Expression, purification, and characterization of cytokinin signaling intermediates: Arabidopsis histidine phosphotransfer protein 1 (AHP1) and AHP2.

We have expressed, purified, and biophysically characterized recombinant AHP1 and AHP2. Also, using computational homology models for AHP1, ARR7, and AHP1–ARR7 complex, we identified threedimensional positioning of key amino acids. Cytokinin signaling involves activation of Arabidopsis Response Regulators (ARRs) by Arabidopsis Histidine Phosphotransfer Proteins (AHPs) by phosphorylation. Type-A ARRs are key regulators of several developmental pathways, but the mechanism underlying this phosphorylation and activation is not known in plants. In this study, we report the successful expression and purification of recombinant AHP1 and AHP2. Biophysical characterization shows that these two recombinant proteins were purified to homogeneity and possess well-defined secondary structures. Brief attempts to purify recombinant ARR7 posed problems during size-exclusion chromatography. Nevertheless, we generated computational homology models for AHP1, ARR7, and AHP1-ARR7 complex using crystal structures of homologous proteins from other organisms. The homology models helped to identify the three-dimensional positioning of the key conserved residues of AHP1 and ARR7 involved in phosphorylation. The similarity in positioning of these residues to other homologous proteins suggests that AHPs and type-A ARRs could be structurally conserved across kingdoms. Thus, our homology models can serve as valuable tools to gain structural insights into the phosphorylation and activation of cytokinin response regulators in plants.