CIISB Research Highlights Archive

Structural characterization of the PSMA/Glu-490 complex

a Molecular formula of the Glu-490. b A stereo view of the Gluo-490 inhibitor. The Fo-Fc omit map (green) is contoured at 3.0 σ and the inhibitor is shown in stick representation with atoms colored red (oxygen), blue (nitrogen), yellow (sulfur), and cyan (carbon). c Details of interactions between residues of the glutarate sensor (green carbons) and Glu-490 (cyan carbons). CH–π interactions are depicted as dashed lines with distances to the ring centers in Angstroms. The active-site zinc ions are shown as orange spheres. d Surface representation of PSMA with residues of the glutarate sensor interaction with the FMR moiety colored blue, PDB code (7BFZ).

Yiguang Wang, Cyril Bařinka & Xing Yang Research Groups

Significance

Surgery is an efficient way to treat localized prostate cancer (PCa), however, it is challenging to demarcate rapidly and accurately the tumor boundary intraoperatively, as existing tumor detection methods are seldom performed in real-time. To overcome those limitations, we develop a fluorescent molecular rotor that specifically targets the prostate-specific membrane antigen (PSMA), an established marker for PCa. The probes have picomolar affinity (IC₅₀= 63-118 pM) for PSMA and generate virtually instantaneous onset of robust fluorescent signal proportional to the concentration of the PSMA-probe complex. In vitro and ex vivo experiments using PCa cell lines and clinical samples, respectively, indicate the utility of the probe for biomedical applications, including real-time monitoring of endocytosis and tumor staging. Experiments performed in a PCa xenograft model reveal suitability of the probe for imaging applications in vivo.

Zhang, J.; Rakhimbekova, A.; Duan, X.; Yin, Q.; Foss, C. A.; Fan, Y.; Xu, Y.; Li, X.; Cai, X.; Kutil, Z.; Wang., P.; Yang, Z.; Zhang, N.; Pomper, M. G.; Wang, Y.; Bařinka, C. & Yang, X.: A prostate-specific membrane antigen activated molecular rotor for real-time fluorescence imaging, Nature Commun. (2021)12:5460, https://doi.org/10.1038/s41467-021-25746-6

Overall architecture of the giant E3 ligase HUWE1_N.

a, Domain architecture of HUWE1_N. ARM repeats 1–34 are numbered, with the four insertions indicated. The positions of human HUWE1 insertions, absent in HUWE1_N, are shown in brackets. b, Crystal structure of HUWE1_N. c, Crystal structure of HUWE1_N shown in cartoon representation from four different views, using the same color coding as in a (catalytic Cys in red). A schematic cartoon illustrates the snake-like organization of the E3 ligase. d, Negative-stain EM analysis of CeHUWE1. The obtained EM density is shown from two viewpoints, with approximate dimensions indicated. e, Organization and increasing complexity of HUWE1.

Tim Clausen Research Group

Significance

HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin–proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of NematocidaHUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure, where the C-terminal HECT domain heads an extended alpha-solenoid body that coils in on itself and houses various protein–protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins.

Grabarczyk, D. B.; Petrova, O. A.; Deszcz, L.; Kurzbauer, R.; Murphy, P.; Ahel, J.; Vogel, A.; Gogova, R.; Faas, V.; Kordic, D.; Schleiffer, A.; Meinhart, A.; Imre, R.; Lehner, A.; Neuhold, J.; Bader, G.; Stolt-Bergner, P.; Böttcher, J.; Wolkerstorfer, B.; Fischer, G.; Grishkovskaya, I.; Haselbach, D.; Kessler, D. & Clausen, T.: HUWE1 employs a giant substrate-binding ring to feed and regulate its HECT E3 domain, Nat. Chem. Biol. (2021) https://doi.org/10.1038/s41589-021-00831-5

Structure of virion of rhinovirus 14 contains resolved density corre- sponding to octanucleotides from its RNA genome. (A) Surface representa- tion of cryo-EM of reconstruction of virion of rhinovirus 14 with front half of the particle removed to show internal structure. Density corresponding to VP1 is shown in blue, VP2 in green, VP3 in red, VP4 in yellow, and RNA segments in pink. Borders of a selected icosahedral asymmetric unit are in- dicated with a dashed triangle and positions of selected twofold, threefold, and fivefold symmetry axes are represented by an oval, triangle, and pen- tagon, respectively. (Scale bar, 5 nm.) (B) Cartoon representation of icosa- hedral asymmetric unit of rhinovirus 14 viewed from the inside of the capsid. The color coding of individual virus components is the same as in A. Positions of twofold, threefold, and fivefold symmetry axes are represented by an oval, triangle, and pentagon, respectively. (C) Two RNA octanucleotides that interact with each other and with VP2 subunits. Protein and RNA coloring is the same as in A. The cryo-EM density of the RNA segments is shown as a pink semitransparent surface. RNA bases and side chains of Trp38 of VP2 are shown in stick representation, in orange, and indicated with black arrow- heads. The position of a twofold symmetry axis is indicated with an oval. (D) Detail of stacking interaction between Gua2 from RNA segment and Trp38 of VP2. The cryo-EM densities of RNA and protein are shown as semitrans- parent surfaces in pink and gray, respectively. (E) Interaction between N ter- minus of VP1 and genome. Capsid proteins are shown in cartoon representation with the same coloring as in A. Cryo-EM densities of individual proteins are shown as semitransparent surfaces colored according to the chain they belong to. The density of the RNA genome is shown in gray. The blue arrow indicates the contact between the N terminus of VP1 and the genome. The position of Thr17, the first modeled residue from the N terminus of VP1, is indicated.

Pavel Plevka Research Group

Significance

Most rhinoviruses, which are the leading cause of the common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. To release their genomes, rhinoviruses convert to acti- vated particles that contain pores in the capsid, lack minor capsid protein VP4, and have an altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here, we present the structures of virion of rhinovirus 14 and its complex with ICAM-1 determined to resolutions of 2.6 and 2.4 Å, respectively. The cryo- electron microscopy reconstruction of rhinovirus 14 virions contains the resolved density of octanucleotide segments from the RNA ge- nome that interact with VP2 subunits. We show that the binding of ICAM-1 to rhinovirus 14 is required to prime the virus for activation and genome release at acidic pH. Formation of the rhinovirus 14– ICAM-1 complex induces conformational changes to the rhinovirus 14 capsid, including translocation of the C termini of VP4 subunits, which become poised for release through pores that open in the capsids of activated particles. VP4 subunits with altered conforma- tion block the RNA–VP2 interactions and expose patches of posi- tively charged residues. The conformational changes to the capsid induce the redistribution of the virus genome by altering the capsid–RNA interactions. The restructuring of the rhinovirus 14 cap- sid and genome prepares the virions for conversion to activated particles. The high-resolution structure of rhinovirus 14 in complex with ICAM-1 explains how the binding of uncoating receptors en- ables enterovirus genome release.

Hrebik, D.; Fuzik, T.; Gondova, M.; Smerdova, L.; Adamopoulos, A.; Sedo, O.; Zdrahal, Z. & Plevka, P.: ICAM-1 induced rearrangements of capsid and genome prime rhinovirus 14 for activation and uncoating, PNAS 2021 Vol. 118 No. 19 e2024251118, https://doi.org/10.1073/pnas.2024251118

Anillin generates tens of pico-Newton forces to slide actin filaments 
a Schematic representation of the experimental setup. b Time-lapse fluorescence micrographs showing an actin bundle attached between two silica microspheres. The bundle is being stretched as the left microsphere is pulled leftwards by an optical trap. c Typical force time-trace (top) and the force-distance curve (bottom) corresponding to stretching of an anillin-actin filament bundle (experimental data points—magenta). Asymptotic forces of individual stretching steps, calculated by fitting an exponential to the force decays (as shown in d), are indicated by black crosses. These increase hyperbolically with increasing distance between the microspheres, and thus with decreasing overlap length L. The green line represents ~1/L fit to the data. d Temporal response of the construct to stretching and relaxation; the left optical trap is moved 100 nm away from the right trap and then, after ~7 s, moved back to the original position. The temporal profile of the longitudinal position of the left optical trap is shown together with the detected distance between the microspheres (top) and the detected force (bottom). e The detected force increased with decreasing overlap length before the filaments slid apart completely (distance to disconnection = 0). All events longer than six steps are plotted (n = 11 experiments indicated by different colours). Inset, anillin density in the overlap (fluorescence intensity of anillin per unit length of the overlap) at the start and the end of the bundle stretching. Grey boxplots represent raw data, green boxplots represent data after photobleaching correction (see Fig. S2e for the photobleaching estimation) (n = 10 experiments). Corresponding data points overlay the boxplots. f, g Force response of a pre-stretched actin-anillin bundle to a decrease (f) or increase (g) of anillin-GFP concentration. Schematic representation of the experiment (top) and temporal experimental data (bottom). Decrease of the concentration (n = 15 events in 14 experiments), increase of the concentration (n = 15 events in 14 experiments). Green curves are the experimental data, mean temporal profile is shown in magenta. Box and whisker plots show a significant decrease or increase in force between time points 0 and 30 s after a decrease (one-sided Wilcoxon test, p = 0.02) or increase (one-sided Wilcoxon test, p = 0.03) of anillin-GFP concentration. In e–gdata were represented as boxplots. Central marks represent median, top and bottom edges of the box indicate the 75th and 25th percentiles, respectively. Whiskers extend the most extreme points that are not considered outliers. Outliers are marked as grey circles.

Zdeněk Lánský Research Group

Significance

Constriction of the cytokinetic ring, a circular structure of actin filaments, is an essential step during cell division. Mechanical forces driving the constriction are attributed to myosin motor proteins, which slide actin filaments along each other. However, in multiple organisms, ring constriction has been reported to be myosin independent. How actin rings constrict in the absence of motor activity remains unclear. Here, we demonstrate that anillin, a non­motor actin crosslinker, indispensable during cytokinesis, autonomously propels the contractility of actin bundles. Anillin generates contractile forces of tens of pico-Newtons to maximise the lengths of overlaps between bundled actin filaments. The contractility is enhanced by actin disassembly. When multiple actin filaments are arranged into a ring, this contractility leads to ring constriction. Our results indicate that passive actin crosslinkers can substitute for the activity of molecular motors to generate contractile forces in a variety of actin networks, including the cytokinetic ring.

Kučera, O.; Siahaan, V.; Janda, D.; Dijkstra, S. H.; Pilátová, E.; Zatecka, E.; Diez, S.; Braun, M. & Lansky, Z.: Anillin propels myosin-independent constriction of actin rings, Nature Commun. (2021)12:4595 | https://doi.org/10.1038/s41467-021-24474-1

Cryo-EM structure of TEL bound to the yeast ribosome. a Transverse section of the cryo-EM map density (gray) of the large (60S) subunit of the yeast G2400A mutant ribosome with TEL (salmon) bound within the NPET. b Isolated cryo-EM density for TEL (gray mesh) with fitted molecular model for TEL. The similarly-oriented chemical structure of TEL is shown for reference. c TEL bound within the NPET with the surrounding nucleotides of the 25S rRNA (gray) and His133 residue of uL22 protein (purple). The N1 of A2400(A2058) forms a hydrogen bond interaction to the hydroxyl group of the desosamine sugar of TEL and a water molecule (W1) mediates interaction of the desosamine’s dimethylamine with the N6 of A2400(A2058), as was also observed in bacteria43. The alkyl–aryl sidechain of TEL stacks upon the base pair A884(A752)-U2978(U2609) and forms bridging interaction with water W2 (light blue) and O6 of G880(G748). d Superposition of TEL bound to the E. coli ribosome (green, PDB ID 4V7S15) with TEL (salmon) in complex with the S. cerevisiae 60S subunit (Sc60S) bearing the G2400A mutation. E. coli rRNA and ribosomal protein residues are light green, yeast rRNA and protein residues are gray.

Daniel N. Wilson and Alexander S. Mankin Research Group

Significance

Macrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukar- yotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and bio- chemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.

Svetlov, M. S.; Koller, T. O.; Meydan, S.; Shankar, V.; Klepacki, D.; Polacek, N.; Guydosh, N. R.; Vazquez-Laslop, N.; Wilson, D. N. & Mankin, A. S.: Context-specific action of macrolide antibiotics on the eukaryotic ribosome, Nature Commun. (2021)12:2803 https://doi.org/10.1038/s41467-021-23068-1

The essential fungal‐specific translation elongation factor 3 (eEF3) has been implicated in tRNA binding and release. Combined in vitro and in vivo analyses show that its critical is in release of E‐site‐tRNA from the ribosome during late steps of translocation.

Daniel N. Wilson Research Group

Significance

In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA–tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl‐tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slowdown in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo‐EM analysis of native eEF3‐ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non‐rotated ribosomal states, as well as by opening the L1 stalk to release the E‐site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.

Ranjan, N.; Pochopien, A.; Chih-Chien Wu, C.; Beckert, B.; Blanchet, S.; Green, R.; Rodnina, M. & Wilson, D. N.: Yeast translation elongation factor eEF3 promotes late stages of tRNA translocation during RF3-mediated recycling of RF1 EMBO J (2021) 40: e106449; https://doi.org/10.15252/embj.2020106449