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 kinaseactivating 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.The aspartate receptor of bacterial chemotaxis is one of the best-characterized members of a large family of cell surface receptors that modulate two-component signaling pathways (reviewed in refs 1-6). Through regulation of the associated histidine kinase CheA, the aspartate receptor controls cellular chemotaxis in response to changing concentrations of the chemoattractant aspartate, allowing the cell to move up an attractant concentration gradient. The receptor is an oligomer composed of three homodimers that assemble to form a trimer of dimers, as illustrated in Figure 1A. The chemotaxis signaling pathway is triggered by aspartate † Support provided by NIH Grant GM ...

Evidence that the Adaptation Region of the Aspartate Receptor Is a Dynamic Four-Helix Bundle: Cysteine and Disulfide Scanning Studies

“…A recent study showed that electrostatic repulsion between negative charges on the surface of the subdomain modulates the packing between the adjacent helices of the two subunits within the homodimer, thereby regulating kinase activity (69). Covalent modification of the receptor adaptation sites is believed to modulate this electrostatic interaction in the same way, because methylation or amidation of the adaptation sites decreases the electrostatic repulsion between subunits, thereby stabilizing the helix interactions and activating the kinase.…”

Bacterial chemoreceptors: high-performance signaling in networked arrays

“…In addition, the adaptation region is highly anionic and is regulated by an electrostatic mechanism involving the adaptation site glutamates and several other anionic side-chains lining the subunit-subunit interface [58]. Covalent neutralization of an interfacial anion by methyl esterification or amidation stabilizes the interface and activates kinase, whereas simultaneous neutralization of all anions locks the receptor in the kinaseactivating state [58] Conformational signaling in the protein interaction region, where the kinase interacts, remains poorly understood. However, like the adaptation region, its subunit interface seems to be crucial for signal transmission because many 'lock-on' mutations occur at interfacial locations [54,59].…”

“…The other is tilting of dimers relative to the central trimer axis, detected by changes in dimer-to-dimer FRET [47,48]. In principle, such macroscopic transitions could be driven by changes in supercoiling, or supercoiling-dependent flexibility of the four-helix bundle of the kinase control module [46,58]. By triggering changes in inter-dimer distances or contacts, macroscopic transitions could have an important role in the strong positive signaling cooperativity between dimers, within or between trimers.…”

“…However, considerable evidence indicates that changes at the subunitsubunit interface within the kinase control module are important in signaling [56][57][58]. This contrasts with the transmembrane sensing domain, in which signaling movements occur between the two helices in a single subunit and the subunit interface is static [49].…”

“…In the otherwise dynamic kinase control module, stabilization of inter-subunit packing by disulfide bonds or amino acid replacements can lock the receptor to a kinase-on state, implying that attractant-generated, kinase-inhibiting signals shift or destabilize the subunit interface via mechanical forces [54,57]. In addition, the adaptation region is highly anionic and is regulated by an electrostatic mechanism involving the adaptation site glutamates and several other anionic side-chains lining the subunit-subunit interface [58]. Covalent neutralization of an interfacial anion by methyl esterification or amidation stabilizes the interface and activates kinase, whereas simultaneous neutralization of all anions locks the receptor in the kinaseactivating state [58] Conformational signaling in the protein interaction region, where the kinase interacts, remains poorly understood.…”