Jana Aupič

The chemical richness of RNAs is greatly enhanced by post-transcriptional modifications with RNA methylation as the most prominent type. RNA modifications modulate the stability, folding and interaction pattern of RNA molecules. Furthermore, emerging data suggests RNA modifications also directly regulate the activity of catalytic RNA molecules, i.e., ribozymes. Here, we employ classical and hybrid quantum-classical (QM/MM) molecular dynamics (MD) simulations to investigate the reaction mechanism of an artificial methyltransferase ribozyme MTR1. Importantly, we pinpoint how 2’- O -methylations of active site nucleotides synergistically enhance ribozyme activity by reducing the conformational flexibility of the ribose rings and rigidifying the active site. Finally, the herein reported crystal structure of the modified MTR1, solved at 2.6 Å resolution, validates the results of our simulations. Taken together, our work supports the purported central role of modified RNA for early RNA catalysis and may guide rational design of more efficient ribozymes.

Vaults are massive ribonucleoprotein complexes, highly conserved and abundant in eukaryotic cells, yet with unclear function. Their thin-walled barrel-shape architecture is composed of two symmetrical, antiparallel half-shells, each containing 39 copies of the major vault protein (MVP). The spacious lumen of the vault suggests a role in cellular transport. Although vaults are thought to undergo conformational changes to facilitate cargo exchange, the molecular basis for their inherent flexibility remains unknown. Here, we integrate cryogenic electron microscopy (cryo-EM) and multi-scale molecular dynamics (MD) simulations to reveal the structural determinants of the human vault particle’s flexibility. Cryo-EM identified two high-resolution alternative conformational states: a symmetric and an asymmetric structure, pointing to the vault shell’s structural plasticity. MD simulations of these conformations revealed that these structures are flexible and exhibit breathing-like motions, and porous solvent-exposed surfaces. Mutagenesis disrupting persistent MD-identified inter-half contacts reduced full MVP shell assembly, confirming the functional relevance of these flexibility determinants. Together, these findings establish the molecular basis for the human vault particle’s conformational plasticity.

Interactions between plants and pathogens pose a major challenge for plant health and agriculture. One type of threat comes from widespread cytolysins necrosis- and ethylene-inducing peptide 1–like proteins (NLPs) secreted by microbial pathogens. NLPs disrupt plant membranes through interactions with glycosylinositol phosphorylceramides (GIPCs), but the specificity of these interactions remains unclear. We studied binding and pore formation of NLPs in model membranes with GIPCs derived from dicotyledonous or monocotyledonous plants. Using the cytotoxic NLP 2 from fungus Moniliophthora perniciosa (MpNEP2), we show that membrane binding and damage extend beyond previously studied oomycetal NLPs. Furthermore, NLP _Pya from the oomycete Pythium aphanidermatum exhibits promiscuous binding with a marked preference for branched GIPC series B lipids. Molecular dynamics simulations demonstrated that the structural plasticity of NLP _Pya provides a rational basis for its interaction with a variety of GIPC headgroups. These results clarify the molecular basis of NLP cytotoxicity and emphasize their role in broad-spectrum pathogenicity.

RNA splicing is orchestrated by a complex and exceptionally dynamic RNA-protein machine, called the spliceosome. Stepwise, large-scale structural and compositional remodeling of the spliceosome enables splicing and ensures its fidelity. While cryogenic electron microscopy provided structural information on numerous splicing cycle intermediates, allowing large-scale rearrangements to be inferred on a comparative basis, all-atom simulations complement and enrich structural studies by capturing the dynamic nature of the spliceosome on a finer but equally important scale. Here, we review the current understanding of the spliceosome's function attained by enriching experimental insights with computation. We focus on splicing factors mediating the spliceosome's dynamic behavior, key for splicing cycle progression, and discuss computational challenges on the path toward more accurate large-scale simulations that could further bridge the gap between computational and experimental data. A synergistic interplay between experiment and computation is vital for obtaining high-accuracy structural ensembles of the spliceosome and its components and for addressing unresolved mechanistic and biological questions related to splicing. Such integrative approaches also hold promise for advancing the design of splicing-targeted therapeutics and gene modulation technologies for treating diseases linked to splicing dysregulation.

Branch-site recognition is a pivotal event in spliceosome assembly, and branch-site antagonists disrupt this process, inducing a splicing rewiring that underlies much of their antitumor activity. Antagonists from the spliceostatin family covalently bind the SF3b complex nearby the branch-site pocket, forming an adduct with Cys26 of the PHF5A zinc finger while remarkably preserving Zn2+; coordination - an unconventional mode of covalent inhibition. Yet the molecular features that activate this normally protected ZnCys4 cysteine and initiate spliceostatin's epoxide ring opening have remained obscure. Here, we combine extensive classical and QM/MM molecular dynamics simulations to map the full reactive trajectory of spliceostatin attachment to PHF5A. We identify two alternative noncovalent spliceostatin binding poses and show that only its thermodynamically favoured conformation pre-organizes the epoxide for nucleophilic attack. The local distortion of the ZnCys4; coordination sphere weakens the Zn-Cys26 bond, enabling water-Cys26 exchange and generating a highly reactive nucleophilic thiolate. Covalent bond formation is subsequently accelerated by an Asp34-Lys29 proton relay that activates the epoxide leaving group. Free-energy calculations confirm that the overall reaction is fast and strongly exergonic. Together, these findings provide a complete mechanistic framework for zinc-assisted inhibition of early spliceosomes by branch-site antagonists and advance our fundamental understanding of zinc finger reactivity.

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.

Intrinsically disordered proteins and regions (IDP/IDRs) are ubiquitous across all domains of life. Characterized by a lack of a stable tertiary structure, IDP/IDRs populate a diverse set of transiently formed structural states that can promiscuously adapt upon binding with specific interaction partners and/or certain alterations in environmental conditions. This malleability is foundational for their role as tunable interaction hubs in core cellular processes such as signaling, transcription, and translation. Tracing the conformational ensemble of an IDP/IDR and its perturbation in response to regulatory cues is thus paramount for illuminating its function. However, the conformational heterogeneity of IDP/IDRs poses several challenges. Here, we review experimental and computational methods devised to disentangle the conformational landscape of IDP/IDRs, highlighting recent computational advances that permit proteome-wide scans of IDP/IDRs conformations. We briefly evaluate selected computational methods using the disordered N-terminal of the human copper transporter 1 as a test case and outline further challenges in IDP/IDRs ensemble prediction.

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.