Crystal structure of Vigna radiata cytokinin-specific binding protein in complex with zeatin.
The cytosolic fraction of Vigna radiata contains a 17-kD protein that binds plant hormones from the cytokinin group, such as zeatin. Using recombinant protein and isothermal titration calorimetry as well as fluorescence measurements coupled with ligand displacement, we have reexamined the K(d) values and show them to range from approximately 10(-6) M (for 4PU30) to 10(-4) M (for zeatin) for 1:1 stoichiometry complexes. In addition, we have crystallized this cytokinin-specific binding protein (Vr CSBP) in complex with zeatin and refined the structure to 1.2 A resolution. Structurally, Vr CSBP is similar to plant pathogenesis-related class 10 (PR-10) proteins, despite low sequence identity (<20%). This unusual fold conservation reinforces the notion that classic PR-10 proteins have evolved to bind small-molecule ligands. The fold consists of an antiparallel beta-sheet wrapped around a C-terminal alpha-helix, with two short alpha-helices closing a cavity formed within the protein core. In each of the four independent CSBP molecules, there is a zeatin ligand located deep in the cavity with conserved conformation and protein-ligand interactions. In three cases, an additional zeatin molecule is found in variable orientation but with excellent definition in electron density, which plugs the entrance to the binding pocket, sealing the inner molecule from contact with bulk solvent.
Pasternak
Oliwia
O
Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61704 Poznan, Poland.
Bujacz
Grzegorz D
GD
Fujimoto
Yasuyuki
Y
Hashimoto
Yuichi
Y
Jelen
Filip
F
Otlewski
Jacek
J
Sikorski
Michal M
MM
Jaskolski
Mariusz
M
D000595:Amino Acid Sequence; D002151:Calorimetry; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003583:Cytokinins; D008024:Ligands; D008969:Molecular Sequence Data; D027805:Phaseolus / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D015026:Zeatin / Q000737:chemistry / Q000378:metabolism*
D002352:Carrier Proteins; D003583:Cytokinins; D008024:Ligands; D010940:Plant Proteins; D015026:Zeatin