CIISB Research Highlights Archive

Structure of the RcGTA particle and organization of segments of the R. capsulatus genome encoding protein components of RcGTA particles. a Cryo-EM reconstruction of a native particle of RcGTA from R. capsulatus strain DE442 calculated from 42,242 particle images. The left part of the panel shows the complete particle, whereas on the right the front half of the particle has been removed to show DNA and internal proteins. Individual proteins in the density map are colored according to the gene map in panel b. Yellow mesh highlights the structural organization of capsid proteins within the RcGTA head. The inset shows an example of a two-dimensional class average and an electron micrograph of an RcGTA particle. The scale bar within the inset represents 20 nm. b Gene map of three genome segments encoding fourteen structural proteins of RcGTA particles. c Cryo-EM reconstruction of an RcGTA particle from R. capsulatus strain DE442 with T = 3 quasi-icosahedral head. The reconstruction is based on 1076 particle images. The structure is at the scale of those shown in panel a. The inset shows an example of a two-dimensional class average and an electron micrograph of RcGTA particle with an icosahedral head. Scale bar represents 20 nm. d Organization of capsomers in the oblate capsid of RcGTA. Capsomers forming one fifth of the capsid are highlighted in different colors and marked with P for pentamer and H for hexamer.

Pavel Plevka Research Group

Significance

Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm.

Bárdy, P.; Füzik, T.; Hrebík, D.; Pantůček, R.; Beatty, T. .J. & Plevka, P: Structure and mechanism of DNA delivery of a gene transfer agent, Nat. Commun. (2020) 11, 3034. doi.org/10.1038/s41467-020-16669-9

Crystal structure of BP39L and CV39L. (A) Crystal structure of BP39L shown in secondary structure representation (β-strands in yellow and loops in green) (B) Surface representation (yellow) of BP39L color-coded according to putative binding sites (blue). (C) Crystal structure of CV39L shown in secondary structure representation (β-strands in violet, α-helices in cyan and loops in pink). (D) Surface representation (violet) of CV39L color-coded according to putative binding sites (blue), α-helices shown in cyan.

Michaela Wimmerova Research Group

Significance

Burkholderia pseudomallei and Chromobacterium violaceum are bacteria of tropical and subtropical soil and water that occasionally cause fatal infections in humans and animals. Microbial lectins mediate the adhesion of organisms to host cells, which is the first phase in the development of infection. Here we report the discovery of two novel lectins from the above-mentioned bacteria – BP39L and CV39L. The crystal structures revealed that the lectins possess a seven-bladed β-propeller fold. Functional studies conducted on a series of oligo- and polysaccharides confirmed the preference of BP39L for mannosylated saccharides and CV39L for rather more complex polysaccharides with a monosaccharide preference for β-l-fucose. The presented data indicate that the proteins belong to a currently unknown family of lectins.

Sykorova, P.; Novotna, J.; Demo, G.; Pompidor, G.; Dubska, E.; Komarek, J.; Fujdiarova, E.; Houser, J.; Haronikova, L.; Varrot, A.; Shilova, N.; Imberty, A.; Bovin, N.; Pokorna, M. & Wimmerova, M.: Characterization of novel lectins from Burkholderia pseudomallei and Chromobacterium violaceum with seven-bladed beta-propeller fold, Int. J. Biol. Macromol. 2019, 152, 1113-1124, doi.org/10.1016/j.ijbiomac.2019.10.200

The NMR structure of the self-processing module (SPM) of the Neisseria meningitidis FrpC protein. (A) Schematic representation of the Ca²⁺-dependent clip-and-link of FrpC. The Ca²⁺-induced folding of FrpC is associated with a Ca²⁺-dependent conformational switch of SPM (residues 415 to 591 of FrpC, in orange), which promotes autocatalytic processing of the D₄₁₄-P₄₁₅ peptide bond and covalent linkage of the released D₄₁₄ residue to an e-amino group of a neighboring lysine residue through an Asp-Lys isopeptide bond. The residues of the putative EF-hand-like Ca²⁺-binding motifs are underlined. (B) Overlay of the ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled SPM in the absence (-) and in the presence (+) of 10 mM CaCl₂. (C) Overlay of backbone traces of the 20 lowest energy structures of Ca-SPM solved by NMR, shown in a rainbow representation from blue (N terminus) to red (C terminus). (D) Topology of secondary structure elements of Ca-SPM generated by Pro-origami.

Lukáš Žídek Research Group and Peter Šebo Research Group

Significance

The posttranslational Ca²⁺-dependent “clip-and-link” activity of large repeat-in-toxin (RTX) proteins starts by Ca²⁺-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free e-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neiseria meningitidis. The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a “twisted-amide” activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca²⁺-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface.

Kuban, V.; Macek, P.; Hritz, J.; Nechvatalova, K.; Nedbalcova, K.; Faldyna, M.; Zidek, L. & Bumba, L.: Structural Basis of Ca2􏰀-Dependent Self-Processing Activity of Repeat-in-Toxin Proteins, mBio. 2020, 11:e00226-20, https://doi.org/10.1128/mBio.00226-20.

(−)-Bactobolin A and selected related natural products.

(−)-Bactobolin A (1) is a polyketide–peptide natural product first isolated in the late 1970s as a secondary metabolite of Pseudomonas sp. Early biological evaluations revealed that 1 exhibits broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens and in vivo antiproliferative effects on certain cancer cell lines. (−)-Bactobolin A (1) was subsequently identified as a strong inhibitor of protein synthesis in intact prokaryotic and eukaryotic cells, and mammalian cell-free systems.

Jakub Švenda Research Group

Significance

A stereoselective synthesis of the ribosome-binding antitumor antibiotic (−)-bactobolin A is reported. The presented approach makes effective use of (−)-quinic acid as a chiral pool starting material and substrate stereocontrol to establish the five contiguous stereocenters of (−)-bactobolin A. The key steps of the synthesis include a stereoselective vinylogous aldol reaction to introduce the unusual dichloromethyl substituent, a completely diastereoselective rhodium(II)-catalyzed C–H amination reaction to set the configuration of the axial amine, and an intramolecular alkoxycarbonylation to build the bicyclic lactone framework. The developed synthetic route was used to prepare 90 mg of (−)-bactobolin A trifluoroacetate in 10% overall yield.

Vojáčková, P.; Michalska, L.; Nečas, M.; Shcherbakov, D.; Böttger, E. C.; Šponer, J.; Šponer, J. E. & Švenda, J.: Stereocontrolled Synthesis of (−)-Bactobolin A,  J. Am. Chem. Soc.  2020, 142, 7306-7311, doi.org/10.1021/jacs.0c01554

Structure of STAU1 dsRBD4 and dsRBD3/4 in complex with sARF1 SBS dsRNA. (A) Structural ensemble of the STAU1 dsRBD4–sARF1 SBS dsRNA complex. Heavy-atom superposition of the ensemble of the 20 lowest-energy structures. The protein backbone is shown in dark green and the RNA heavy atoms of the bases in orange and those of the ribose-phosphodiester backbone are shown in gold (omitting phosphate and 2’-OH oxygens). (B) Schematic representation of sARF1 SBS dsRNA showing interactions of dsRBD3 as well as dsRBD4 as dotted lines. Interactions with the ribose-phosphodiester backbone are circled in dark green for dsRBD4 and light green for dsRBD3 while base interactions are shown as filled circles.

Peter J. Lukavsky Research Group

Significance

Staufen1 (STAU1) is a dsRNA binding protein mediating mRNA transport and localization, translational control and STAU1-mediated mRNA decay (SMD). The STAU1 binding site (SBS) within human ADP-ribosylation factor1 (ARF1) 3’ UTR binds STAU1 and this downregulates ARF1 cytoplasmic mRNA levels by SMD. However, how STAU1 recognizes specific mRNA targets is still under debate. The structure of the ARF1 SBS–STAU1 complex, presented in this study, uncovers target recognition by STAU1. STAU1 dsRNA binding domain (dsRBD) 4 interacts with two pyrimidines and one purine from the minor groove side via helix a1, the b1–b 2 loop anchors the dsRBD at the end of the dsRNA and lysines in helix a2 bind to the phosphodiester backbone from the major groove side. STAU1 dsRBD3 displays the same binding mode with specific recognition of one guanine base. Mutants disrupting minor groove recognition of ARF1 SBS affect in vitrobinding and reduce SMD in vivo. Our data thus reveal how STAU1 recognizes minor groove features in dsRNA relevant for target selection.

Yadav, D. K.; Zigáčková, D.; Zlobina, M.; Klumpler, T.; Beaumont, C.; Kubíčková, M.; Vaňáčová, Š. & Lukavsky, P. J.: Staufen1 reads out structure and sequence features in ARF1 dsRNA for target recognition,  Nucleic Acids Res. 2020, 48, 2091-2106, doi:10.1093/nar/gkz1163

Crystal structures of (A) PBD anthramycin covalently bound to DNA strands and (B) lincomycin targeting the peptidyl transferase center in the 50S ribosomal subunit of Staphylococcus aureus.

Jiří Janata Research Group

Significance

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorumsensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of L -tyrosine- or L -leucine-derived 4-alkyl-L -proline derivatives (APDs) in their structures. In this study, the authors report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420 H2 -dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosynthesis of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4- substituted Δ 1-pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. Furthermore, the differences in the reaction specificity of the Apd6 reductases determine the formation of the fully saturated APD moiety of lincomycin versus the unsaturated APD moiety of PBDs, providing molecules with optimal shapes to bind their distinct biological targets. Moreover, the Apd6 reductases establish the first F420 H2-dependent enzymes from the luciferase-like hydride transferase protein superfamily in the biosynthesis of bioactive molecules. Finally, bioinformatics analysis demonstrates that Apd6 and their homologues, widely distributed within several bacterial phyla, play a role in the formation of novel yet unknown natural products with incorporated L-proline-like precursors and likely in the microbial central metabolism.

Steiningerova, L.; Kamenik, Z.*; Gazak, R.; Kadlcik, S.; Bashiri, G.; Man, P.; Kuzma, M.; Pavlikova, M. & Janata, J.: Different Reaction Specificities of F420H2‑Dependent Reductases Facilitate Pyrrolobenzodiazepines and Lincomycin To Fit Their Biological Targets, J. Am. Chem. Soc.  2020, 142, 3440-3448, https://dx.doi.org/10.1021/jacs.9b11234