Interaction between the H2 sensor HupUV and the histidine kinase HupT controls HupSL hydrogenase synthesis in Rhodobacter capsulatus.
The photosynthetic bacterium Rhodobacter capsulatus contains two [NiFe]hydrogenases: an energy-generating hydrogenase, HupSL, and a regulatory hydrogenase, HupUV. The synthesis of HupSL is specifically activated by H(2) through a signal transduction cascade comprising three proteins: the H(2)-sensing HupUV protein, the histidine kinase HupT, and the transcriptional regulator HupR. Whereas a phosphotransfer between HupT and HupR was previously demonstrated, interaction between HupUV and HupT was only hypothesized based on in vivo analyses of mutant phenotypes. To visualize the in vitro interaction between HupUV and HupT proteins, a six-His (His(6))-HupU fusion protein and the HupV protein were coproduced by using a homologous expression system. The two proteins copurified as a His(6)-HupUHupV complex present in dimeric and tetrameric forms, both of which had H(2) uptake activity. We demonstrated that HupT and HupUV interact and form stable complexes that could be separated on a native gel. Interaction was also monitored with surface plasmon resonance technology and was shown to be insensitive to salt concentration and pH changes, suggesting that the interactions involve hydrophobic residues. As expected, H(2) affects the interaction between HupUV and HupT, leading to a weakening of the interaction, which is independent of the phosphate status of HupT. Several forms of HupT were tested for their ability to interact with HupUV and to complement hupT mutants. Strong interaction with HupUV was obtained with the isolated PAS domain of HupT and with inactive HupT mutated in the phosphorylable histidine residue, but only the wild-type HupT protein was able to restore normal H(2) regulation.
Elsen
Sylvie
S
Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, UMR 5092 CNRS-CEA-UJF, Département Réponse et Dynamique Cellulaires, 38054 Grenoble Cedex 9, France.
Duché
Ophélie
O
Colbeau
Annette
A
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D000071677:Histidine Kinase; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D016296:Mutagenesis; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; C084201:HupT protein, Rhodobacter capsulatus; D009097:Multienzyme Complexes; D006859:Hydrogen; D006864:Hydrogenase; D011494:Protein Kinases; D000071677:Histidine Kinase