title: Medicago truncatula histidine-containing phosphotransfer protein: structural and biochemical insights into the cytokinin transduction pathway in plants.
abstract: Histidine-containing phosphotransfer proteins (HPts) take part in hormone signal transduction in higher plants. The overall pathway of this process is reminiscent of the two-component system initially identified in prokaryotes. HPts function in histidine-aspartate phosphorelays in which they mediate the signal from sensory kinases (usually membrane proteins) to RRs in the nucleus. Here, we report the crystal structure of an HPt protein from Medicago truncatula (MtHPt1) determined at 1.45 Å resolution and refined to an R-factor of 16.7% using low-temperature synchrotron-radiation X-ray diffraction data. There is one MtHPt1 molecule in the asymmetric unit of the crystal lattice with P2(1)2(1)2(1) symmetry. The protein fold consists of six α helices, four of which form a C-terminal helix bundle. The coiled-coil structure of the bundle is stabilized by a network of S-aromatic interactions involving highly conserved sulfur-containing residues. The structure reveals a solvent-exposed side chain of His79, which is the phosphorylation site, as demonstrated by autoradiography combined with site-directed mutation. It is surrounded by highly conserved residues present in all plant HPts. These residues form a putative docking interface for either the receiver domain of the sensory kinase, or for the RR. The biological activity of MtHPt1 was tested by autoradiography. It demonstrated phosphorylation by the intracellular kinase domain of the cytokinin receptor MtCRE1. Complex formation between MtHPt1 and the intracellular fragment of MtCRE1 was confirmed by thermophoresis, with a dissociation constant K(d) of 14 μM.
authors.affiliation: Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Milosz
authors.initials: M
authors.lastname: Ruszkowski
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Brzezinski
authors.forename: Robert
authors.initials: R
authors.lastname: Jedrzejczak
authors.forename: Miroslawa
authors.initials: M
authors.lastname: Dauter
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Dauter
authors.forename: Michal
authors.initials: M
authors.lastname: Sikorski
authors.forename: Mariusz
authors.initials: M
authors.lastname: Jaskolski
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003583:Cytokinins / Q000502:physiology; D006860:Hydrogen Bonding; D046913:Medicago truncatula / Q000201:enzymology*; D008958:Models, Molecular; D010766:Phosphorylation; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000737:chemistry* / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D040681:Structural Homology, Protein
chemical_list: D003583:Cytokinins; D010940:Plant Proteins; D011956:Receptors, Cell Surface; D017852:Phosphotransferases (Nitrogenous Group Acceptor)
keywords: HPt; MtCRE1; cytokinin hormone receptor; histidine-aspartate two-component phosphorelay; phytohormone signal transduction