structured regions of Spt6 which regulates nucleosome and Pol II CTD binding.

Richard Štefl Research Group

Significance

Transcription elongation factor Spt6 associates with RNA polymerase II (Pol II) and acts as a histone chaperone, which promotes the reassembly of nucleosomes following the passage of Pol II. The precise mechanism of nucleosome reassembly mediated by Spt6 remains unclear. In this study, we used a hybrid approach combining cryo-electron microscopy and small-angle X-ray scattering to visualize the architecture of Spt6 from Saccharomyces cerevisiae. The reconstructed overall architecture of Spt6 reveals not only the core of Spt6, but also its flexible N- and C-termini, which are critical for Spt6’s function. We found that the acidic N-terminal region of Spt6 prevents the binding of Spt6 not only to the Pol II CTD and Pol II CTD-linker, but also to pre-formed intact nucleosomes and nucleosomal DNA. The N-terminal region of Spt6 self-associates with the tSH2 domain and the core of Spt6 and thus controls binding to Pol II and nucleosomes. Furthermore, we found that Spt6 promotes the assembly of nucleosomes in vitro. These data indicate that the cooperation between the intrinsically disordered and structured regions of Spt6 regulates nucleosome and Pol II CTD binding, and also nucleosome assembly.

Kasiliauskaite, A., Kubicek, K., Klumpler, T., Zanova, M., Zapletal, D., Koutna, E., Novacek, J., and Stefl. R.: Cooperation between intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly, Nucleic Acids Res. 2022, 50 (10), 5961–5973, https://doi.org/10.1093/nar/gkac451

Cryo-electron density map of CV-A6 virion (a), altered particle (b), and empty particle (c), viewed along the twofold icosahedral symmetry axis, coloured according to distance from centre of particle. The insets show central slices through the particle interior (central slice, thickness 5 Å). Virion and altered particle contain genomic RNA (a, b). Positions of icosahedral symmetry axes are indicated by dashed lines in a. Scale bar 5 nm.

Pavel Plevka Research Group

Significance

Coxsackievirus A6 (CV-A6) has recently overtaken enterovirus A71 and CV-A16 as the primary causative agent of hand, foot, and mouth disease worldwide. Virions of CV-A6 were not identified in previous structural studies, and it was speculated that the virus is unique among enteroviruses in using altered particles with expanded capsids to infect cells. In contrast, the virions of other enteroviruses are required for infection. Here we used cryo-electron microscopy (cryo-EM) to determine the structures of the CV-A6 virion, altered particle, and empty capsid. We show that the CV-A6 virion has features characteristic of virions of other enteroviruses, including a compact capsid, VP4 attached to the inner capsid surface, and fatty acid-like molecules occupying the hydrophobic pockets in VP1 subunits. Furthermore, we found that in a purified sample of CV-A6, the ratio of infectious units to virions is 1 to 500. Therefore, it is likely that virions of CV-A6 initiate infection, like those of other enteroviruses. Our results provide evidence that future vaccines against CV-A6 should target its virions instead of the antigenically distinct altered particles. Furthermore, the structure of the virion provides the basis for the rational development of capsid-binding inhibitors that block the genome release of CV-A6

Büttner, C.R., Spurný, R., Füzik, T., and Plevka, P.: Cryo-electron microscopy and image classification reveal the existence and structure of the coxsackievirus A6 virio

Commun. Biol. 2022, 5, 898, https://doi.org/10.1038/s42003-022-03863-2

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