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Autophosphorylation by the Histidine kinase CheA (LB222)
Author(s) -
Greenswag Anna,
Crane Brian
Publication year - 2014
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.28.1_supplement.lb222
Subject(s) - autophosphorylation , histidine kinase , thermotoga maritima , chemotaxis , biophysics , protein kinase domain , biology , allosteric regulation , periplasmic space , histidine , biochemistry , crystallography , chemistry , kinase , protein kinase a , receptor , enzyme , gene , mutant , escherichia coli
Chemotaxis is the signal transduction mechanism that allows bacteria to change direction in response to an external stimulus. Detection of an environmental stimulus by the receptors has a cascading effect leading to the flagellar motor changing direction. This results in the tumbling of the bacterium. The receptors form hexagonal lattices at the poles of cells. The histidine kinase CheA and coupling protein CheW are localized to the lattice to form the ternary complex. CheA, the principal enzyme in the chemotaxis signaling pathway, is a multi domain protein comprised of: P1‐histidine phosphotransfer, P2‐regulatory, P3‐dimerization, P4‐kinase, and P5‐regulatory. From previous studies it is known that the transfer of the γ‐phosphate of ATP occurs between the P1 and P4 domain, however how autophosphorylation activity is regulated is not completely understood. To comprehend this transient interaction, variants (ΔP2) of Thermotoga maritima CheA were generated. Small angle X‐ray scattering (SAXS) data has shown that spatial changes occur upon addition of an ATP analog. To determine relative activity of the ΔP2 variants or CheA domains radioisotope assays were used. Point mutations on the full length and ΔP2 CheA showed whether autophosphorylation occurs cis or trans in the dimer. Crosslinking between residues near the active sites in the P4 and P1 domains enabled isolation of the autophosphorylation transfer conformation. The crystal structure of P3P4 domains reveals a more active configuration of the P4 domain. Progress towards understanding how the domains interact to facilitate the transfer of the ATP γ‐phosphate is not clear and will be discussed.