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Computational prediction of amino acid residues essential for ligand-binding selectivity of cytokinin receptors Mikhail Yu. Steklov, Sergey N. Lomin, Georgy A. Romanov
Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia; gar@ippras.ru

Dmitry I. Osolodkin
Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1/3, 119991 Moscow, Russia

Cytokinins are essent ial plant hormones regulat ing plant growth and development. Natural cytokinins are a group of adenine derivat ives having various side chains at N6 posit ion of the purine mo iet y [1]. Several cytokinin receptors were ident ified in higher plants, differing in expressio n pattern and hormone preference [2]. The crystal structure of the ligand-binding module fro m typical cytokinin receptor AtHK4 (CRE1/AHK4) fro m Arabidopsis thaliana was recent ly disclosed [3] allowing the co mparison o f different receptors by a co mputational approach. In the present study ho mology models for closely related receptors AtHK2, AtHK3 (fro m Arabidopsis thaliana), ZmHK1, ZmHK2 and ZmHK3 (fro m Zea mays) were built based on AtHK4 template. Molecular docking of natural and synt hetic cytokinins was performed, and an attempt was made to explain the structural basis of different ial hormone select ivit y. Due to high ho mology o f ligand-binding modules of cytokinin receptors, all the models built closely resemble the template. Altogether ligand binding sites consists o f around 20 amino acids, half o f them being conservative and other half to some extent variable. Both conservative and variable residues contact to adenine- or side chain mo ieties of natural cytokinins. Noticeable difference between the receptors is a presence o f ~15 residue insertion between the positions 229 and 230 of AtHK4 that resides both in AtHK2/ZmHK3 and AtHK3/ZmHK2 receptor ortholog pairs [4]. These insert ions appear as non-structured loops in our models, but it is also possible that they form a -hairpin. Despite they are located in the vicinit y of the hormone binding site, they do not affect its conformat ion and cannot form contacts with the hormone mo lecule. Trans-zeatin (tZ) is the most interesting cytokinin as it is widespread natural hormone wit h high affinit y to receptors and bio logical act ivit y. AtHK2, AtHK4 and ZmHK3 have similar affinit y for tZ whereas AtHK3 and ZmHK1 bind it one order of magnitude stronger and weaker, respectively. The difference between AtHK4 and


ZmHK1 is intriguing because the binding sites of these two ortholog receptors are very similar. Gly229 of AtHK4 is deleted in ZmHK1, possibly affect ing the conformation of the loop formed by residues 225-235. The most similar ligand specificit y profile is observed for AtHK2 and AtHK4. The binding site composit io n is also very similar for these receptors: subst itutions appear only at the periphery o f the binding site (Tyr250His) or out of plane of adenine mo iet y (Leu251Ile, Ala322Thr) and affinit y pattern must be defined by the delicate balance of these three subst itutions. On the basis of the mo lecular modelling and docking study we can conclude that the backbone conformation o f the cytokinin binding site is rather similar in all tested receptors though delet ion of so me amino acids adjacent to binding site may influence site vo lume and shape. The difference in ligand specificit y of receptors might be attributed to the difference in defined amino acids in binding sites. We have shown that a few noted amino acid subst itutions lead to variat ions in vo lume and properties of cytokinin-binding pockets belo nging to different receptors. To clarify the question o f ligand-binding preferences of cytokinin receptors a series o f receptor mutants was produced by site-directed mutagenesis and tested for hormone select ivit y in binding assays. Binding energy calculat ion based on docking results for these mutants allowed us to rationalize experimental findings. We acknowledge the financial support from the Program o f Presidium o f RAS and from grants of RFBR, projects 11-04-00614, 12-04-33282. 1. G.A.Romanov (2012) Cytokinins, McGra w Hill Encyclopedia of Science & Technol., 5:205-207. 2. S.N.Lomin et al. (2012) Receptor properties and features of cytokinin signaling, Acta Naturae, 4(3):31-45. 3. M.Hothorn, T.Dabi, J.Chory (2011) Structural basis for cytokinin recognit ion by Arabidopsis thaliana histidine kinase 4, Nature Chem Biol, 7:766-768. 4. K.Yonekura-Sakakibara, M. Ko jima, T.Yamaya, H.Sakakibara (2004) Molecular characterizat ion o f cytokinin responsive hist idine kinases in maize. Differential ligand preferences and response to cis-zeatin, Plant Physiol, 134:1654-1661.