CIISB Research Highlights Archive

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

Cryo-EM structure of pT=4 quasi-icosahedral BDP and its penatameric and hexameric components. a Surface model of pT = 4 quasi-icosahedral BDP particle, displayed on the left side. A ribbon model of a cmcD pentamer and three cmcC′ hexamers is displayed on the right side. Pentameric cmcD protein is colored in yellow and hexameric cmcC′ is colored in green. Note that the fivefold symmetry axis is located at the center of cmcD pentamer and threefold axis is located in the middle between three cmcC′ hexamers. b Electrostatic surface potential of pentameric cmcD and hexameric cmcC′. Note the pores in the centers of pentamers and hexamers.

Kaspas Tars Research Group

Significance

Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. BMCs are involved in several biochemical processes, such as choline, glycerol and ethanolamine degradation and carbon fixation. Since non-native enzymes can also be encapsulated in BMCs, an improved understanding of BMC shell assembly and encapsulation processes could be useful for synthetic biology applications. Here we report the isolation and recombinant expression of BMC structural genes from the Klebsiella pneumoniae GRM2 locus, the investigation of mechanisms behind encapsulation of the core enzymes, and the characterization of shell particles by cryo-EM. We conclude that the enzymatic core is encapsulated in a hierarchical manner and that the CutC choline lyase may play a secondary role as an adaptor protein. We also present a cryo-EM structure of a pT = 4 quasi-symmetric icosahedral shell particle at 3.3 Å resolution, and demonstrate variability among the minor shell forms.

Kalnins, G.; Cesle, E-E.; Jansons, J.; Liepins, J.; Filimonenko, A. & Tars, K.: Encapsulation mechanisms and structural studies of GRM2 bacterial microcompartment particles, Nature Comm. (2020) 11 (1), No. 388, doi.org/10.1038/s41467-019-14205-y

The unstructured C-terminal domain of delta subunit of bacterial RNA Polymerase is 90 aa long and highly charged. The charge distribution of this domain is distinct, with a conserved stretch of 9 residues (96−104) containing 7 positive charges followed by the rest of the domain with 51 acidic residues (K-D/E motif). A previous study demonstrated that the two parts of the motif transiently interact, and this affects the spatiotemporal properties of this domain. From the biological point of view, δ increases cell fitness and virulence of pathogens and was previously proposed to function as a nucleic acid mimic and affect RNAP− nucleic acid interactions.

Lukáš Žídek Research Group

Significance

Electrostatic interactions play important roles in the functional mechanisms exploited by intrinsically disordered proteins (IDPs). The atomic resolution description of long-range and local structural propensities that can both be crucial for the function of highly charged IDPs presents significant experimental challenges. Here, we investigate the conformational behavior of the δ subunit of RNA polymerase from Bacillus subtilis whose unfolded domain is highly charged, with 7 positively charged amino acids followed by 51 acidic amino acids. Using a specifically designed analytical strategy, we identify transient contacts between the two regions using a combination of NMR paramagnetic relaxation enhancements, residual dipolar couplings (RDCs), chemical shifts, and small-angle scattering. This strategy allows the resolution of long-range and local ensemble averaged structural contributions to the experimental RDCs, and reveals that the negatively charged segment folds back onto the positively charged strand, compacting the conformational sampling of the protein while remaining highly flexible in solution. Mutation of the positively charged region abrogates the long-range contact, leaving the disordered domain in an extended conformation, possibly due to local repulsion of like-charges along the chain. Remarkably, in-vitro studies show that this mutation also has a significant effect on transcription activity, and results in diminished cell fitness of the mutated bacteria in vivo. This study highlights the importance of accurately describing electrostatic interactions for understanding the functional mechanisms of IDPs.

Kuban, V.; Srb, P.; Stegnerova, H.; Padrta, P.; Zachrdla, M.; Jasenakova, Z.; Sanderova, H.; Vitovska, D.; Krasny, L.; Koval, T.; Dohnalek, J.; Ziemska-Legiecka, J.; Grynberg, M.; Jarnot, P.; Gruca, A.; Jensen, M. R.; Blackledge, M. & Zidek, L.: Quantitative Conformational Analysis of Functionally Important Electrostatic Interactions in the Intrinsically Disordered Region of Delta Subunit of Bacterial RNA Polymerase,  J. Am. Chem. Soc.  2019, 141, 16817-16828, DOI:10.1021/jacs.9b07837