Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.

<h4>Background</h4>The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by three proteins, KaiA, KaiB and KaiC. Homo-hexameric KaiC displays kinase, phosphatase and ATPase activities; KaiA enhances KaiC phosphorylation and KaiB antagonizes KaiA....

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Autores principales:	Rekha Pattanayek, Tetsuya Mori, Yao Xu, Sabuj Pattanayek, Carl H Johnson, Martin Egli
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2009
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e22edcfd75044aa38efec1607945d156
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spelling	oai:doaj.org-article:e22edcfd75044aa38efec1607945d1562021-11-25T06:27:46ZStructures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.1932-620310.1371/journal.pone.0007529https://doaj.org/article/e22edcfd75044aa38efec1607945d1562009-11-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19956664/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by three proteins, KaiA, KaiB and KaiC. Homo-hexameric KaiC displays kinase, phosphatase and ATPase activities; KaiA enhances KaiC phosphorylation and KaiB antagonizes KaiA. Phosphorylation and dephosphorylation of the two known sites in the C-terminal half of KaiC subunits, T432 and S431, follow a strict order (TS-->pTS-->pTpS-->TpS-->TS) over the daily cycle, the origin of which is not understood. To address this void and to analyze the roles of KaiC active site residues, in particular T426, we determined structures of single and double P-site mutants of S. elongatus KaiC.<h4>Methodology and principal findings</h4>The conformations of the loop region harboring P-site residues T432 and S431 in the crystal structures of six KaiC mutant proteins exhibit subtle differences that result in various distances between Thr (or Ala/Asn/Glu) and Ser (or Ala/Asp) residues and the ATP gamma-phosphate. T432 is phosphorylated first because it lies consistently closer to Pgamma. The structures of the S431A and T432E/S431A mutants reveal phosphorylation at T426. The environments of the latter residue in the structures and functional data for T426 mutants in vitro and in vivo imply a role in dephosphorylation.<h4>Conclusions and significance</h4>We provide evidence for a third phosphorylation site in KaiC at T426. T426 and S431 are closely spaced and a KaiC subunit cannot carry phosphates at both sites simultaneously. Fewer subunits are phosphorylated at T426 in the two KaiC mutants compared to phosphorylated T432 and/or S431 residues in the structures of wt and other mutant KaiCs, suggesting that T426 phosphorylation may be labile. The structures combined with functional data for a host of KaiC mutant proteins help rationalize why S431 trails T432 in the loss of its phosphate and shed light on the mechanisms of the KaiC kinase, ATPase and phosphatase activities.Rekha PattanayekTetsuya MoriYao XuSabuj PattanayekCarl H JohnsonMartin EgliPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 4, Iss 11, p e7529 (2009)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Rekha Pattanayek Tetsuya Mori Yao Xu Sabuj Pattanayek Carl H Johnson Martin Egli Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
description	<h4>Background</h4>The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by three proteins, KaiA, KaiB and KaiC. Homo-hexameric KaiC displays kinase, phosphatase and ATPase activities; KaiA enhances KaiC phosphorylation and KaiB antagonizes KaiA. Phosphorylation and dephosphorylation of the two known sites in the C-terminal half of KaiC subunits, T432 and S431, follow a strict order (TS-->pTS-->pTpS-->TpS-->TS) over the daily cycle, the origin of which is not understood. To address this void and to analyze the roles of KaiC active site residues, in particular T426, we determined structures of single and double P-site mutants of S. elongatus KaiC.<h4>Methodology and principal findings</h4>The conformations of the loop region harboring P-site residues T432 and S431 in the crystal structures of six KaiC mutant proteins exhibit subtle differences that result in various distances between Thr (or Ala/Asn/Glu) and Ser (or Ala/Asp) residues and the ATP gamma-phosphate. T432 is phosphorylated first because it lies consistently closer to Pgamma. The structures of the S431A and T432E/S431A mutants reveal phosphorylation at T426. The environments of the latter residue in the structures and functional data for T426 mutants in vitro and in vivo imply a role in dephosphorylation.<h4>Conclusions and significance</h4>We provide evidence for a third phosphorylation site in KaiC at T426. T426 and S431 are closely spaced and a KaiC subunit cannot carry phosphates at both sites simultaneously. Fewer subunits are phosphorylated at T426 in the two KaiC mutants compared to phosphorylated T432 and/or S431 residues in the structures of wt and other mutant KaiCs, suggesting that T426 phosphorylation may be labile. The structures combined with functional data for a host of KaiC mutant proteins help rationalize why S431 trails T432 in the loss of its phosphate and shed light on the mechanisms of the KaiC kinase, ATPase and phosphatase activities.
format	article
author	Rekha Pattanayek Tetsuya Mori Yao Xu Sabuj Pattanayek Carl H Johnson Martin Egli
author_facet	Rekha Pattanayek Tetsuya Mori Yao Xu Sabuj Pattanayek Carl H Johnson Martin Egli
author_sort	Rekha Pattanayek
title	Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
title_short	Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
title_full	Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
title_fullStr	Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
title_full_unstemmed	Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.
title_sort	structures of kaic circadian clock mutant proteins: a new phosphorylation site at t426 and mechanisms of kinase, atpase and phosphatase.
publisher	Public Library of Science (PLoS)
publishDate	2009
url	https://doaj.org/article/e22edcfd75044aa38efec1607945d156
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Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase.

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