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M edicago truncatula histidine‐containing phosphotransfer protein
Author(s) -
Ruszkowski Milosz,
Brzezinski Krzysztof,
Jedrzejczak Robert,
Dauter Miroslawa,
Dauter Zbigniew,
Sikorski Michal,
Jaskolski Mariusz
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12363
Subject(s) - histidine , helix bundle , chemistry , signal transduction , kinase , phosphorylation , histidine kinase , protein kinase domain , biochemistry , protein structure , biology , biophysics , crystallography , enzyme , mutant , gene
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 RR s in the nucleus. Here, we report the crystal structure of an HPt protein from M edicago truncatula ( M t HP t1) 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 M t HP t1 molecule in the asymmetric unit of the crystal lattice with P 2 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 H is79, 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 M t HP t1 was tested by autoradiography. It demonstrated phosphorylation by the intracellular kinase domain of the cytokinin receptor M t CRE 1. Complex formation between M t HP t1 and the intracellular fragment of M t CRE 1 was confirmed by thermophoresis, with a dissociation constant K d of 14 μ m . Database The atomic coordinates and structure factors for the crystal structure of histidine‐containing phosphotransfer protein MtHPt1 from Medicago truncatula have been deposited with the RCSB Protein Data Bank under the accession code 3us6 .