From PII signaling to metabolite sensing: a novel 2-oxoglutarate sensor that details PII-NAGK complex formation.
The widespread PII signal transduction proteins are known for integrating signals of nitrogen and energy supply and regulating cellular behavior by interacting with a multitude of target proteins. The PII protein of the cyanobacterium Synechococcus elongatus forms complexes with the controlling enzy...
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2013-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3861474?pdf=render |
| _version_ | 1817986127945007104 |
|---|---|
| author | Jan Lüddecke Karl Forchhammer |
| author_facet | Jan Lüddecke Karl Forchhammer |
| author_sort | Jan Lüddecke |
| collection | DOAJ |
| description | The widespread PII signal transduction proteins are known for integrating signals of nitrogen and energy supply and regulating cellular behavior by interacting with a multitude of target proteins. The PII protein of the cyanobacterium Synechococcus elongatus forms complexes with the controlling enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK) in a 2-oxoglutarate- and ATP/ADP-dependent manner. Fusing NAGK and PII proteins to either CFP or YFP yielded a FRET sensor that specifically responded to 2-oxoglutarate. The impact of the fluorescent tags on PII and NAGK was evaluated by enzyme assays, surface plasmon resonance spectroscopy and isothermal calorimetric experiments. The developed FRET sensor provides real-time data on PII - NAGK interaction and its modulation by the effector molecules ATP, ADP and 2-oxoglutarate in vitro. Additionally to its utility to monitor 2-oxoglutarate levels, the FRET assay provided novel insights into PII - NAGK complex formation: (i) It revealed the formation of an encounter-complex between PII and NAGK, which holds the proteins in proximity even in the presence of inhibitors of complex formation; (ii) It revealed that the PII T-loop residue Ser49 is neither essential for complex formation with NAGK nor for activation of the enzyme but necessary to form a stable complex and efficiently relieve NAGK from arginine inhibition; (iii) It showed that arginine stabilizes the NAGK hexamer and stimulates PII - NAGK interaction. |
| first_indexed | 2024-04-14T00:06:22Z |
| format | Article |
| id | doaj.art-05cb5ad000cc4f9287bb18f11436b36e |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-14T00:06:22Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-05cb5ad000cc4f9287bb18f11436b36e2022-12-22T02:23:31ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-01812e8318110.1371/journal.pone.0083181From PII signaling to metabolite sensing: a novel 2-oxoglutarate sensor that details PII-NAGK complex formation.Jan LüddeckeKarl ForchhammerThe widespread PII signal transduction proteins are known for integrating signals of nitrogen and energy supply and regulating cellular behavior by interacting with a multitude of target proteins. The PII protein of the cyanobacterium Synechococcus elongatus forms complexes with the controlling enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK) in a 2-oxoglutarate- and ATP/ADP-dependent manner. Fusing NAGK and PII proteins to either CFP or YFP yielded a FRET sensor that specifically responded to 2-oxoglutarate. The impact of the fluorescent tags on PII and NAGK was evaluated by enzyme assays, surface plasmon resonance spectroscopy and isothermal calorimetric experiments. The developed FRET sensor provides real-time data on PII - NAGK interaction and its modulation by the effector molecules ATP, ADP and 2-oxoglutarate in vitro. Additionally to its utility to monitor 2-oxoglutarate levels, the FRET assay provided novel insights into PII - NAGK complex formation: (i) It revealed the formation of an encounter-complex between PII and NAGK, which holds the proteins in proximity even in the presence of inhibitors of complex formation; (ii) It revealed that the PII T-loop residue Ser49 is neither essential for complex formation with NAGK nor for activation of the enzyme but necessary to form a stable complex and efficiently relieve NAGK from arginine inhibition; (iii) It showed that arginine stabilizes the NAGK hexamer and stimulates PII - NAGK interaction.http://europepmc.org/articles/PMC3861474?pdf=render |
| spellingShingle | Jan Lüddecke Karl Forchhammer From PII signaling to metabolite sensing: a novel 2-oxoglutarate sensor that details PII-NAGK complex formation. PLoS ONE |
| title | From PII signaling to metabolite sensing: a novel 2-oxoglutarate sensor that details PII-NAGK complex formation. |
| title_full | From PII signaling to metabolite sensing: a novel 2-oxoglutarate sensor that details PII-NAGK complex formation. |
| title_fullStr | From PII signaling to metabolite sensing: a novel 2-oxoglutarate sensor that details PII-NAGK complex formation. |
| title_full_unstemmed | From PII signaling to metabolite sensing: a novel 2-oxoglutarate sensor that details PII-NAGK complex formation. |
| title_short | From PII signaling to metabolite sensing: a novel 2-oxoglutarate sensor that details PII-NAGK complex formation. |
| title_sort | from pii signaling to metabolite sensing a novel 2 oxoglutarate sensor that details pii nagk complex formation |
| url | http://europepmc.org/articles/PMC3861474?pdf=render |
| work_keys_str_mv | AT janluddecke frompiisignalingtometabolitesensinganovel2oxoglutaratesensorthatdetailspiinagkcomplexformation AT karlforchhammer frompiisignalingtometabolitesensinganovel2oxoglutaratesensorthatdetailspiinagkcomplexformation |