Information on protonation in biomolecular structures

J. Dohnálek¹, K. Adámková¹, M. Trundová¹, B. Husťáková¹, J. Dušková¹, P. Kolenko^1,2, L. Gajdoš³, T. Skálová¹, T. Kovaľ¹

¹Institute of Biotechnology, Czech Academy of Sciences, Průmyslová 595, 25250 Vestec
²Czech Technical University in Prague, Břehová 7, 115 19, Prague
³Institut Laue-Langevin, 71 avenue des Martyrs, 38042 Grenoble

dohnalek@ibt.cas.cz

Hydrogen plays a key role in structure and function of biomolecules. Intermolecular interactions rely amongst others on a finely tuned concert of hydrogen bonds formation and typical protonation patterns. Structural information on hydrogen/proton presence and localization in structural biology is not easily accessible.

S1-P1 nucleases are coded for by fungi, trypanosomatids, plants and some pathogenic bacteria [1]. The active site relies on the metal cluster (typically containing zinc) and the nucleobase-binding site 1 stabilizing the –1 nucleotide with respect to the cleaved O3’-P3’ bond. The enzymes cleave DNA, RNA, single strands, double strands, viroids, some modified nucleotides, oligonucleotides and genomic DNA [1]. While their fold does not change across the species, their activity profiles differ dramatically.

Our structure-function studies of S1-P1 nucleases from plants, fungus, and two bacterium species [2-5], including crystal structures, mutagenesis, numerous product/ligand complexes helped us better understand the structure-function questions, such as active site remodelling and key mobility elements in the active site. In a recent study we have identified SmNuc1 from opportunistic pathogen Stenotrophomonas maltophilia with unusually high catalytic rates for this enzyme class. We could identify the key region for RNA/DNA preference and discovered its high activity towards cyclic-di-GMP, the bacterial second messenger [6]. Our crystallographic studies answered key questions regarding non-specificity of S1-P1 nucleases and brought us closer to understanding protonation details of the protein-nucleic acid interactions.

The excellent diffraction properties of S1 and SmNuc1 nuclease crystals enable atomic resolution studies with the promise of mapping of important protonation patterns with the use of neutron radiation and sub-Ångstrom resolution X-ray crystallography.

1. T. Koval, J. Dohnalek Biotechnol. Adv. (2017) Epub 2017 Dec 14, 10.1016/j.biotechadv.2017.12.007.

2. T. Kovaľ T, L.H. Østergaard, J. Lehmbeck, et al. PLOS One 11, (2016), e0168832.

3. M. Trundová, T. Kovaľ, R.J. Owens, et al. Int J Biol Macromol 114, (2018), 776.

4. K. Adámková, T. Koval', L.H. Østergaard, et al. Acta Crystallogr D78, (2022), 1194.

5. B. Husťáková, M. Trundová, K. Adámková, et al. FEBS Lett 597, (2023), 2103.

6. K. Adámková, M. Trundová, T. Kovaľ, B. Husťáková, et al. FEBS J., 292, (2025), 129.

This work was supported by the Czech Science Foundation (25-17546S), and by the Czech Academy of Sciences (86652036). CIISB, Instruct-CZ Centre of Instruct-ERIC EU consortium, funded by MEYS CR infrastructure project LM2023042 and European Regional Development Fund-Project No. CZ.02.01.01/00/23_015/0008175 is acknowledged for providing access to all facilities at CMS in BIOCEV for this project.