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Abstract Cleavage and formation of phosphodiester bonds in nucleic acids is accomplished by large cellular machineries composed of both protein and RNA. Long thought to rely on a two-metal-ion mechanism for catalysis, structure comparisons revealed many contain highly spatially conserved second-shell monovalent cations, whose precise function remains elusive. A recent high-resolution structure of the spliceosome, essential for pre-mRNA splicing in eukaryotes, revealed a potassium ion in the active site. Here, we employ biased quantum mechanics/ molecular mechanics molecular dynamics to elucidate the function of this monovalent ion in splicing. We discover that the K+ ion regulates the kinetics and thermodynamics of the first splicing step by rigidifying the active site and stabilizing the substrate in the pre- and post-catalytic state via formation of key hydrogen bonds. Our work supports a direct role for the K+ ion during catalysis and provides a mechanistic hypothesis likely shared by other nucleic acid processing enzymes.

Abstract Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism of viral spike protein-mediated fusion or screening for viral inhibitors under biosafety level 1 conditions.

Coiled-coil protein origami (CCPO) is a strategy for the design of polyhedral cage-shaped protein folds based on coiled-coil (CC) dimer-forming peptides. Here, the authors show that CCPO proteins fold in a multistep process governed by the spatial distance between CC modules in the primary sequence and subsequent folding intermediates, which enables the use of identical CC modules for the CCPO tetrahedron design.

Coiled-coil protein origami is a strategy for the de novo design of polypeptide nanostructures based on coiled-coil dimer forming peptides, where a single chain protein folds into a polyhedral cage. Here, the authors design a single-chain triangular bipyramid and also demonstrate that the bipyramid can be self-assembled as a heterodimeric complex, comprising pre-defined subunits.

Copper is a trace element vital to many cellular functions. Yet its abnormal levels are toxic to cells, provoking a variety of severe diseases. The high affinity copper transporter 1 (CTR1), being the main in-cell copper [Cu(I)] entry route, tightly regulates its cellular uptake via a still elusive mechanism. Here, all-atoms simulations unlock the molecular terms of Cu(I) transport in eukaryotes disclosing that the two methionine (Met) triads, forming the selectivity filter, play an unprecedented dual role both enabling selective Cu(I) transport and regulating its uptake rate thanks to an intimate coupling between the conformational plasticity of their bulky side chains and the number of bound Cu(I) ions. Namely, the Met residues act as a gate reducing the Cu(I) import rate when two ions simultaneously bind to CTR1. This may represent an elegant autoregulatory mechanism through which CTR1 protects the cells from excessively high, and hence toxic, in-cell Cu(I) levels. Overall, our outcomes resolve fundamental questions in CTR1 biology and open new windows of opportunity to tackle diseases associated with an imbalanced copper uptake.

Recently an artificial protein named Pizza6 was reported, which possesses six identical tandem repeats and adopts a monomeric β-propeller fold with sixfold structural symmetry. Pizza2, a truncated form that consists of a double tandem repeat, self-assembles into a trimer reconstructing the same propeller architecture as Pizza6. The ability of pizza proteins to self-assemble to form complete propellers makes them interesting building blocks to engineer larger symmetrical protein complexes such as symmetric nanoparticles. Here we have explored the self-assembly of Pizza2 fused to homo-oligomerizing peptides. In total, we engineered five different fusion proteins, of which three appeared to assemble successfully into larger complexes. Further characterization of these proteins showed one monodisperse designer protein with a structure close to the intended design. This protein was further fused to eGFP to investigate functionalization of the nanoparticle. The fusion protein was stable and could be expressed in high yield, showing that Pizza-based nanoparticles may be further decorated with functional domains

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and β-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small β-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The β-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the β-annulus peptide for vaccine design.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-21969-9