CIISB Research Highlights Archive

(D–F) Ribbon diagrams for the complete cryo-EM structures. Shown are (D) the A/H7N9 TRX structure stalled by CMPcPP, (E) T1106-stalled and backtracked A/H7N9 structure, and (F) T1106-stalled and backtracked FluB structure. The polymerase color code is as follows: PA endonuclease (forest green), PA-C-terminal domain (green), PB1 (cyan), PB2-N (red), PB2-midlink domain (magenta), PB2-cap-binding domain (orange), PB2-627 domain (deep pink), and PB2-NLS domain (firebrick). The RNA is colored as in (A)–(C), with (D) the CMPcPP shown in red and (E and F) the observed backtracked nucleotides shown in purple-blue.

(G–I) Determined structures of the RNA moieties. Shown are (G) the A/H7N9 TRX structure stalled by CMPcPP (red) with template (orange), 18-mer capped product (slate blue), and 5′ end (violet); (H) T1106-stalled and backtracked A/H7N9 structure with singly incorporated T1106 (magenta), template (pale orange), 21-mer capped product (pale blue), and 5′ end (violet); and (I) the T1106-stalled and backtracked FluB structure with doubly incorporated T1106 (magenta), template (yellow), 21-mer capped product (blue), and 5′ end (violet). In each case, the conformation of the priming loop residues 632–660 in A/H7N9 (631–659 in FluB) are shown in cyan, fully extruded in (G) and (I), partially extruded in (H), and with the position of Tyr657 (Tyr656 in FluB) highlighted.

Stephen Cusack Research Group

Significance

The antiviral pseudo-base T705 and its de-fluoro analog T1106 mimic adenine or guanine and can be competitively incorporated into nascent RNA by viral RNA-dependent RNA polymerases. Although dispersed, single pseudo-base incorporation is mutagenic, consecutive incorporation causes polymerase stalling and chain termination. Using a template encoding single and then consecutive T1106 incorporation four nucleotides later, we obtained a cryogenic electron microscopy structure of stalled influenza A/H7N9 polymerase. This shows that the entire product-template duplex backtracks by 5 nt, bringing the singly incorporated T1106 to the +1 position, where it forms an unexpected T1106:U wobble base pair. Similar structures show that influenza B polymerase also backtracks after consecutive T1106 incorporation, regardless of whether prior single incorporation has occurred. These results give insight into the unusual mechanism of chain termination by pyrazinecarboxamide base analogs. Consecutive incorporation destabilizes the proximal end of the product-template duplex, promoting irreversible backtracking to a more energetically favourable overall configuration.

Kouba, T., et.al.: Direct observation of backtracking by influenza A and B polymerases upon consecutive incorporation of the nucleoside analog T1106 Cell Rep. 2023, 42, 111901, https://doi.org/10.1016/j.celrep.2022.111901

a, Schematic illustration of the in silico design of libraries of de novo and unevolved random-sequence proteins. A de novo library (DN) was built from putative de novo proteins identified in human and fly. Subsequently, a library of unevolved random sequences (R) was designed to mirror the length and amino acid frequencies of library DN. The two libraries were synthesized by oligonucleotide library synthesis ready for experimental study. b, Approaches used to profile solubility and structure content of each library. Following amplification, each library was expressed in a chaperone-assisted cell-free format and structural content was quantified using a proteolytic assay. In parallel, subcloned libraries were expressed in E. coli to screen for soluble and folded variants that did not disrupt periplasmic export.

Klára Hlouchová and Erich Bornberg-Bauer Research Groups

Significance

De novo gene emergence provides a route for new proteins to be formed from previously non-coding DNA. Proteins born in this way are considered random sequences and typically assumed to lack defined structure. While it remains unclear how likely a de novo protein is to assume a soluble and stable tertiary structure, intersecting evidence from random sequence and de novo-designed proteins suggests that native-like biophysical properties are abundant in sequence space. Taking putative de novo proteins identified in human and fly, we experimentally characterize a library of these sequences to assess their solubility and structure propensity. We compare this library to a set of synthetic random proteins with no evolutionary history. Bioinformatic prediction suggests that de novo proteins may have remarkably similar distributions of biophysical properties to unevolved random sequences of a given length and amino acid composition. However, upon expression in vitro, de novo proteins exhibit moderately higher solubility which is further induced by the DnaK chaperone system. We suggest that while synthetic random sequences are a useful proxy for de novo proteins in terms of structure propensity, de novo proteins may be better integrated in the cellular system than random expectation, given their higher solubility.

Brennen, H. et. al.: Experimental characterization of de novo proteins and their unevolved random-sequence counterparts Nat. Ecol. Evol. (2023), 7, 570-580: https://doi.org/10.1038/s41559-023-02010-2

Detection of SARS-CoV-2 N protein. (A) UCNP label: Alkyne-PEG-neridronate strongly binds via two phosphonate groups to surface lanthanide ions of UCNPs, and a click reaction binds the conjugate to azide-modified streptavidin. (B) Scheme of sandwich ULISA: A microtiter plate is coated with two monoclonal antibodies that capture the N protein. Then, two biotinylated detection antibodies bind to the N protein. The sandwich immune complex is finally detected by using the UCNP label.

Hans H. Gorris Research Group

Significance

The COVID-19 crisis requires fast and highly sensitive tests for the early stage detection of the SARS-CoV-2 virus. For detecting the nucleocapsid protein (N protein), the most abundant viral antigen, we have employed upconversion nanoparticles that emit short-wavelength light under near-infrared excitation (976 nm). The anti-Stokes emission avoids autofluorescence and light scattering and thus enables measurements without optical background interference. The sandwich upconversion-linked immunosorbent assay (ULISA) can be operated both in a conventional analog mode and in a digital mode based on counting individual immune complexes. We have investigated how different antibody combinations affect the detection of the wildtype N protein and the detection of SARS-CoV-2 (alpha variant) in lysed culture fluid via the N protein. The ULISA yielded a limit of detection (LOD) of 1.3 pg/mL (27 fM) for N protein detection independent of the analog or digital readout, which is approximately 3 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays or commercial lateral flow assays for home testing. In the case of SARS-CoV-2, the digital ULISA additionally improved the LOD by a factor of 10 compared to the analog readout.

Brandmeier, J.C., Jurga, N., Grzyb, T., Hlaváček, A., Obořilová, R., Skládal, P., Farka, Z., and Gorris, H.H.: Digital and Analog Detection of SARS-CoV-2 Nucleocapsid Protein via an Upconversion-Linked Immunosorbent Assay, Anal. Chem. 2023 95, 51, 4753 – 4759, https://doi.org/10.1021/acs.analchem.2c05670

This study experimentally determines the properties that would have accompanied some of the most feasible ncAA candidates from the prebiotic pool. We incorporated the selected ncAAs into combinatorial peptide libraries along with (or replacing) other early amino acids to evaluate their effect on fundamental physicochemical properties such as solubility and secondary structure formation.

Stephen D. Fried and Klara Hlouchová Research Groups

Significance

Whereas modern proteins rely on a quasi-universal repertoire of 20 canonical amino acids (AAs), numerous lines of evidence suggest that ancient proteins relied on a limited alphabet of 10 “early” AAs and that the 10 “late” AAs were products of biosynthetic pathways. However, many nonproteinogenic AAs were also prebiotically available, which begs two fundamental questions: Why do we have the current modern amino acid alphabet, and would proteins be able to fold into globular structures as well if different amino acids comprised the genetic code? Here, we experimentally evaluate the solubility and secondary structure propensities of several prebiotically relevant amino acids in the context of synthetic combinatorial 25-mer peptide libraries. The most prebiotically abundant linear aliphatic and basic residues were incorporated along with or in place of other early amino acids to explore these alternative sequence spaces. The results show that foldability was likely a critical factor in the selection of the canonical alphabet. Unbranched aliphatic amino acids were purged from the proteinogenic alphabet despite their high prebiotic abundance because they generate polypeptides that are oversolubilized and have low packing efficiency. Surprisingly, we find that the inclusion of a short-chain basic amino acid also decreases polypeptides’ secondary structure potential, for which we suggest a biophysical model. Our results support the view that, despite lacking basic residues, the early canonical alphabet was remarkably adaptive at supporting protein folding and explain why basic residues were only incorporated at a later stage of protein evolution.

Makarov M. et al.: Early Selection of the Amino Acid Alphabet Was Adaptively Shaped by Biophysical Constraints of Foldability, J. Amer. Chem. Soc. (2023), 145, 5320-5329, https://doi.org/10.1021/jacs.2c12987

Overview of the dormant ribosome structure from 1 hpf zebrafish (a) and Xenopus egg (b). Ribosome-associated factors are shown as surface representations; eEF2b and eIF5a correspond to Protein Data Bank (PDB) accessions 6MTE and 5DAT, respectively.

Andrea Pauli Research Group

Significance

Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4–eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.

Leesch, F. et. al.: A molecular network of conserved factors keeps ribosomes dormant in the egg, Nature (2023), 613, 712-720: https://doi.org/10.1038/s41586-022-05623-y

Interaction of Myxovalargin with the E. coli ribosome. (a) Relative position of P-site tRNA (green) and erythromycin (Ery, cyan) to 23S rRNA nucleotides protected from DMS by 10 μM (light blue) or 100 μM (dark blue) MyxB.

Andreas Kirschning, Daniel N. Wilson, and R. Müller Research Groups

Significance

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.

Koller, T.O., Scheid, U., et al.: The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition, J. Amer. Chem. Soc. (2023), 145, 851-863 https://doi.org/10.1021/jacs.2c08816