Hydrogen Exchange Reveals a Stable and Expandable Core within the Aspartate Receptor Cytoplasmic Domain

• Published in: The Journal of biological chemistry Vol. 276; no. 46; pp. 43262 - 43269
• Main Authors: Murphy, 3rd, O J, Yi, X, Weis, R M, Thompson, L K
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 16-11-2001
• Subjects: Calorimetry; Chromatography, Gel; Cytoplasm - chemistry; Dimerization; Glutamic Acid - chemistry; Histidine - chemistry; Hot Temperature; Hydrogen - chemistry; Hydrogen - metabolism; Hydrogen-Ion Concentration; Kinetics; Ligands; Models, Chemical; Models, Molecular; Mutation; Plasmids - metabolism; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Receptors, Amino Acid - chemistry; Temperature; Time Factors; Tritium - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M105585200

Intensive study of bacterial chemoreceptors has not yet revealed how receptor methylation and ligand binding alter the interactions
between the receptor cytoplasmic domain and the CheA kinase to control kinase activity. Both monomeric and dimeric forms of
an Asp receptor cytoplasmic fragment have been shown to be highly dynamic, with a small core of slowly exchanging amide hydrogens
(Seeley, S. K., Weis, R. M., and Thompson, L. K. (1996) Biochemistry 35, 5199–5206). Hydrogen exchange studies of the wild-type cytoplasmic fragment and an S461L mutant thought to mimic the
kinase-inactivating state are used to investigate the relationship between the stable core and dimer dissociation. Our results
establish that (i) decreasing pH stabilizes the dimeric state, (ii) the stable core is present also in the transition state
for dissociation, and (iii) this core is expanded significantly by small changes in electrostatic and hydrophobic interactions.
These kinase-inactivating changes stabilize both the monomeric and the dimeric states of the protein, which has interesting implications for the mechanism of kinase activation.
We conclude that the cytoplasmic domain is a flexible region poised for stabilization by small changes in electrostatic and
hydrophobic interactions such as those caused by methylation of glutamate residues and by ligand-induced conformational changes
during signaling.