| Abstract
| - The aspartate receptor of the bacterial chemotaxis pathway serves as a scaffold for the formationof a multiprotein signaling complex containing the receptor and the cytoplasmic pathway components.Within this complex, the receptor regulates the autophosphorylation activity of histidine kinase CheA,thereby controlling the signals sent to the flagellar motor and the receptor adaptation system. The receptorcytoplasmic domain, which controls the on−off switching of CheA, possesses 14 glycine residues thatare highly conserved in related receptors. In principle, these conserved glycines could be required forstatic turns, bends, or close packing in the cytoplasmic domain, or they could be required for conformationaldynamics during receptor on−off switching. To determine which glycines are essential and to probe theirfunctional roles, we have substituted each conserved glycine with both alanine and cysteine, and thenmeasured the effects on receptor function in vivo and in vitro. The results reveal a subset of six glycineswhich are required for receptor function during cellular chemotaxis. Two of these essential glycines (G388and G391) are located at a hairpin turn at the distal end of the folded cytoplasmic domain, where they arerequired for the tertiary fold of the signaling subdomain and for CheA kinase activation. Three otheressential glycines (G338, G339, and G437) are located at the border between the adaptation and signalingsubdomains, where they play key roles in CheA kinase activation and on−off switching. These threeglycines form a ring around the four-helix bundle that comprises the receptor cytoplasmic domain, yieldinga novel architectural feature termed a bundle hinge. The final essential glycine (G455) is located in theadaptation subdomain where it is required for on−off switching. Overall, the findings confirm that six ofthe 14 conserved cytoplasmic glycines are essential for receptor function because they enable helix turnsand bends required for native receptor structure, and in some cases for switching between the on and offsignaling states. An initial working model proposes that the novel bundle hinge enables the four-helixbundle to bend, perhaps during the assembly of the receptor trimer of dimers or during on−off switching.More generally, the findings predict that certain human disease states, including specific cancers, couldbe triggered by lock-on mutations at essential glycine positions that control the on−off switching ofreceptors and signaling proteins.
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