title: The Crp regulator of Pseudomonas putida: evidence of an unusually high affinity for its physiological effector, cAMP.
abstract: Although the genome of Pseudomonas putida KT2440 encodes an orthologue of the crp gene of Escherichia coli (encoding the cAMP receptor protein), the regulatory scope of this factor seems to be predominantly co-opted in this bacterium for controlling non-metabolic functions. In order to investigate the reasons for such a functional divergence in otherwise nearly identical proteins, the Crp regulator of P. putida (Crp(P. putida)) was purified to apparent homogeneity and subject to a battery of in vitro assays aimed at determining its principal physicochemical properties. Analytical ultracentrifugation indicated effector-free Crp(P. putida) to be a dimer in solution that undergoes a significant change in its hydrodynamic shape in the presence of cAMP. Such a conformational transition was confirmed by limited proteolysis of the protein in the absence or presence of the inducer. Thermodynamic parameters calculated by isothermal titration calorimetry revealed a tight cAMP-Crp(P. putida) association with an apparent K(D) of 22.5 ± 2.8 nM, i.e. much greater affinity than that reported for the E. coli's counterpart. The regulator also bound cGMP, but with a K(D) = 2.6 ± 0.3 µM. An in vitro transcription system was then set up with purified P. putida's RNA polymerase for examining the preservation of the correct protein-protein architecture that makes Crp to activate target promoters. These results, along with cognate gel retardation assays indicated that all basic features of the reference Crp(E. coli) protein are kept in the P. putida's counterpart, albeit operating under a different set of parameters, the extraordinarily high affinity for cAMP being the most noticeable.
authors: Systems Biology Program, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain.
authors: Alejandro
authors: A
authors: Arce-Rodríguez
authors: Gonzalo
authors: G
authors: Durante-Rodríguez
authors: Raúl
authors: R
authors: Platero
authors: Tino
authors: T
authors: Krell
authors: Belén
authors: B
authors: Calles
authors: Víctor
authors: V
authors: de Lorenzo
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002097:C-Reactive Protein / Q000502:physiology; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D029968:Escherichia coli Proteins; D002097:C-Reactive Protein; D000242:Cyclic AMP; D012321:DNA-Directed RNA Polymerases