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 plant tissue differentiation . Furthermore, TBN1 exhibits anticancerogenic properties . The enzyme possesses endonuclease and exonuclease-like activity on single stranded and double stranded RNA and DNA and on structured RNA, with production of mono- and oligonucleotides from 3'-end of nucleic acids . Based on the crystal structure of TBN1, the phospholipase activity of the enzyme was discovered . TBN1 consists of 277 aminoacids with a molecular mass of 31.6 kDa (about 37 kDa when glycosylated).
Three crystal structures of TBN1 were solved in our group: one structure of wild type TBN1 and two structures of mutant N211D . The common motif conserved among all known structures is formation of super-helices, where protein molecules are related by 31 screw axis. The contacts are provided by the active site of one molecule and a surface loop (SDR-loop) of a neighboring molecule. However, packing of super-helices to final crystal differs across different crystallization conditions. The conserved formation of intermolecular contacts in crystals suggests the way of assembly of molecules into oligomers in solution, which were observed by dynamic light scattering. 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 behavior of TBN1 in phosphate buffer. The phosphate ion binds differently than corresponding ions in known structures of AtBFN2 from Arabidopsis thaliana . Also presence of phosphate in the zinc cluster leads to more open TBN1 active center.
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.
This publication is supported by the project „BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund.“, by the Ministry of Education, Youth and Sports of the Czech Republic (grant No. EE2.3.30.0029 and No. LG14009) and by the Grant Agency of the Czech Technical University in Prague, grant No. SGS13/219/OHK4/3T/14.