CIISB Research Highlights Archive

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

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