Biology subjects

Henning-Knechtel, A.

Publications and source records attributed to Henning-Knechtel, A..

4 recordsLinked to original sources

bioRxiv2025

Cat_Wiz: A stereochemistry-guided toolkit for locating, diagnosing and annotating Mg2+ ions in RNA structures.

Misassigned Mg2+ ions are pervasive in RNA structural databases, obscuring mechanistic interpretation, undermining comparative analyses and compromising machine-learning training sets. Here, we present Cat_Wiz, a Coot-integrated, stereochemistry guided toolkit that facilitates the localization, diagnosis, correction and annotation of Mg2+ binding sites. Cat_Wiz comprises three modules: MG_diagnosis which validates and regularizes existing assignments; MG_detect which identifies unmodelled ion binding sites; and MG_clamp which classifies recurrent Mg2+ clamp motifs. Cat_Wiz also includes a complete binding site annotation system. The stereochemical principles implemented in Cat_Wiz were derived from an earlier analysis of the 1.55 [A] resolution Escherichia coli ribosome and from surveys of the Cambridge Structural Database. These principles provide a robust experimental foundation for characterizing Mg2+ binding sites. Applications to ribosomes, hammerhead ribozymes, group I introns, and quaternary RNA assemblies demonstrate that Cat_Wiz rapidly locates overlooked ions, corrects misassignments, and improves stereochemical fidelity in hours rather than days. Beyond refinement, Cat_Wiz generates curated data that can seed diverse machine-learning and AI models. This transparent, cost-effective framework establishes reproducible standards for RNA-ion assignments and will drive progress in the design of RNA 3D architectures through the identification of unique Mg2+-dependent backbone folds. Cat_Wiz is also applicable to Mg2+ binding sites in proteins and all related biomolecular systems since it is based on universal stereochemical principles.

molecular biology↗

bioRxiv2025

Protein Crowders Remodel RNA Electrostatics, Hydration, and Dynamics: A Challenge to Steric Crowding Models

The intracellular environment is densely populated with macromolecules, creating crowded conditions. Whether in vitro environments or synthetic crowders like polyethylene glycol (PEG) accurately capture the complexity of RNA interactions in vivo remains unclear. Using extensive all-atom molecular dynamics simulations, we investigated the HIV-1 TAR RNA hairpin in dilute, PEG-crowded, and realistic protein-crowded solutions. We found that PEG primarily exerts excluded-volume effects, maintaining RNA hydration and Na+ ion condensation similar to dilute conditions. In contrast, protein crowders significantly altered RNA electrostatics, reducing Na+ condensation by nearly 60%, displacing surface hydration water, and forming chemically specific contacts dominated by positively charged residues, notably arginine and lysine. These interactions led to local RNA expansion and reshaped its conformational landscape without disrupting the global fold. Moreover, protein crowding dramatically slowed RNA translational and rotational dynamics, as well as local water and ion mobility, effects minimally observed with PEG. Our findings emphasize that crowder identity critically determines RNA behavior and challenge the use of PEG as a universal model for intracellular conditions, providing mechanistic predictions for RNA studies in biologically relevant environments.

biophysics↗

bioRxiv2024

Principles of ion binding to RNA inferred from the analysis of a 1.55 A resolution bacterial ribosome structure - Part I: Mg2+

The importance of Mg2+ ions for RNA structure and function can difficultly be overstated. Several attempts were made to establish a comprehensive Mg2+ binding site classification. However, such descriptions were hampered by poorly modelled ion binding sites. Recently, ribosome cryo-EM structures with resolutions < 2.0 [A] allowed better descriptions of site-bound ions. However, in a recent cryo-EM 1.55 [A] E. coli ribosome structure, incomplete ion assignments prevented a full understanding of their binding modes. We revisited this model to establish general binding principles applicable to any RNA of sufficient resolution. These principles rely on the 2.9 [A] distance separating two Mg2+-bound cis-water molecules. By applying these rules, we could assign all Mg2+ ions bound with 2 to 4 non-water oxygens. We also uncovered unanticipated motifs where up to five adjacent nucleotides wrap around a single ion. The formation of these complex motifs involves a hierarchical dehydration of the Mg2+ ions, a process that plays a significant role in ribosome biogenesis and in the folding of large RNAs. These binding principles enhance our understanding of the roles of ions in RNA structure and will help refining the solvation shell of different ribosomes and of RNAs with complex topologies.

molecular biology↗

bioRxiv2022

Differences in Ion-RNA binding modes due to charge density variations explain the stability of RNA in monovalent salts

The stability of RNA increases as the charge density of the alkali metal cations increases. The molecular mechanism for the stability does not exist. To fill this gap, we performed all-atom molecular dynamics (MD) pulling simulations to dissect the microscopic origin of this phenomenon. We first established that the free energy landscape obtained in the simulations is in excellent agreement with the single-molecule optical tweezer experiments. The origin of the stronger stability in Na+ compared to K+ is found to be due to the differences in the charge-density related binding modes. The smaller hydrated Na+ ion preferentially binds to the highly charged phosphates. In contrast, the larger K+ ions interact with the major grooves. As a result, the electrostatic repulsion between the phosphate groups is reduced more effectively by Na+ ions. Because the proposed mechanism is generic, we predict that the same conclusions are valid for divalent alkaline earth metal cations.

biophysics↗