title: Adaptation mechanism of the aspartate receptor: electrostatics of the adaptation subdomain play a key role in modulating kinase activity.
abstract: The aspartate receptor of the Escherichia coli and Salmonella typhimurium chemotaxis pathway generates a transmembrane signal that regulates the activity of the cytoplasmic kinase CheA. Previous studies have identified a region of the cytoplasmic domain that is critical to receptor adaptation and kinase regulation. This region, termed the adaptation subdomain, contains a high density of acidic residues, including specific glutamate residues that serve as receptor adaptation sites. However, the mechanism of signal propagation through this region remains poorly understood. This study uses site-directed mutagenesis to neutralize each acidic residue within the subdomain to probe the hypothesis that electrostatics in this region play a significant role in the mechanism of kinase activation and modulation. Each point mutant was tested for its ability to regulate chemotaxis in vivo and kinase activity in vitro. Four point mutants (D273N, E281Q, D288N, and E477Q) were found to superactivate the kinase relative to the wild-type receptor, and all four of these kinase-activating substitutions are located along the same intersubunit interface as the adaptation sites. These activating substitutions retained the wild-type ability of the attractant-occupied receptor to inhibit kinase activity. When combined in a quadruple mutant (D273N/E281Q/D288N/E477Q), the four charge-neutralizing substitutions locked the receptor in a kinase-superactivating state that could not be fully inactivated by the attractant. Similar lock-on character was observed for a charge reversal substitution, D273R. Together, these results implicate the electrostatic interactions at the intersubunit interface as a major player in signal transduction and kinase regulation. The negative charge in this region destabilizes the local structure in a way that enhances conformational dynamics, as detected by disulfide trapping, and this effect is reversed by charge neutralization of the adaptation sites. Finally, two substitutions (E308Q and E463Q) preserved normal kinase activation in vitro but blocked cellular chemotaxis in vivo, suggesting that these sites lie within the docking site of an adaptation enzyme, CheR or CheB. Overall, this study highlights the importance of electrostatics in signal transduction and regulation of kinase activity by the cytoplasmic domain of the aspartate receptor.
authors.affiliation: Molecular Biophysics Program and Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
authors.forename: Diane J
authors.initials: DJ
authors.lastname: Starrett
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Falke
mesh_terms: D000222:Adaptation, Physiological; D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002633:Chemotaxis / Q000235:genetics / Q000502:physiology*; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D019281:Dimerization; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D000071677:Histidine Kinase; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D010957:Plasmids; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D017459:Receptors, Amino Acid / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D055672:Static Electricity
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D017459:Receptors, Amino Acid; C031959:aspartic acid receptor; D001224:Aspartic Acid; D011494:Protein Kinases; D000071677:Histidine Kinase; C000607608:cheA protein, E coli