Cargando…

Evidence That Both Ligand Binding and Covalent Adaptation Drive a Two-State Equilibrium in the Aspartate Receptor Signaling Complex

The transmembrane aspartate receptor of bacterial chemotaxis regulates an associated kinase protein in response to both attractant binding to the receptor periplasmic domain and covalent modification of four adaptation sites on the receptor cytoplasmic domain. The existence of at least 16 covalent m...

Descripción completa

Detalles Bibliográficos
Autores principales: Bornhorst, Joshua A., Falke, Joseph J.
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2001
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2229510/
https://www.ncbi.nlm.nih.gov/pubmed/11723162
_version_ 1782150145386741760
author Bornhorst, Joshua A.
Falke, Joseph J.
author_facet Bornhorst, Joshua A.
Falke, Joseph J.
author_sort Bornhorst, Joshua A.
collection PubMed
description The transmembrane aspartate receptor of bacterial chemotaxis regulates an associated kinase protein in response to both attractant binding to the receptor periplasmic domain and covalent modification of four adaptation sites on the receptor cytoplasmic domain. The existence of at least 16 covalent modification states raises the question of how many stable signaling conformations exist. In the simplest case, the receptor could have just two stable conformations (“on” and “off”) yielding the two-state behavior of a toggle-switch. Alternatively, covalent modification could incrementally shift the receptor between many more than two stable conformations, thereby allowing the receptor to function as a rheostatic switch. An important distinction between these models is that the observed functional parameters of a toggle-switch receptor could strongly covary as covalent modification shifts the equilibrium between the on- and off-states, due to population-weighted averaging of the intrinsic on- and off-state parameters. By contrast, covalent modification of a rheostatic receptor would create new conformational states with completely independent parameters. To resolve the toggle-switch and rheostat models, the present study has generated all 16 homogeneous covalent modification states of the receptor adaptation sites, and has compared their effects on the attractant affinity and kinase activity of the reconstituted receptor–kinase signaling complex. This approach reveals that receptor covalent modification modulates both attractant affinity and kinase activity up to 100-fold, respectively. The regulatory effects of individual adaptation sites are not perfectly additive, indicating synergistic interactions between sites. The three adaptation sites at positions 295, 302, and 309 are more important than the site at position 491 in regulating attractant affinity and kinase activity, thereby explaining the previously observed dominance of the former three sites in in vivo studies. The most notable finding is that covalent modification of the adaptation sites alters the receptor attractant affinity and the receptor-regulated kinase activity in a highly correlated fashion, strongly supporting the toggle-switch model. Similarly, certain mutations that drive the receptor into the kinase activating state are found to have correlated effects on attractant affinity. Together these results provide strong evidence that chemotaxis receptors possess just two stable signaling conformations and that the equilibrium between these pure on- and off-states is modulated by both attractant binding and covalent adaptation. It follows that the attractant and adaptation signals drive the same conformational change between the two settings of a toggle. An approach that quantifies the fractional occupancy of the on- and off-states is illustrated.
format Text
id pubmed-2229510
institution National Center for Biotechnology Information
language English
publishDate 2001
publisher The Rockefeller University Press
record_format MEDLINE/PubMed
spelling pubmed-22295102008-04-21 Evidence That Both Ligand Binding and Covalent Adaptation Drive a Two-State Equilibrium in the Aspartate Receptor Signaling Complex Bornhorst, Joshua A. Falke, Joseph J. J Gen Physiol Original Article The transmembrane aspartate receptor of bacterial chemotaxis regulates an associated kinase protein in response to both attractant binding to the receptor periplasmic domain and covalent modification of four adaptation sites on the receptor cytoplasmic domain. The existence of at least 16 covalent modification states raises the question of how many stable signaling conformations exist. In the simplest case, the receptor could have just two stable conformations (“on” and “off”) yielding the two-state behavior of a toggle-switch. Alternatively, covalent modification could incrementally shift the receptor between many more than two stable conformations, thereby allowing the receptor to function as a rheostatic switch. An important distinction between these models is that the observed functional parameters of a toggle-switch receptor could strongly covary as covalent modification shifts the equilibrium between the on- and off-states, due to population-weighted averaging of the intrinsic on- and off-state parameters. By contrast, covalent modification of a rheostatic receptor would create new conformational states with completely independent parameters. To resolve the toggle-switch and rheostat models, the present study has generated all 16 homogeneous covalent modification states of the receptor adaptation sites, and has compared their effects on the attractant affinity and kinase activity of the reconstituted receptor–kinase signaling complex. This approach reveals that receptor covalent modification modulates both attractant affinity and kinase activity up to 100-fold, respectively. The regulatory effects of individual adaptation sites are not perfectly additive, indicating synergistic interactions between sites. The three adaptation sites at positions 295, 302, and 309 are more important than the site at position 491 in regulating attractant affinity and kinase activity, thereby explaining the previously observed dominance of the former three sites in in vivo studies. The most notable finding is that covalent modification of the adaptation sites alters the receptor attractant affinity and the receptor-regulated kinase activity in a highly correlated fashion, strongly supporting the toggle-switch model. Similarly, certain mutations that drive the receptor into the kinase activating state are found to have correlated effects on attractant affinity. Together these results provide strong evidence that chemotaxis receptors possess just two stable signaling conformations and that the equilibrium between these pure on- and off-states is modulated by both attractant binding and covalent adaptation. It follows that the attractant and adaptation signals drive the same conformational change between the two settings of a toggle. An approach that quantifies the fractional occupancy of the on- and off-states is illustrated. The Rockefeller University Press 2001-12-01 /pmc/articles/PMC2229510/ /pubmed/11723162 Text en © 2001 The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Original Article
Bornhorst, Joshua A.
Falke, Joseph J.
Evidence That Both Ligand Binding and Covalent Adaptation Drive a Two-State Equilibrium in the Aspartate Receptor Signaling Complex
title Evidence That Both Ligand Binding and Covalent Adaptation Drive a Two-State Equilibrium in the Aspartate Receptor Signaling Complex
title_full Evidence That Both Ligand Binding and Covalent Adaptation Drive a Two-State Equilibrium in the Aspartate Receptor Signaling Complex
title_fullStr Evidence That Both Ligand Binding and Covalent Adaptation Drive a Two-State Equilibrium in the Aspartate Receptor Signaling Complex
title_full_unstemmed Evidence That Both Ligand Binding and Covalent Adaptation Drive a Two-State Equilibrium in the Aspartate Receptor Signaling Complex
title_short Evidence That Both Ligand Binding and Covalent Adaptation Drive a Two-State Equilibrium in the Aspartate Receptor Signaling Complex
title_sort evidence that both ligand binding and covalent adaptation drive a two-state equilibrium in the aspartate receptor signaling complex
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2229510/
https://www.ncbi.nlm.nih.gov/pubmed/11723162
work_keys_str_mv AT bornhorstjoshuaa evidencethatbothligandbindingandcovalentadaptationdriveatwostateequilibriumintheaspartatereceptorsignalingcomplex
AT falkejosephj evidencethatbothligandbindingandcovalentadaptationdriveatwostateequilibriumintheaspartatereceptorsignalingcomplex