Maryna Žygalko1, Naďa Špačková2,  Jana Šefčíková3, Jiří Šponer2


1 National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic


2Institute of Biophysics, Academy of Sciences of the Czech Republic and National Centre for  

  Biomolecular Research, Kralovopolska 135, 61265 Brno, Czech Republic


3Department of Chemistry, The University of Michigan, 930 North University, Ann Arbor,

 Michigan 48109-1055



The hepatitis delta virus (HDV) ribozymes are self-cleaving RNA sequences critical to the replication of small RNA genomes. The cleavage activity of the genomic and antigenomic ribozymes can be mapped to minimal ~85 nt motifs. Molecular dynamics simulations of the cleavage product form of genomic cis-acting HDV ribozyme were performed with explicit inclusion of solvent and counterions. Simulation of the native structure with protonated C41 residue was carried out for 15 ns, and exhibited stable trajectories retaining all Watson-Crick and non-Watson-Crick basepairs, except fluctuations and transient disruptions at specific sites. Three additional simulations were carried out for ~45 ns in all to clarify the structural and dynamic effect of selected mutations.  In one of them C41 was left unprotonated. Significant local rearrangements occur during this simulation, such as irreversible disruption of C41·C73 basepair as well as rearrangement of nucleotides close to the catalytic pocket, thus confirming N3-protonation of C41 in the native molecule. The most considerable event is rotation of G76 residue and formation of numerous hydrogen binding interactions of the base of G76 inside the catalytic pocket, which are not present in the crystal structure. However, the prolongation of this simulation after protonation of C41 base did not result in recovery of the original structure of disrupted non-Watson-Crick basepairs as well as extrusion of G76 outside the catalytic pocket. This fact is an evidence of significant height of energy barrier between considered conformational states and gives rise to the question about a sufficiency of sampling in both simulations. One of the commonly considered self-cleavage reaction mechanism include C75(N+) as a general acid catalyst. The simulation included protonated C75 was performed to obtain insight into the possible protonation state of C75 base at the product form of the ribozyme under investigation. In this simulation interaction of C75 with cleavage site occurred unstable and irreversibly broke after 1 ns simulation. Therefore, C75 is not likely to be protonated at the ribozyme product form.

The simulation revealed several critical, highly ordered hydration sites with close to 100% occupancies and long residency times of individual water molecules which form water bridges in non-Watson-Crick base pairs or participate in formation of catalytic pocket environment. Sodium cation coordination sites with occupancies above 50% were also found.