Specificity Residues Determine Binding Affinity for Two-Component Signal Transduction Systems

ABSTRACT Two-component systems (TCS) comprise histidine kinases and their cognate response regulators and allow bacteria to sense and respond to a wide variety of signals. Histidine kinases (HKs) phosphorylate and dephosphorylate their cognate response regulators (RRs) in response to stimuli. In gen...

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Autores principales:	Jonathan W. Willett, Nitija Tiwari, Susanne Müller, Katherine R. Hummels, Jon C. D. Houtman, Ernesto J. Fuentes, John R. Kirby
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2013
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/44cbad0c79704a19909f22850bfc00aa
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spelling	oai:doaj.org-article:44cbad0c79704a19909f22850bfc00aa2021-11-15T15:42:32ZSpecificity Residues Determine Binding Affinity for Two-Component Signal Transduction Systems10.1128/mBio.00420-132150-7511https://doaj.org/article/44cbad0c79704a19909f22850bfc00aa2013-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00420-13https://doaj.org/toc/2150-7511ABSTRACT Two-component systems (TCS) comprise histidine kinases and their cognate response regulators and allow bacteria to sense and respond to a wide variety of signals. Histidine kinases (HKs) phosphorylate and dephosphorylate their cognate response regulators (RRs) in response to stimuli. In general, these reactions appear to be highly specific and require an appropriate association between the HK and RR proteins. The Myxococcus xanthus genome encodes one of the largest repertoires of signaling proteins in bacteria (685 open reading frames [ORFs]), including at least 127 HKs and at least 143 RRs. Of these, 27 are bona fide NtrC-family response regulators, 21 of which are encoded adjacent to their predicted cognate kinases. Using system-wide profiling methods, we determined that the HK-NtrC RR pairs display a kinetic preference during both phosphotransfer and phosphatase functions, thereby defining cognate signaling systems in M. xanthus. Isothermal titration calorimetry measurements indicated that cognate HK-RR pairs interact with dissociation constants (Kd) of approximately 1 µM, while noncognate pairs had no measurable binding. Lastly, a chimera generated between the histidine kinase, CrdS, and HK1190 revealed that residues conferring phosphotransfer and phosphatase specificity dictate binding affinity, thereby establishing discrete protein-protein interactions which prevent cross talk. The data indicate that binding affinity is a critical parameter governing system-wide signaling fidelity for bacterial signal transduction proteins. IMPORTANCE Using in vitro phosphotransfer and phosphatase profiling assays and isothermal titration calorimetry, we have taken a system-wide approach to demonstrate specificity for a family of two-component signaling proteins in Myxococcus xanthus. Our results demonstrate that previously identified specificity residues dictate binding affinity and that phosphatase specificity follows phosphotransfer specificity for cognate HK-RR pairs. The data indicate that preferential binding affinity is the basis for signaling fidelity in bacterial two-component systems.Jonathan W. WillettNitija TiwariSusanne MüllerKatherine R. HummelsJon C. D. HoutmanErnesto J. FuentesJohn R. KirbyAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 4, Iss 6 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Jonathan W. Willett Nitija Tiwari Susanne Müller Katherine R. Hummels Jon C. D. Houtman Ernesto J. Fuentes John R. Kirby Specificity Residues Determine Binding Affinity for Two-Component Signal Transduction Systems
description	ABSTRACT Two-component systems (TCS) comprise histidine kinases and their cognate response regulators and allow bacteria to sense and respond to a wide variety of signals. Histidine kinases (HKs) phosphorylate and dephosphorylate their cognate response regulators (RRs) in response to stimuli. In general, these reactions appear to be highly specific and require an appropriate association between the HK and RR proteins. The Myxococcus xanthus genome encodes one of the largest repertoires of signaling proteins in bacteria (685 open reading frames [ORFs]), including at least 127 HKs and at least 143 RRs. Of these, 27 are bona fide NtrC-family response regulators, 21 of which are encoded adjacent to their predicted cognate kinases. Using system-wide profiling methods, we determined that the HK-NtrC RR pairs display a kinetic preference during both phosphotransfer and phosphatase functions, thereby defining cognate signaling systems in M. xanthus. Isothermal titration calorimetry measurements indicated that cognate HK-RR pairs interact with dissociation constants (Kd) of approximately 1 µM, while noncognate pairs had no measurable binding. Lastly, a chimera generated between the histidine kinase, CrdS, and HK1190 revealed that residues conferring phosphotransfer and phosphatase specificity dictate binding affinity, thereby establishing discrete protein-protein interactions which prevent cross talk. The data indicate that binding affinity is a critical parameter governing system-wide signaling fidelity for bacterial signal transduction proteins. IMPORTANCE Using in vitro phosphotransfer and phosphatase profiling assays and isothermal titration calorimetry, we have taken a system-wide approach to demonstrate specificity for a family of two-component signaling proteins in Myxococcus xanthus. Our results demonstrate that previously identified specificity residues dictate binding affinity and that phosphatase specificity follows phosphotransfer specificity for cognate HK-RR pairs. The data indicate that preferential binding affinity is the basis for signaling fidelity in bacterial two-component systems.
format	article
author	Jonathan W. Willett Nitija Tiwari Susanne Müller Katherine R. Hummels Jon C. D. Houtman Ernesto J. Fuentes John R. Kirby
author_facet	Jonathan W. Willett Nitija Tiwari Susanne Müller Katherine R. Hummels Jon C. D. Houtman Ernesto J. Fuentes John R. Kirby
author_sort	Jonathan W. Willett
title	Specificity Residues Determine Binding Affinity for Two-Component Signal Transduction Systems
title_short	Specificity Residues Determine Binding Affinity for Two-Component Signal Transduction Systems
title_full	Specificity Residues Determine Binding Affinity for Two-Component Signal Transduction Systems
title_fullStr	Specificity Residues Determine Binding Affinity for Two-Component Signal Transduction Systems
title_full_unstemmed	Specificity Residues Determine Binding Affinity for Two-Component Signal Transduction Systems
title_sort	specificity residues determine binding affinity for two-component signal transduction systems
publisher	American Society for Microbiology
publishDate	2013
url	https://doaj.org/article/44cbad0c79704a19909f22850bfc00aa
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Specificity Residues Determine Binding Affinity for Two-Component Signal Transduction Systems

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