Dataset Information

Oxidant sensor in the cGMP-binding pocket of PKGI? regulates nitroxyl-mediated kinase activity.

ABSTRACT: Despite the mechanisms for endogenous nitroxyl (HNO) production and action being incompletely understood, pharmacological donors show broad therapeutic promise and are in clinical trials. Mass spectrometry and site-directed mutagenesis showed that chemically distinct HNO donors 1-nitrosocyclohexyl acetate or Angeli's salt induced disulfides within cGMP-dependent protein kinase I-alpha (PKGI?), an interdisulfide between Cys42 of the two identical subunits of the kinase and a previously unobserved intradisulfide between Cys117 and Cys195 in the high affinity cGMP-binding site. Kinase activity was monitored in cells transfected with wildtype (WT), Cys42Ser or Cys117/195Ser PKGI? that cannot form the inter- or intradisulfide, respectively. HNO enhanced WT kinase activity, an effect significantly attenuated in inter- or intradisulfide-deficient PKGI?. To investigate whether the intradisulfide modulates cGMP binding, real-time imaging was performed in vascular smooth muscle cells expressing a FRET-biosensor comprising the cGMP-binding sites of PKGI?. HNO induced FRET changes similar to those elicited by an increase of cGMP, suggesting that intradisulfide formation is associated with activation of PKGI?. Intradisulfide formation in PKGI? correlated with enhanced HNO-mediated vasorelaxation in mesenteric arteries in vitro and arteriolar dilation in vivo in mice. HNO induces intradisulfide formation in PKGI?, inducing the same effect as cGMP binding, namely kinase activation and thus vasorelaxation.

SUBMITTER: Donzelli S

PROVIDER: S-EPMC5577323 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Oxidant sensor in the cGMP-binding pocket of PKGIα regulates nitroxyl-mediated kinase activity.

Donzelli Sonia S Goetz Mara M Schmidt Kjestine K Wolters Markus M Stathopoulou Konstantina K Diering Simon S Prysyazhna Oleksandra O Polat Volkan V Scotcher Jenna J Dees Christian C Subramanian Hariharan H Butt Elke E Kamynina Alisa A Schobesberger Sophie S King S Bruce SB Nikolaev Viacheslav O VO de Wit Cor C Leichert Lars I LI Feil Robert R Eaton Philip P Cuello Friederike F

Scientific reports 20170830 1

Despite the mechanisms for endogenous nitroxyl (HNO) production and action being incompletely understood, pharmacological donors show broad therapeutic promise and are in clinical trials. Mass spectrometry and site-directed mutagenesis showed that chemically distinct HNO donors 1-nitrosocyclohexyl acetate or Angeli's salt induced disulfides within cGMP-dependent protein kinase I-alpha (PKGIα), an interdisulfide between Cys42 of the two identical subunits of the kinase and a previously unobserved ...[more]

PMID: 28855531

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Project description:UnlabelledThe two-component system BvgAS controls the expression of the virulence regulon of Bordetella pertussis. BvgS is a prototype of bacterial sensor kinases with extracytoplasmic Venus flytrap perception domains. Following its transmembrane segment, BvgS harbors a cytoplasmic Per-Arnt-Sim (PAS) domain and then a predicted 2-helix coiled coil that precede the dimerization-histidine-phosphotransfer domain of the kinase. BvgS homologs have a similar domain organization, or they harbor only a predicted coiled coil between the transmembrane and the dimerization-histidine-phosphotransfer domains. Here, we show that the 2-helix coiled coil of BvgS regulates the enzymatic activity in a mechanical manner. Its marginally stable hydrophobic interface enables a switch between a state of great rotational dynamics in the kinase mode and a more rigid conformation in the phosphatase mode in response to signal perception by the periplasmic domains. We further show that the activity of BvgS is controlled in the same manner if its PAS domain is replaced with the natural α-helical sequences of PAS-less homologs. Clamshell motions of the Venus flytrap domains trigger the shift of the coiled coil's dynamics. Thus, we have uncovered a general mechanism of regulation for the BvgS family of Venus flytrap-containing two-component sensor kinases.ImportanceThe two-component system BvgAS of the whooping cough agent Bordetella pertussis regulates the virulence factors necessary for infection in a coordinated manner. BvgS is the prototype of a family of sensor kinase proteins found in major bacterial pathogens. When BvgS functions as a kinase, B. pertussis is virulent, and the bacterium shifts to an avirulent phase after BvgS senses chemicals that make it switch to phosphatase. Our goal is to decipher the signaling mechanisms of BvgS in order to understand virulence regulation in Bordetella, which may lead to new antimicrobial treatments targeting those two-component systems. We discovered that the activity of BvgS is regulated in a mechanical manner. A short region of the protein that precedes the enzymatic domain switches between two states in response to signal perception by other BvgS domains. This switch region is conserved among BvgS homologs, and thus, the regulation uncovered here will likely be relevant for the family.

Dataset Information

Oxidant sensor in the cGMP-binding pocket of PKGI? regulates nitroxyl-mediated kinase activity.

Publications

Oxidant sensor in the cGMP-binding pocket of PKGIα regulates nitroxyl-mediated kinase activity.

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