Crystal Structure of Vigna radiata Cytokinin-Specific Binding Protein in Complex with Zeatin

Oliwia Pasternak ¹, Grzegorz D. Bujacz ², Yasuyuki Fujimoto ³, Yuichi Hashimoto ⁴, Filip Jelen ⁵, Jacek Otlewski ⁵, Michal M. Sikorski ¹, and Mariusz Jaskolski ⁶^*

¹ Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61704 Poznan, Poland
² Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61704 Poznan, Poland; Institute of Technical Biochemistry, Technical University of Lodz, 90924 Lodz, Poland
³ Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa 1990195, Japan
⁴ Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 1130032, Japan
⁵ Laboratory of Protein Engineering, Institute of Biochemistry and Molecular Biology, University of Wroclaw, 50137 Wroclaw, Poland
⁶ Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61704 Poznan, Poland; Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, 60780 Poznan, Poland

^* To whom correspondence should be addressed. E-mail: mariuszj{at}amu.edu.pl.

The cytosolic fraction of Vigna radiata contains a 17-kD proteinthat binds plant hormones from the cytokinin group, such aszeatin. Using recombinant protein and isothermal titration calorimetryas well as fluorescence measurements coupled with ligand displacement,we have reexamined the K_d values and show them to range from $~$ 10^-6 M (for 4PU30) to 10^-4 M (for zeatin) for 1:1 stoichiometrycomplexes. In addition, we have crystallized this cytokinin-specificbinding protein (Vr CSBP) in complex with zeatin and refinedthe structure to 1.2 Å resolution. Structurally, Vr CSBPis similar to plant pathogenesis-related class 10 (PR-10) proteins,despite low sequence identity (<20%). This unusual fold conservationreinforces the notion that classic PR-10 proteins have evolvedto bind small-molecule ligands. The fold consists of an antiparallel ${beta}$ -sheet wrapped around a C-terminal ${alpha}$ -helix, with two short ${alpha}$ -helicesclosing a cavity formed within the protein core. In each ofthe four independent CSBP molecules, there is a zeatin ligandlocated deep in the cavity with conserved conformation and protein-ligandinteractions. In three cases, an additional zeatin moleculeis found in variable orientation but with excellent definitionin electron density, which plugs the entrance to the bindingpocket, sealing the inner molecule from contact with bulk solvent.

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