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Towards a Crystal Structure of Complete Histidine Kinase E in Bacillus
Author(s) -
Sergeeva Oksana,
Bick Matthew J,
Darst Seth A
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.879.6
Subject(s) - autophosphorylation , histidine kinase , thermophile , protein kinase domain , kinase , dimer , biochemistry , chemistry , protein kinase a , biology , histidine , enzyme , organic chemistry , mutant , gene
Sporulation in Bacillus is controlled by five histidine kinases (HKs), KinA‐KinE, which are composed of an N‐terminal sensor domain and a C‐terminal kinase domain. Signals sensed in the sensor domain are transduced to the kinase domain, resulting in an autophosphorylation event on a conserved histidine. The crystal structure of the kinase domain from KinB in complex with the autophosphorylation inhibitor Sda, from the moderate thermophile G eobacillus stearothermophilis , has previously been solved in the Darst laboratory. The structure reveals a KinB dimer with Sda bound to a single monomer. Binding of Sda stabilizes the KinB dimer and prevents autophosphorylation. In order to gain insights into how signals are communicated in HKs, we focused on the crystallization of a complete HK. Currently, a structure of an HK containing both sensor and kinase domains is unavailable. Availing ourselves of the fact that two of the five sporulation HKs in Bacillus are soluble, we choose one of these, KinE, for structural studies. A double expression construct containing KinE and Sda was made from three different thermophilic Geobacillus species. Of the three constructs tested for expression, one from G. thermodenitrificans gave considerable amounts of soluble protein following expression in E. coli Rosetta2 cells. We have purified the KinE/Sda complex via nickel affinity, ion exchange, and size‐exclusion chromatography. Biochemical assays have shown that autophosphorylation of KinE, like KinB, is inhibited by Sda. Initial crystallization trials in the presence of ADP have given several promising leads. This work was supported by NIH GM081697 to S.A.Darst.