Jana Aupič’s research while affiliated with Istituto Officina dei Materiali, Italian National Research Council and other places

Versatile DNA and polypeptide‐based structures have been designed based on complementary modules. However, polypeptides can also form higher oligomeric states. We investigated the introduction of tetrameric modules as a substitute for coiled‐coil dimerization units used in previous modular nanostructures. Tetramerizing helical bundles can run in parallel or antiparallel orientation, expanding the number of topological solutions for modular nanostructures. Furthermore, this strategy facilitates the construction of nanostructures from two identical polypeptide chains. Importantly, tetrameric modules substantially stabilized protein nanostructures against air–water interface denaturation, enabling the determination of the first cryo‐electron microscopy three‐dimensional structure of a coiled‐coil‐based nanostructure, confirming the designed agreement of the modules forming a tetrahedral cage.

Versatile DNA and polypeptide‐based structures have been designed based on complementary modules. However, polypeptides can also form higher oligomeric states. We investigated the introduction of tetrameric modules as a substitute for coiled‐coil dimerization units used in previous modular nanostructures. Tetramerizing helical bundles can run in parallel or antiparallel orientation, expanding the number of topological solutions for modular nanostructures. Furthermore, this strategy facilitates the construction of nanostructures from two identical polypeptide chains. Importantly, tetrameric modules substantially stabilized protein nanostructures against air‐water interface denaturation, enabling the determination of the first cryo‐electron microscopy three‐dimensional structure of a coiled‐coil‐based nanostructure, confirming the designed agreement of the modules forming a tetrahedral cage.

RNA-membrane interactions are starting to emerge as an important organizing force in both natural and synthetic biological systems. Notably, RNA molecules were recently discovered to be present on the extracellular surface of living cells, where they mediate intercellular signalling. Furthermore, RNA-membrane interactions influence the efficacy of lipid-based RNA delivery systems. However, the molecular terms driving RNA localisation at the membrane remain poorly understood. In this work, we investigate how RNA-phospholipid membrane interactions occur, by means of all-atom simulations. We find that among the four RNA nucleobases guanine exhibits the strongest interaction with the membrane due to extensive hydrogen bond formation. Additionally, we show that intra-RNA base pairing present in organised RNA structures significantly hinders RNA binding to the membrane. Elucidating the molecular details of RNA-membrane association will importantly contribute to improving the design of RNA-based drugs as well as lipid-based RNA delivery systems and to parsing out RNA transport and localisation mechanisms.

Nucleic acid processing enzymes use a two‐Mg²⁺‐ion motif to promote the formation and cleavage of phosphodiester bonds. Yet, recent evidence demonstrates the presence of spatially conserved second‐shell cations surrounding the catalytic architecture of proteinaceous and RNA‐dependent enzymes. The RNase mitochondrial RNA processing (MRP) complex, which cleaves the ribosomal RNA (rRNA) precursor at the A3 cleavage site to yield mature 5′‐end of 5.8S rRNA, hosts in the catalytic core one atypically‐located Mg²⁺ ion, in addition to the ions forming the canonical catalytic motif. Here, we employ biased quantum classical molecular dynamics simulations of RNase MRP to discover that the third Mg²⁺ ion inhibits the catalytic process. Instead, its displacement in favour of a second‐shell monovalent K⁺ ion propels phosphodiester bond cleavage by enabling the formation of a specific hydrogen bonding network that mediates the essential proton transfer step. This study points to a direct involvement of a transient K⁺ ion in the catalytic cleavage of the phosphodiester bond and implicates cation trafficking as a general mechanism in nucleic acid processing enzymes and ribozymes.

Nucleic acid processing enzymes use a two‐Mg2+‐ion motif to promote the formation and cleavage of phosphodiester bonds. Yet, recent evidence demonstrates the presence of spatially conserved second‐shell cations surrounding the catalytic architecture of proteinaceous and RNA‐dependent enzymes. The RNase mitochondrial RNA processing (MRP) complex, which cleaves the ribosomal RNA (rRNA) precursor at the A3 cleavage site to yield mature 5'‐end of 5.8S rRNA, hosts in the catalytic core one atypically‐located Mg2+ ion, in addition to the ions forming the canonical catalytic motif. Here, we employ biased quantum classical molecular dynamics simulations of RNase MRP to discover that the third Mg2+ ion inhibits the catalytic process. Instead, its displacement in favour of a second‐shell monovalent K+ ion propels phosphodiester bond cleavage by enabling the formation of a specific hydrogen bonding network that mediates the essential proton transfer step. This study points to a direct involvement of a transient K+ ion in the catalytic cleavage of the phosphodiester bond and implicates cation trafficking as a general mechanism in nucleic acid processing enzymes and ribozymes.

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.

Two new 'hybrid' metallodrugs of Au(III) (AuTAML) and Cu(II) (CuTAML) were designed featuring a tamoxifen-derived pharmacophore to ideally synergize the anticancer activity of both the metal center and the organic ligand. The compounds have antiproliferative effects against human MCF-7 and MDA-MB 231 breast cancer cells. Molecular dynamics studies suggest that the compounds retain the binding activity to estrogen receptor (ERα). In vitro and in silico studies showed that the Au(III) derivative is an inhibitor of the seleno-enzyme thioredoxin reductase, while the Cu(II) complex may act as an oxidant of different intracellular thiols. In breast cancer cells treated with the compounds, a redox imbalance characterized by a decrease in total thiols and increased reactive oxygen species production was detected. Despite their different reactivities and cytotoxic potencies, a great capacity of the metal complexes to induce mitochondrial damage was observed as shown by their effects on mitochondrial respiration, membrane potential, and morphology.

The spliceosome machinery catalyzes precursor-messenger RNA (pre-mRNA) splicing by undergoing at each splicing cycle assembly, activation, catalysis, and disassembly processes, thanks to the concerted action of specific RNA-dependent ATPases/helicases. Prp2, a member of the DExH-box ATPase/helicase family, harnesses the energy of ATP hydrolysis to translocate a single pre-mRNA strand in the 5' to 3' direction, thus promoting spliceosome remodeling to its catalytic-competent state. Here, we established the functional coupling between ATPase and helicase activities of Prp2. Namely, extensive multi-μs molecular dynamics simulations allowed us to unlock how, after pre-mRNA selection, ATP binding, hydrolysis, and dissociation induce a functional typewriter-like rotation of the Prp2 C-terminal domain. This movement, endorsed by an iterative swing of interactions established between specific Prp2 residues with the nucleobases at 5'- and 3'-ends of pre-mRNA, promotes pre-mRNA translocation. Notably, some of these Prp2 residues are conserved in the DExH-box family, suggesting that the translocation mechanism elucidated here may be applicable to all DExH-box helicases.