Comparison of V-shaped packing in ApoE4 and ApoE3 structures. The conformational change of the loop connecting helices H2 and H3 in ApoE3 structures (PDB IDs: 1OR2 and 1OR3) leads to V-shaped packing but to a different residue arrangement at the self-association interface than in ApoE4 structures.

Martin Marek Research Group

Significance

Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer’s Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. Here, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE ε3/ε3 and ε4/ε4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. We found that C112R substitution in ApoE4 induces long-distance (> 15 Å) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE ε4/ε4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. Our results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing.

Nemergut, M., Marques, S.M., Uhrik, L. et al.: Domino-like effect of C112R mutation on ApoE4 aggregation and its reduction by Alzheimer’s Disease drug candidate.

Mol. Neurodegeneration 2023, 18, 38 (2023). https://doi.org/10.1186/s13024-023-00620-9

Comparison of the structure of Sen1 helicase domain (PBD ID 5MZN -(52)) and the helicase domain of SETX, predicted by AI-mediated modelling using AlphaFold2. RecA1 and RecA2 domains are depicted in yellow, β-barrel in orange, and the ‘prong’ subdomain in red. In purple are highlighted the key residues responsible for ATP hydrolysis (K1969, within the Walker A motif), as well as for Mg²⁺ coordination (D2181, E2182, within the Walker B motif).

Richard Štefl Research Group

Significance

Prolonged pausing of the transcription machinery may lead to the formation of three-stranded nucleic acid structures, called R-loops, typically resulting from the annealing of the nascent RNA with the template DNA. Unscheduled persistence of R-loops and RNA polymerases may interfere with transcription itself and other essential processes such as DNA replication and repair. Senataxin (SETX) is a putative helicase, mutated in two neurodegenerative disorders, which has been implicated in the control of R-loop accumulation and in transcription termination. However, understanding the precise role of SETX in these processes has been precluded by the absence of a direct characterisation of SETX biochemical activities. Here, we purify and characterise the helicase domain of SETX in parallel with its yeast orthologue, Sen1. Importantly, we show that SETX is a bona fide helicase with the ability to resolve R-loops. Furthermore, SETX has retained the transcription termination activity of Sen1 but functions in a species-specific manner. Finally, subsequent characterisation of two SETX variants harbouring disease-associated mutations shed light into the effect of such mutations on SETX folding and biochemical properties. Altogether, these results broaden our understanding of SETX function in gene expression and the maintenance of genome integrity and provide clues to elucidate the molecular basis of SETX-associated neurodegenerative diseases.

Hasanová, Z., et. al.: Human senataxin is a bona fide R-loop resolving enzyme and transcription termination factor.

Nucleic Acids Res. 2023, 51, 2818–2837, https://doi.org/10.1093/nar/gkad092

(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