Structural and functional characterization of Ralstonia solanacearum lectin mutant

Daniel Pokorný¹, Josef Houser^2,3, Michaela Wimmerová^1,2,3

¹Department of Biochemistry, Faculty of Science, Masaryk University, Brno 611 37, Czech Republic

²National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno 611 37, Czech Republic

³ Central European Institute of Technology, Masaryk University, Brno 625 00, Czech Republic

pokec@mail.muni.cz

Ralstonia solanacearum is a gram-negative plant pathogen with wide radius of hosts (potato, tomato, banana, …). Among other ways it uses carbohydrate-binding proteins as a strategy to invade a plant. Understanding of this interaction can help in prevention or treatment of infections caused by R. solanacearum.

Sugar binding protein RSL from Ralstonia solanacearum was previously characterized in our group [1]. In this project, we examined a two-point mutant RSL_S15QH60Q produced in the E.coli host cells. FPLC on the mannose-agarose column was used to purify the protein, which was subsequently subjected to in-depth analysis. Isothermal titration calorimetry was used to assess thermodynamic parameters of protein binding to various ligands. Protein showed the highest affinity towards fucose-containing saccharides. Also, near-UV CD spectroscopy revealed changes of tryptophan peaks while being titrated with L-fucose. The protein was crystallized by a hanging drop technique and crystals were subsequently used to determine the structure using X-ray crystallography. Diffraction data were collected at high resolution of 1.0 Å and structure was solved by molecular replacement using the native protein structure.

At the same time, other mutants of the lectin are studied in our group. This combined approach enables us to deeper understand the relationship between lectin sequence-structure and its binding properties including fine specificity towards various ligands.

[1] N. Kostlánová et al.: The Fucose-binding Lectin from Ralstonia solanacearum, 2005, THE JOURNAL OF BIOLOGICAL CHEMISTRY, 27839–27849.

The research leading to these results obtained financial contribution from the European Union under the Seventh Framework Programme by CEITEC (CZ.1.05/1.1.00/02.0068) project from European Regional Development Fund, SYLICA (Contract No. 286154 under ‘‘Capacities’’ specific programme), Czech Ministry of Education (LH13055) and the Czech Science Foundation (GA13-25401S).