Identification of a Site Critical for Kinase Regulation on the Central Processing Unit (CPU) Helix of the Aspartate Receptor

• Published in: Biochemistry (Easton) Vol. 38; no. 1; pp. 329 - 336
• Main Authors: Trammell, Matthew A, Falke, Joseph J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 05.01.1999
• Subjects: Amino Acid Substitution - genetics; Aspartic Acid - metabolism; Bacterial Proteins - physiology; Binding Sites; Chemotaxis - genetics; Escherichia coli; Escherichia coli Proteins; Membrane Proteins - physiology; Methyl-Accepting Chemotaxis Proteins; Mutagenesis, Site-Directed; Protein Binding - genetics; Protein Kinases - metabolism; Protein Structure, Secondary; Receptors, Amino Acid - biosynthesis; Receptors, Amino Acid - chemistry; Receptors, Amino Acid - genetics; Receptors, Amino Acid - physiology; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemical synthesis; Salmonella typhimurium
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi981964u

Ligand binding to the homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium generates a transmembrane signal that regulates the activity of a cytoplasmic histidine kinase, thereby controlling cellular chemotaxis. This receptor also senses intracellular pH and ambient temperature and is covalently modified by an adaptation system. A specific helix in the cytoplasmic domain of the receptor, helix α6, has been previously implicated in the processing of these multiple input signals. While the solvent-exposed face of helix α6 possesses adaptive methylation sites known to play a role in kinase regulation, the functional significance of its buried face is less clear. This buried region lies at the subunit interface where helix α6 packs against its symmetric partner, helix α6‘. To test the role of the helix α6−helix α6‘ interface in kinase regulation, the present study introduces a series of 13 side-chain substitutions at the Gly 278 position on the buried face of helix α6. The substitutions are observed to dramatically alter receptor function in vivo and in vitro, yielding effects ranging from kinase superactivation (11 examples) to complete kinase inhibition (one example). Moreover, four hydrophobic, branched side chains (Val, Ile, Phe, and Trp) lock the kinase in the superactivated state regardless of whether the receptor is occupied by ligand. The observation that most side-chain substitutions at position 278 yield kinase superactivation, combined with evidence that such facile superactivation is rare at other receptor positions, identifies the buried Gly 278 residue as a regulatory hotspot where helix packing is tightly coupled to kinase regulation. Together, helix α6 and its packing interactions function as a simple central processing unit (CPU) that senses multiple input signals, integrates these signals, and transmits the output to the signaling subdomain where the histidine kinase is bound. Analogous CPU elements may be found in other receptors and signaling proteins.