Plant nuclease TBN1 involved in apoptotic processes blocks its active site
by a surface loop – sign of regulatory function?

J. Stránský^{1, 2}, T. Kovaľ¹, T. Podzimek³, P. Lipovová³, J. Matoušek⁴, P. Kolenko¹, J. Dušková¹, T. Skálová¹, J. Hašek¹, J. Dohnálek¹

¹Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovského nám. 2, 162 06 Praha 6, Czech Republic

²Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University, Břehová 7, 115 19 Praha 1, Czech Republic

³Institute of Chemical Technology, Technická 5, 166 28 Praha 6, Czech Republic

⁴Institute of Plant Molecular Biology, Biology Centre, AS CR, v.v.i., Branišovská 31, 370 05 České Budějovice, Czech Republic,

stransky@imc.cas.cz

Tomato multifunctional nuclease (TBN1; UniProt accession no. Q0KFV0), which belongs to the nuclease type I family, plays an important role in specific apoptotic functions, vascular system development, stress response, and tissue differentiation in plants [1]. Furthermore, TBN1 exhibits anticancerogenic properties [2]. The enzyme posseses endonuclease and exonuclease-like activity on ds and ss RNA and DNA and on structured RNA, with production of 5’-mono- and oligonucleotides [3]. TBN1 consists of 277 aminoacids with a molecular mass of 31.6 kDa (about 37 kDa when glycosylated).

Structures of wild type TBN1 and mutant N211D were solved by our group by the means of X-ray crystallography [4]. Molecules of TBN1 form super-helices generated by crystal symmetry, where contacts are provided by the active site of one molecule and a surface loop of a neighboring molecule. This motif is conserved in all the known crystal structures of the enzyme but the rest of crystal packing differs across different crystallization conditions. Formation of intermolecular contacts in crystals suggests the way of assembly of molecules into oligomers in solution. The interaction of the active site and the surface loop is best resolved in the currently reported structure, where the active centre at the zinc cluster is occupied by phosphate ion. It correlates with the behavior of TBN1 in phosphate buffer, observed with dynamic light scattering. The phosphate ion binds in the same fashion as the corresponding part of a substrate analog in the structure of Phospholipase C [5] with highly homologous active centre.

Properties of mutants, designed to modify dimerization and activity of TBN1, suggest that deliberate disruption of the loop-active site contacts by mutations limits expression of the active enzyme. Therefore formation of TBN1 oligomers together with phosphate binding are hypothesized to have regulatory roles in apoptotic-like and senescense processes in plant cells.

The work on this project was supported by the Czech Science Foundation, projects no. P302/11/0855, 202/06/0757 and 521/09/1214, by the EC under ELISA grant agreement number 226716 (synchrotron access, projects 09.2.90262 and 10.1.91347), by the Institution research plan AV0Z50510513 of the Institute of Plant Molecular Biology, Biology Centre. We acknowledge support of the Ministry of Education, Youth and Sports of the Czech Republic (grant No. CZ.1.07/2.3.00/30.0029). The authors wish to thank Dr. U. Müller of the Helmholtz-Zentrum Berlin, for support at the beam line.

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